compositions and methods for the treatment of hemoglobinopathies

专利摘要:
The present invention is directed to genome editing systems, reagents and methods for the treatment of hemoglobinopathies.
公开号:BR112019016205A2
申请号:R112019016205-4
申请日:2018-02-05
公开日:2020-07-14
发明作者:Craig Stephen Mickanin；Christian SCHMEDT；Jennifer SNEAD；Susan C. Stevenson；Yi Yang
申请人:Novartis Ag；Intellia Therapeutics, Inc.；
IPC主号:

专利说明:

[0001] [0001] This application claims priority for US Provisional Patent Application 62 / 455,464, filed on February 6, 2017, the content of which is incorporated herein by reference in its entirety. SEQUENCE LISTING
[0002] [0002] The present application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 30, 2018, is called PATOS57603-WO-PCT SL.txt and is 258,837 bytes in size. BACKGROUND
[0003] [0003] CRISPRs (Short Palindromic Repetitions Grouped Regularly Interleaved) evolved in bacteria as an adaptive immune system to defend against viral attacks. After exposure to a virus, small segments of viral DNA are integrated into the CRISPR locus of the bacterial genome. The RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. This RNA, which contains the sequence complementary to the viral genome, mediates the targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thus silences the viral target.
[0004] [0004] Recently, the CRISPR / Cas system was adapted for genome editing in eukaryotic cells. The introduction of single site-specific breaks (SSBs) or double-strand breaks (DSBs) allows for the alteration of the target sequence through, for example, non-homologous edge splicing (NHEJ) or homology-directed repair (HDR). SUMMARY OF THE INVENTION
[0005] [0005] Without being limited by theory, the invention is based in part on the discovery that CRISPR systems, for example Cas9 CRISPR systems, for example, as described herein, can be used to modify cells (for example, stem cells and hematopoietic progenitors (HSPCs)), for example, in a non-deletion HPFH region, as described herein, to increase fetal hemoglobin (HbF) expression and / or decrease beta globin expression (for example, a beta globin gene with a disease-causing mutation), for example, in the offspring, for example, offspring of the red blood cells, of the modified cells, and that the modified cells (for example, modified HSPCs) can be used to treat hemoglobinopathies, for example, sickle cell disease and beta-thalassemia.
[0006] [0006] Thus, in one aspect, the invention provides CRISPR systems (for example, Cas CRISPR systems, for example, Cas9 CRISPR systems, for example, Cas9 CRISPR systems of S. pyogenes) comprising one or more, for example, a molecule of gRNA as described herein. Any of the gRNA molecules described herein can be used in such systems, and in the methods and cells described herein.
[0007] [0007] In one aspect, the invention provides a gRNA molecule, including a tracr and cr »RNA, wherein the crRNA includes a target domain that: a) is complementary to a target sequence from a non-deletion HFPH region ( for example, a non-human deletion HPFH region); b) is complementary to a target sequence in the genomic nucleic acid sequence at Chr11: 5,249,833 to Chr11: 5,250,237, - strand, ho38; c) is complementary to a target sequence in the genomic nucleic acid sequence at Chr11: 5,254,738 to Chr11: 5,255,164, - strand, ho38; d) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,250,094-5,250,237, - strand, ho38; e) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,255,022-5,255,164, - strand, hg38; f) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,249,833-5,249,927, - strand, hg38;
[0008] [0008] In modalities, the target domain includes, for example, consists of any one of SEQ ID NO: 1 to SEQ ID NO: 72. In modalities, the target domain includes, for example, consists of any SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO : 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO:
[0009] [0009] In any of the aspects and modalities mentioned above, the gRNA molecule can still have regions and / or properties described here. In embodiments, the gRNA molecule includes a fragment from any of the aforementioned target domains. In modalities, the target domain includes, for example,
[0010] [0010] In one aspect, including in any of the above mentioned aspects and modalities, the crRNA portion and a tracr portion hybridize to form a mast, including SEQ ID NO: 182 or
[0011] [0011] In one aspect, including in any of the aspects and modalities mentioned above, the tracr includes SEQ ID NO: 224, or a SEQ ID NO: 225. In one aspect, including in any of the aspects and modalities mentioned above, tracr includes SEQ ID NO: 232, optionally additionally including, at the 3 ', 1, 2, 3, 4, 5, 6 or 7 additional uracil (U) nucleotides. In one aspect, including any of the aspects and modalities mentioned above, the crRNA includes, from 5 'to 3', [target domain] 1: a) SEQ ID NO: 182; b) SEQ ID NO: 183; c) SEQ ID NO: 199; d) SEQ ID NO: 200; e) SEQ ID NO: 201; f) SEQ ID NO: 202; or g) SEQ ID NO: 226.
[0012] [0012] In one aspect, including any of the aspects and modalities mentioned above, the tracr includes, from 5 'to 3: a) SEQ ID NO: 187; b) SEQ ID NO: 188; c) SEQ ID NO: 203; d) SEQ ID NO: 204; e) SEQ ID NO: 224; f) SEQ ID NO: 225; g) SEQ ID NO: 232; h) SEQ ID NO: 227; i) (SEQ ID NO: 228; j) SEQ ID NO: 229 ;; k) any of a) to j), above, including, at the 3 'end, at least 1, 2,3,4,5,6 or 7 nucleotides of uracil (U), for example, 1, 2, 3, 4, 5, 6 or 7 uracil nucleotides (U); |) any of a) to k), above, also including, at the 3 'end, at least 1, 2, 3, 4, 5, 6 or 7 adenine nucleotides (A), for example, 1, 2, 3, 4, 5, 6 or 7 nucleotides of adenine (A); or m) any of a) to |), above, further including, at the 5 'end (for example, at the 5' end), at least 1, 2,3,4,5,6 or 7 adenine nucleotides (A ), for example, 1, 2, 3, 4, 5, 6 or 7 adenine nucleotides (A).
[0013] [0013] In one aspect, including any of the aspects and modalities mentioned above, the target domain and tracr are arranged in separate nucleic acid molecules. In one aspect, including any of the above-mentioned aspects and modalities, the target domain and tracr are arranged in separate nucleic acid molecules and the nucleic acid molecule including the target domain includes SEQ ID NO: 201, optionally arranged immediately 3 'to the target domain, and the nucleic acid molecule including tracr includes, for example, consists of, SEQ ID NO:
[0014] [0014] In one aspect, including any of the aspects and modalities mentioned above, the target domain and tracr are arranged in a single nucleic acid molecule, for example, where the tracr is arranged 3 'in the target domain . In one aspect, the gRNA molecule includes a loop, arranged 3 "in relation to the target domain and 5" in relation to the tracr. In embodiments, the loop includes SEQ ID NO: 186. In one aspect, including in any of the aspects and modalities mentioned above, the gRNA molecule includes, from 5 'to 3', [target domain] -: (a ) SEQ ID NO: 195; (b) SEQ ID NO: 196; (c) SEQ ID NO: 197; (d) SEQ ID NO: 198; (e) SEQ ID NO: 231; or (f) any one of (a) to (e), above, further including, at the 3 'end, 1,2,3, 4, 5, 6 or 7 uracil nucleotides (U).
[0015] [0015] In one aspect, including any of the aspects and modalities mentioned above, the target domain and tracr are arranged in a single nucleic acid molecule and in which said nucleic acid molecule includes, for example, consists of, said target domain and SEQ ID NO: 231, optionally immediately arranged 3 'with respect to said target domain.
[0016] [0016] In one aspect, including any of the aspects and modalities mentioned above, one, or optionally more than one, of the nucleic acid molecules including the gRNA molecule includes:
[0017] [0017] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 74; (b) SEQ ID NO: 75; or (c) SEQ ID NO: 76.
[0018] [0018] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 77 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 77 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQID NO: 78 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 78 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0019] [0019] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 79; (b) SEQ ID NO: 80; or (c) SEQ ID NO: 81.
[0020] [0020] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 82 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 82 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 83 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 83 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0021] [0021] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 84; (b) SEQ ID NO: 85; or (c) SEQ ID NO: 86.
[0022] [0022] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 87 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 87 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 88 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 88 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0023] [0023] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 89; (b) SEQ ID NO: 90; or (c) SEQ ID NO: 91.
[0024] [0024] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 92 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 92 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 93 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of SEQ ID NO: 93 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0025] [0025] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 94; (b) SEQ ID NO: 95; or (c) SEQ ID NO: 96.
[0026] [0026] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 97 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 97 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 98 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of SEQ ID NO: 98 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0027] [0027] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 99; (b) SEQ ID NO: 100; or (c) SEQ ID NO: 101.
[0028] [0028] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 102 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 102 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 103 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 103 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0029] [0029] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 104; (b) SEQ ID NO: 105; or (c) SEQ ID NO: 106.
[0030] [0030] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 107 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 107 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 108 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 108 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0031] [0031] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 109; (b) SEQ ID NO: 110; or (c) SEQ ID NO: 111.
[0032] [0032] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 112 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 112 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 113 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of SEQ ID NO: 113 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0033] [0033] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 114; (b) SEQ ID NO: 115; or (c) SEQ ID NO: 116.
[0034] [0034] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 117 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 117 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 118 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 118 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0035] [0035] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 119; (b) SEQ ID NO: 120; or (c) SEQ ID NO: 121.
[0036] [0036] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 122 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 122 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 123 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 123 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0037] [0037] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 124; (b) SEQ ID NO: 125; or (c) SEQ ID NO: 126.
[0038] [0038] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 127 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 127 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 128 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 128 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0039] [0039] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 129; (b) SEQ ID NO: 130; or (c) SEQ ID NO: 131.
[0040] [0040] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 132 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 132 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 133 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of SEQ ID NO: 133 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0041] [0041] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 134; (b) SEQ ID NO: 135; or (c) SEQ ID NO: 136.
[0042] [0042] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 137 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 137 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 138 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 138 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0043] [0043] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 139; (b) SEQ ID NO: 140; or (c) SEQ ID NO: 141.
[0044] [0044] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 142 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 142 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 143 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 143 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0045] [0045] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 144; (b) SEQ ID NO: 145; or (c) SEQ ID NO: 146.
[0046] [0046] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 147 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 147 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 148 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of SEQ ID NO: 148 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0047] [0047] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 149; (b) SEQ ID NO: 150; or (c) SEQ ID NO: 151.
[0048] [0048] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 152 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 152 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 153 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 153 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0049] [0049] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 154; (b) SEQ ID NO: 155; or (c) SEQ ID NO: 156.
[0050] [0050] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 157 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 157 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 158 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 158 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0051] [0051] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 159; (b) SEQ ID NO: 160; or (c) SEQ ID NO: 161.
[0052] [0052] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 162 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 162 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 163 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 163 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0053] [0053] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 164; (b) SEQ ID NO: 165; or (c) SEQ ID NO: 166.
[0054] [0054] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 167 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 167 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 168 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 168 and a tracer including, for example, consisting of, SEQ ID NO: 73.
[0055] [0055] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 169; (b) SEQ ID NO: 170; or (c) SEQ ID NO: 171.
[0056] [0056] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 172 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 172 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 173 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of SEQ ID NO: 173 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0057] [0057] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ ID NO: 176.
[0058] [0058] In one aspect, the invention provides a gRNA molecule including, for example, consisting of, the sequence: (a) a crRNA including, for example, consisting of, SEQ ID NO: 177 and a tracr including, for example example, consisting of, SEQ ID NO: 224; (b) a crRNA including, for example, consisting of, SEQ ID NO: 177 and a tracer including, for example, consisting of, SEQ ID NO: 73; (c) a crRNA including, for example, consisting of, SEQ ID NO: 178 and a tracer including, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA including, for example, consisting of, SEQ ID NO: 178 and a tracer including, for example, consisting of SEQ ID NO: 73.
[0059] [0059] In one aspect, including in any of the aspects and modalities mentioned above, the invention provides a gRNA molecule in which: a) when a CRISPR system (for example, an RNP as described here) including a gRNA molecule is introduced into a cell, an indel is formed at or near the target sequence complementary to the target domain of the gRNA molecule; and / or b) when a CRISPR system (for example, an RNP as described herein) including the gRNA molecule is introduced into a cell, a deletion including the sequence is created, for example, including substantially the entire sequence, between a sequence complementary to the gRNA target domain (for example, at least 90% complementary to the gRNA target domain, for example, fully complementary to the gRNA target domain) in the HBG1 promoter region and a complementary sequence to the gRNA target domain ( for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region. In embodiments, the indel does not include a nucleotide from a transcription factor or non-deletion HPFH binding site.
[0060] [0060] In one aspect, including in any of the aspects and modalities mentioned above, the invention provides a gRNA molecule, in which a CRISPR system (e.g., an RNP as described here) including the gRNA molecule is introduced in a population of cells, an indel is formed at or near the target sequence complementary to the target domain of the gRNA molecule by at least about 15%, e.g., at least about 17%, e.g. at least about 20%, eg at least about 30%, eg at least about 40%, eg at least about 50%, eg at least at least about 55%, e.g., at least about 60%, e.g., at least about 70%, e.g., at least about 75% of the population cells. In one respect,
[0061] [0061] In one aspect, including in any of the aspects and modalities mentioned above, the invention provides a gRNA molecule, wherein, when a CRISPR system (e.g., an RNP as described here) including the gRNA molecule is introduced into a cell, fetal hemoglobin expression is increased in said cell or its progeny, eg, its erythroid progeny, eg, its red blood cell progeny. In embodiments, when a CRISPR system (for example, an RNP, as described herein), including the gRNA molecule, is introduced into a population of cells, the percentage of F cells in that population, or population of its offspring, by example, your erythroid progeny, for example, your red blood cell progeny is increased by at least about 15%, for example, at least about 17%, for example, at least about 20%, for example, by at least about 25%, for example, at least about 30%, for example, at least about 35%, for example, at least about 40%, relative to the percentage of F cells in a population of cells to which gRNA molecule has not been introduced or a population of its progeny, for example, its erythroid progeny, for example, its progeny of red blood cells. In embodiments, said cell or its progeny, eg, its erythroid progeny, eg, its red blood cell progeny, produces at least about 6 picograms (eg, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or about 8 to about 9 picograms or about 9 to about 10 picograms) of fetal hemoglobin per cell.
[0062] [0062] In one aspect, including in any of the aspects and modalities mentioned above, the invention provides a gRNA molecule, in which a CRISPR system (e.g., an RNP as described here), including a gRNA molecule, is introduced into a cell, for example, indels outside the target do not form outside the promoter regions of HBG1 and / or HBG 2 (for example, within a gene, for example, a coding region for a gene), for example, as detectable by next generation sequencing and / or nucleotide insertion assay.
[0063] [0063] In one aspect, including in any of the aspects and modalities mentioned above, the invention provides a gRNA molecule, in which a CRISPR system (e.g., an RNP as described here), including a gRNA molecule, is introduced into a cell population, no indel outside the target, for example, no indel outside the target outside the promoter regions of HBG1 and / or HBG 2 (for example, within a gene, for example, a coding region for a gene), is detected in more than about 5%, for example, more than about 1%, for example, more than about 0.1%, for example, more than about 0.01% of the cells of the cell population, for example, as detectable by next generation sequencing and / or nucleotide insertion assay.
[0064] [0064] In one aspect, including in any of the aspects and modalities mentioned above, the cell is (or the population of cells includes) a mammalian, primate or human cell, for example, it is a human cell, for example, the cell is (or population of cells includes) an HSPC, for example, HSPC is CD34 +, for example, HSPC is CD34 + CD90 +. In embodiments, the cell is autologous with respect to a patient to be administered with said cell. In other embodiments, the cell is allogeneic with respect to a patient to be administered with said cell.
[0065] [0065] In one aspect, gRNA molecules, genome editing systems (for example, CRISPR systems) and / or methods described here refer to cells, for example, as described herein, that include or result in one or more more of the following properties: (a) at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the cells in a cell population described herein, comprise an indel in or near of a genomic DNA sequence complementary to the target domain of the gRNA molecule described here, optionally where the indel is selected from an indel listed in Table 2-7, optionally where no cell in the population comprises a deletion of a nucleotide disposed between 5,250,092 and 5,249,833, - chain (hg38); (b) a cell (eg, cell population) described herein is capable of differentiating into a cell differentiated from an erythroid lineage (eg, a red blood cell) and in which said differentiated cell exhibits an increased level of fetal hemoglobin , for example, in relation to an unchanged cell (for example, cell population); (c) a population of cells described herein is able to differentiate into a population of differentiated cells, for example, a population of cells of an erythroid lineage (for example, a population of red blood cells) and in which said population of cells differentiated cells have an increased percentage of F cells (for example, at least about 15%, at least about 20%, at least about 25%, at least about 30% or at least about 40% more F cells high), for example, in relation to a population of unchanged cells; (d) a cell (for example, a population of cells) described herein is capable of differentiating into a differentiated cell, for example, a cell of an erythroid lineage (for example, a red blood cell) and in which said cell is differentiated (for example, a population of differentiated cells) produces at least about 6 picograms (for example, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about picograms or about from 8 to about 9 picograms or from 9 to about 10 picograms) of fetal hemoglobin per cell; (e) no off-target indels form in a cell described herein, for example, off-target indels do not form outside the HBG1 and / or HBG2 promoter regions (for example, a gene, for example, a coding region for a gene), for example, as detectable by next generation sequencing and / or nucleotide insertion assay; (f) indel is not detected outside the target, for example, indel is not detected outside the target outside the HBG1 and / or HBG2 promoter regions (for example, within a gene, for example, a coding region for a gene) in more than about 5%, for example, more than about 1%, for example, more than about 0.1%, for example, more than about 0.01% of the cells of a population of cells described herein, for example, as detectable by next generation sequencing and / or a nucleotide insertion assay; (g) a cell described herein or its progeny is detectable, for example, detectable in bone marrow or detectable in peripheral blood, in a patient who has been transplanted for more than 16 weeks, for more than 20 weeks or for more than 24 weeks after transplantation, optionally detected by detecting an indel in one or near a genomic DNA sequence complementary to the target domain of a gRNA molecule, as defined in any of claims 1-22, optionally where the indel is selected from an indel listed in Table 2-7, optionally where the indel is a large deletion indel. (h) a population of cells described herein is able to differentiate into a population of differentiated cells, for example, a population of cells of an erythroid lineage (for example, a population of red blood cells) and in which said population of cells differentiated cells includes a reduced percentage of sickle cells (for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% less sickle cell percentage), for example, in relation to a population of unchanged cells; and / or (i) a cell or cell population described herein is capable of differentiating into a population of differentiated cells, for example, a cell population of an erythroid lineage (for example, a population of red blood cells) and in which said population of differentiated cells includes cells that produce a low level (for example, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40 %, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the lowest level) of sickle hemoglobin (HbS), for example , in relation to a population of unchanged cells.
[0066] [0066] In one aspect, the invention provides a composition including: 1) one or more gRNA molecules (including a first gRNA molecule) described herein, for example, of any of the aspects and modalities of the aforementioned gRNA molecule, and a Cas9 molecule, for example, described herein; 2) one or more gRNA molecules (including a first gRNA molecule) described herein, for example, from any of the aspects and modalities of the gRNA molecule mentioned above, and nucleic acid encoding a Cas9 molecule, for example, described herein; 3) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule) described herein, for example, from any of the aspects and modalities of the aforementioned gRNA molecule, and a Cas9 molecule, for example, described herein; 4) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule) described herein, for example, from any of the aspects and modalities of the gRNA molecule mentioned above, and nucleic acid encoding a Cas9 molecule, for example, described herein; or 5) any one of 1) to 4), above, and a template nucleic acid; or 6) any one of 1) to 4) above, and nucleic acid including sequence encoding a template nucleic acid.
[0067] [0067] In one aspect, the invention provides a composition including a first gRNA molecule described herein, for example, of any of the above mentioned aspects and modalities of gRNA, further including a Cas9 molecule, for example, described herein, for example , where the Cas9 molecule is active or inactive s9. pyogenes, for example, in which the Cas9 molecule includes SEQ ID NO: 205. In aspects, the Cas9 molecule includes, for example, consists of: (a) SEQ ID NO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e) SEQ ID NO: 237; (f) SEQ ID NO: 238; (g) SEQ ID NO: 239; (h) SEQ ID NO: 240; (i) SEQ ID NO: 241; (]) SEQ ID NO: 242; (k) SEQ ID NO: 243 or (1) SEQ ID NO: 244.
[0068] [0068] In one aspect, including any of the aspects and modalities of the composition mentioned above, the first gRNA molecule and the Cas9 molecule are present in a ribonuclear protein complex (RNP).
[0069] [0069] In one aspect, including in any of the above-mentioned aspects and modalities of the composition, the invention provides a composition further comprising a second gRNA molecule; a second gRNA molecule and a third gRNA molecule; or a second gRNA molecule, optionally a third gRNA molecule and, optionally, a fourth gRNA molecule, wherein the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule are a gRNA molecule described herein, for example, are a gRNA molecule of any of the aspects and modalities described above of the gRNA molecule and wherein each gRNA molecule of the composition is complementary to a different target sequence. In embodiments, two or more of the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule and the optional fourth gRNA molecule are complementary with target sequences within the same gene or region.
[0070] [0070] In one aspect, including any of the above-mentioned aspects and modalities of the composition, the composition includes a first gRNA molecule and a second gRNA molecule, wherein: a) the first gRNA molecule is complementary to a sequence target including at least 1 nucleotide (for example, including 20 consecutive nucleotides) in: i) Chr11: 5,249,833 to Chr11: 5,250,237 (hg38); ii) Chr11: 5,250,094-5,250,237 (hg38); iii) Chr11: 5,249,833-5,249,927 (hg38); or iv) Chr11: 5,250,139-5,250,237 (hg38); b) the second gRNA molecule is complementary to a target sequence including at least 1 nucleotide (for example, comprising 20 consecutive nucleotides) in: i) Chr11: 5,254,738 to Chr11: 5,255,164 (hg38); ii) Chr11: 5,255,022-5,255,164 (hg38); or iii) Chr11: 5,254,738-5,254,851 (hg38).
[0071] [0071] In one aspect, with respect to the components of the composition's gRNA molecule, the composition consists of a first gRNA molecule and a second gRNA molecule.
[0072] [0072] In one aspect, including in any of the above mentioned aspects and modalities of the composition, each of said gRNA molecules is found in a ribonuclear protein (RNP) complex with a Cas9 molecule, for example, described herein.
[0073] [0073] In one aspect, including in any of the above-mentioned aspects and modalities of the composition, the composition includes a template nucleic acid, wherein the template nucleic acid includes a nucleotide that corresponds to a nucleotide in or near the target sequence of the first gRNA molecule. In embodiments, the template nucleic acid includes nucleic acid that encodes: (a) human beta globin, e.g., human beta globin including one or more of the G16D, E22A and T87Q mutations or fragment thereof; or (b) human gamma globin, or fragment thereof.
[0074] [0074] In one aspect, including any of the above mentioned aspects and modalities of the composition, the composition is formulated in a suitable medium for electroporation.
[0075] [0075] In one aspect, including in any of the above mentioned aspects and modalities of the composition, each of said gRNA molecules of said composition is in an RNP with a Cas9 molecule described here and in which each of said RNP is a concentration of less than about 10 µM, e.g., less than about 3 µM, e.g., less than about 1 µM, e.g., less than about 0.5 µM, e.g., less than about 0.3 µM, e.g., less than about 0.1 µM. In modalities the RNP is at a concentration of about 1 µM. In modalities the RNP is at a concentration of about 2 µM. In embodiments, said concentration is the concentration of RNP in a composition comprising cells, for example, as described herein, optionally, in which the composition comprising cells and RNP is suitable for electroporation.
[0076] [0076] In one aspect, the invention provides a nucleic acid sequence that encodes one or more gRNA molecules described herein, for example, from any of the above mentioned aspects and modalities of gRNA molecules. In embodiments, the nucleic acid that includes a promoter operably linked to the sequence encoding one or more gRNA molecules, for example, the promoter is a promoter recognized by an I RNA polymerase or RNA polymerase III or, for example, the promoter is a U6 promoter or an HI promoter.
[0077] [0077] In one aspect, including any of the above-mentioned aspects and modalities of the nucleic acid, the nucleic acid further encodes a Cas9 molecule, for example, a Cas9 molecule that includes, for example, consists of any of SEQ ID NO : 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240 , SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO: 244. In embodiments, said nucleic acid includes a promoter operably linked to the sequence encoding a Cas9 molecule, for example, a promoter EF-1, a CMV IE gene promoter, an EF-1a promoter, a ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter.
[0078] [0078] In one aspect, the invention provides a vector including nucleic acid of any of the above-mentioned aspects and modalities of nucleic acid. In modalities, the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid and a vector of RNA.
[0079] [0079] In one aspect, the invention provides a method of altering a cell (e.g., a population of cells), (e.g., altering the structure (e.g., sequence) of nucleic acid) on or near a target sequence within said cell, including contacting (e.g., introduction into) said cell (e.g., cell population) with: 1) one or more gRNA molecules described herein ( for example, of any of the aspects and modalities of the gRNA molecule mentioned above) and a Cas9 molecule, for example, described herein; 2) one or more gRNA molecules described herein (for example, from any of the aspects and modalities of the gRNA molecule mentioned above) and nucleic acid encoding a Cas9 molecule, for example, described herein; 3) nucleic acid encoding one or more gRNA molecules described herein (for example, from any of the aspects and modalities of the gRNA molecule mentioned above) and a Cas9 molecule, for example, described herein; 4) nucleic acid encoding one or more gRNA molecules described herein (for example, from any of the aspects and modalities of the gRNA molecule mentioned above) and nucleic acid encoding a Cas9 molecule, for example, described herein; 5) any one of 1) to 4), above, and a template nucleic acid; 6) any one of 1) to 4) above, and nucleic acid including sequence encoding a template nucleic acid; 7) a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities of the composition; or 8) a vector described herein, for example, a vector of any of the above-mentioned aspects and modalities of the vector.
[0080] [0080] In one aspect, including in any of the above aspects and modalities of the method, the gRNA molecule or nucleic acid encoded the gRNA molecule, and the Cas9 molecule or nucleic acid encoding the Cas9 molecule, are formulated in a single composition. In another aspect, the gRNA or nucleic acid molecule encoding the gRNA molecule, and the Cas9 molecule or nucleic acid encoding the Cas9 molecule, are formulated in more than one composition. In one aspect, more than one composition is administered simultaneously or sequentially.
[0081] [0081] In one aspect of the methods described herein, including in any of the above mentioned aspects and modalities of the method, the cell is an animal cell, for example, the cell is a mammalian, primate or human cell, for example, the cell is a hematopoietic stem cell or progenitor (HSPC) (for example, a population of HSPCs), for example, the cell is a CD34 + cell, for example, the cell is a CD34 + CD90 + cell. In embodiments of the methods described herein, the cell is arranged in a composition including a population of cells that has been enriched for CD34 + cells. In modalities of the methods described herein, the cell (e.g., cell population) was isolated from bone marrow, mobilized peripheral blood, or umbilical cord blood. In embodiments of the methods described herein, the cell is autologous or allogeneic, for example, autologous with respect to a patient to be administered with said cell.
[0082] [0082] In one aspect of the methods described herein, including in any of the aforementioned aspects and modalities of the method, a) results in altering an indel in or near a genomic DNA sequence complementary to the target domain of one or more gRNA molecules; or b) the change results in a deletion including the sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of one or more gRNA molecules (for example, at least 90% complementary to the target domain of gRNA, for example, completely complementary to the gRNA target domain) in the HBG1 promoter region and a sequence complementary to the target domain of one or more gRNA molecules (for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region. In aspects of the method, the indel is an insertion or deletion of less than about 40 nucleotides, eg, less than 30 nucleotides, eg, less than 20 nucleotides, eg, less than that 10 nucleotides, for example, is a single nucleotide deletion.
[0083] [0083] In one aspect of the methods described herein, including any of the above-mentioned aspects and modalities of the method, the method results in a population of cells in which at least about 15%, for example, at least about 17% , for example, at least about 20%, for example, at least about 30%, for example, at least about 40%, for example, at least about 50%, for example, at least about 55% , for example, at least 60%, for example, at least about 70%, at least about 75% of the population has been changed, for example, to include an indel.
[0084] [0084] In one aspect of the methods described here, including in any of the above mentioned aspects and modalities of the method, the change results in a cell (eg cell population) that is capable of differentiating into a differentiated cell of an erythroid lineage (eg, a red blood cell) and in which said differentiated cell exhibits an increased level of fetal hemoglobin, eg, relative to an unchanged cell (eg, cell population) .
[0085] [0085] In one aspect of the methods described herein, including in any of the above mentioned aspects and modalities of the method, the change results in a population of cells that is capable of differentiating into a population of differentiated cells, e.g. a population of cells of an erythroid lineage (eg, a population of red blood cells) and where said population of differentiated cells has an increased percentage of F cells (eg, at least about 15%, at least about 20%, at least about 25%, at least about 30% or at least about 40% higher percentage of F cells), e.g., relative to an unchanged cell population.
[0086] [0086] In one aspect of the methods described here, including in any of the above mentioned aspects and modalities of the method, the change results in a cell that is capable of differentiating into a differentiated cell, eg, a cell of a erythroid lineage (eg, a red blood cell) and wherein said differentiated cell produces at least about 6 picograms (eg, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or about 8 to about 9 picograms or about 9 to about 10 picograms) of fetal hemoglobin per cell.
[0087] [0087] In one aspect, the invention provides a cell, altered by a method described herein, for example, a method of any of the above-mentioned aspects and modalities of the method.
[0088] [0088] In one aspect, the invention provides a cell, obtainable by a method described herein, for example, a method of any of the above mentioned aspects and modalities of the method.
[0089] [0089] In one aspect, the invention provides a cell, including a first gRNA molecule, described herein, for example, of any of the above mentioned aspects or modalities of the gRNA molecule, or a composition, described herein, for example, of any of the above-mentioned aspects or modalities of the composition, a nucleic acid, described herein, for example, of any of the above-mentioned aspects or modalities of nucleic acid, or a vector, described, for example, any of the aspects or above mentioned vector modalities.
[0090] [0090] In one aspect of the cell described herein, including in any of the above-mentioned aspects and modalities of the cell, the cell further includes a Cas9 molecule, for example, described herein, for example, a Cas9 molecule that includes any of the SEQs ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO : 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO: 244.
[0091] [0091] In one aspect of the cell described herein, including in any of the above-mentioned aspects and modalities of the cell, the cell includes, included or will include, a second gRNA molecule described herein, for example, of any of the aspects or modalities mentioned above of the gRNA molecule, or a nucleic acid encoding said gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule include non-identical target domains.
[0092] [0092] In one aspect of the cell described here, including in any of the above-mentioned aspects and modalities of the cell, fetal hemoglobin expression is increased in said cell or its progeny (eg, its erythroid progeny, eg its progeny of red blood cells) in relation to a cell or its progeny of the same type of cell that has not been modified to include a gRNA molecule.
[0093] [0093] In one aspect of the cell described herein, including in any of the above mentioned aspects and modalities of the cell, the cell is able to differentiate into a differentiated cell, e.g., a cell of an erythroid lineage (e.g. a red blood cell) and wherein said differentiated cell exhibits an increased level of fetal hemoglobin, e.g., relative to a cell of the same type that has not been modified to include a gRNA molecule.
[0094] [0094] In one aspect of the cell described here, including in any of the above-mentioned aspects and modalities of the cell, the differentiated cell (eg, cell of an erythroid lineage, eg,
[0095] [0095] In one aspect of the cell described here, including in any of the above-mentioned aspects and modalities of the cell, the cell was brought into contact, for example, brought into contact ex vivo, with a stem cell expander, for example, a stem cell expander selected from: a) (1r, 4r) -N '- (2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b ] indol-4-yl) cyclohexane-1,4-diamine; b) methyl 4- (3-piperidin-1-ylpropylamino) -9H-pyrimido [4,5-b] indole-7-carboxylate; c) 4- (2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol; d) (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1- 01; or e) their combinations (for example, a combination of (1r, 4r) -N '- (2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5- b] indol-4-yl) cyclohexane-1,4-diamine and (S) - 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3 -il) -9H-purin-9- iNpropan-1-ol. In embodiments, the stem cell expander is (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) - 9H-purin-9-yl) propan-1-ol.
[0096] [0096] In one aspect of the cell described herein, including any of the above-mentioned aspects and modalities of the cell, the cell includes: a) an indel in or near a genomic DNA sequence, complementary to the target domain of a molecule of gRNA described herein, for example, from any of the above mentioned aspects or modalities of the gRNA molecule; or b) a deletion including sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of a gRNA molecule described herein, for example, of any of the above mentioned aspects or modalities of the gRNA molecule (for example, example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG1 promoter region and a sequence complementary to the target domain of a gRNA molecule described herein, for example , of any of the aforementioned aspects or modalities of the gRNA molecule, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region. In one aspect, the indel is an insertion or deletion of less than about 40 nucleotides, eg, less than 30 nucleotides, eg, less than 20 nucleotides, eg, less than 10 nucleotides, for example, the indel is a single nucleotide deletion.
[0097] [0097] In one aspect of the cell described herein, including in any of the aspects and modalities of cells mentioned above, the cell is an animal cell, for example, the cell is a mammalian, primate or human cell. In one aspect, the cell is a hematopoietic stem cell or progenitor (HSPC) (for example, a population of HSPCs), for example, the cell is a CD34 + cell, for example, the cell is a CD34 + CD90 + cell. In modalities, the cell (for example, cell population) was isolated from bone marrow, mobilized peripheral blood, or umbilical cord blood. In embodiments, the cell is autologous with respect to a patient to be administered with said cell. In embodiments, the cell is allogeneic with respect to a patient to be administered with said cell.
[0098] [0098] In one aspect, the invention provides a population of cells described herein, for example, a population of cells that includes a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell. In aspects, the invention provides a population of cells, in which at least about 50%, e.g., at least about 60%, e.g., at least about 70%, e.g. at least about 80%, eg, at least about 90% (eg, at least about 95%, at least about 96%, at least about 97%, at least about 98 % or at least about 99%) of the cells in the population are a cell described herein, for example, a cell according to any of the above mentioned aspects and modalities of the cell. In aspects, the cell population (for example, a cell in the cell population) is able to differentiate into a population of differentiated cells, for example, a cell population of an erythroid lineage (for example, a population of red blood cells ) and wherein said population of differentiated cells has an increased percentage of F cells (for example, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least at least about 30% or at least about 40% higher percentage of F cells), for example, in relation to a population of unmodified cells of the same type. In some respects, the F cells of the differentiated cell population produce an average of at least about 6 picograms (eg, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or about 8 to about 9 picograms or about 9 to about 10 picograms) of fetal hemoglobin per cell.
[0099] [0099] In one aspect, including any of the aforementioned aspects and modalities of the cell population, the invention provides a cell population, including: 1) at least 166 CD34 + cells / kg body weight of the patient to whom cells must be administered; 2) at least 2 and 6 CD34 + cells / kg of body weight of the patient to whom the cells should be administered; 3) at least 3 and 6 CD34 + cells / kg of body weight of the patient to whom the cells should be administered; 4) at least 4 € 6 CD34 + cells / kg of body weight of the patient to whom the cells should be administered; or 5) from 2e6 to 10e6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered. In embodiments, at least about 40%, eg at least about 50% (eg, at least about 60%, at least about 70%, at least about 80% or at least about 90%) of the population's cells are CD34 + cells. In embodiments, at least about 5%, for example, at least about 10%, eg, at least about 15%, eg, at least about 20%, eg, at least at least about 30% of the population cells are CD34 + CD90 + cells. In modalities, the cell population is derived from umbilical cord blood, peripheral blood (eg, mobilized peripheral blood) or bone marrow, for example, is derived from bone marrow. In embodiments, the cell population includes, for example, consists of mammalian cells, for example, human cells. In modalities, the cell population is autologous in relation to a patient to whom it is to be administered. In other modalities, the cell population is allogeneic in relation to a patient to whom it is to be administered.
[0100] [0100] In one aspect, the invention provides a composition including a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell, or a population of cells described herein, for example, a cell population of any of the above mentioned aspects of the population. In one aspect, the composition includes a pharmaceutically acceptable medium, e.g., a pharmaceutically acceptable medium suitable for cryopreservation.
[0101] [0101] In one aspect, the invention provides a method of treating a hemoglobinopathy, including administering to a patient a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell, a population of cells described herein, for example, a cell population of any of the above mentioned aspects and modalities of the cell population, or a composition described herein, for example, a composition of any of the above mentioned aspects and modalities of the composition.
[0102] [0102] In one aspect, the invention provides a method of increasing fetal hemoglobin expression in a mammal, including administering to a patient a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of cell, a population of cells described herein, for example, a cell population of any of the above-mentioned aspects and modalities of the cell population, or a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities composition. In aspects, hemoglobinopathy is beta thalassemia. In aspects, hemoglobinopathy is the sickle cell disease.
[0103] [0103] In one aspect, the invention provides a method of preparing a cell (for example, a population of cells) including: (a) providing a cell (for example, a population of cells) (for example, an HSPC ( for example, a population of HSPCs)); (b) culturing said cell (e.g., said cell population) ex vivo in a cell culture medium including a stem cell expander; and (c) introducing into said cell a first gRNA molecule, for example, described herein, for example, a first gRNA molecule of any of the above mentioned aspects and modalities of the gRNA molecule; a nucleic acid molecule that encodes a first gRNA molecule; a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities of the composition; or a vector described herein, for example, a vector of any of the aspects and modalities mentioned above.
[0104] [0104] In aspects of the method of preparing a cell (for example, a cell population), the culture of step (b) includes a culture period before the introduction of step (c), for example, the culture period before the introduction of step (c) it is at least 12 hours, for example, it is for a period of about 1 day to about 12 days, for example, it is for a period of about 1 day to about 6 days, for example, it is for a period of about 1 day to about 3 days, for example, for a period of about 1 day to about 2 days, for example, it is for a period of about 2 days. In aspects of the method of preparing a cell (for example, a population of cells), including in any of the above mentioned aspects and modalities of the method, the culture of step (b) includes a culture period after the introduction of the step ( c), for example, the culture period after the introduction of step (c) is at least 12 hours, for example, it is during a period of about 1 day to about 12 days, eg, it is over a period of about 1 day to about 6 days, eg, is over a period of about 2 days to about 4 days, eg is over a period of about 2 days or is over a period of about 3 days or over a period of about 4 days. In aspects of the method of preparing a cell (for example, a cell population), including in any of the above mentioned aspects and modalities of the method, the cell population is expanded at least 4 times, for example, at least 5 times , for example, at least 10 times, for example, in relation to cells that are not cultured according to step (b).
[0105] [0105] In aspects of the method of preparing a cell (for example, a population of cells), including in any of the above mentioned aspects and modalities of the method, the introduction of step (c) includes an electroporation. In some respects, electroporation includes 1 to 5 pulses, eg 1 pulse, and each pulse is a pulse voltage ranging from 700 volts to 2000 volts and has a pulse duration ranging from 10 ms to 100 ms. In some respects, electroporation includes, for example, it consists of 1 pulse. In aspects, the pulse voltage (or more than one pulse) varies from 1500 to 1900 volts, for example, it is 1700 volts. In aspects, the pulse duration of a pulse or more than one pulse varies from 10 ms to 40 ms, for example, it is 20 ms.
[0106] [0106] In aspects of the method of preparing a cell (for example, a cell population), including in any of the above mentioned aspects and modalities of the method, the cell (for example, cell population) provided in step (a ) is a human cell (for example, a population of human cells). In aspects of the method of preparing a cell (for example, a cell population), including in any of the above mentioned aspects and modalities of the method, the cell (for example, cell population) provided in step (a) is isolated bone marrow, peripheral blood (for example, mobilized peripheral blood) or umbilical cord blood. In aspects of the method of preparing a cell (for example, a cell population), including in any of the above mentioned aspects and modalities of the method, the cell (for example, cell population) provided in step (a) is isolated bone marrow, for example, is isolated from the bone marrow of a patient suffering from hemoglobinopathy.
[0107] [0107] In aspects of the method of preparing a cell (for example, a cell population), including in any of the above mentioned aspects and modalities of the method, the cell population provided in step (a) is enriched with CD34 + cells .
[0108] [0108] In aspects of the method of preparing a cell (for example, a population of cells), including in any of the above mentioned aspects and modalities of the method, subsequent to the introduction of step (c), the cell (for example, cell population) is cryopreserved.
[0109] [0109] In aspects of the method of preparing a cell (for example, a population of cells), including in any of the above mentioned aspects and modalities of the method, after the introduction of step (c), the cell (for example, cell population) includes: a) an indelible at or near a genomic DNA sequence complementary to the target domain of the first gRNA molecule; or b) a deletion including sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of the first gRNA molecule (for example, at least 90% complementary to the target domain of gRNA, for example, completely complementary) to the gRNA target domain) in the HBG1 promoter region and a sequence complementary to the target domain of the first gRNA molecule (for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the target domain of gRNA) in the HBG2 promoter region.
[0110] [0110] In aspects of the method of preparing a cell (for example, a cell population), including in any of the above mentioned aspects and modalities of the method, after the introduction of step (c), at least about 40% at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the cells in the cell population include an indel in or close to a genomic DNA sequence complementary to the target domain of the first gRNA molecule .
[0111] [0111] In one aspect, the invention provides a cell (for example, a population of cells) obtainable by a method of preparing a cell (for example, a population of cells) described herein, for example, described in any one of the aspects and modalities mentioned above methods of preparing a cell.
[0112] [0112] In one aspect, the invention provides a method of treating a hemoglobinopathy in a human patient, including administering to a human patient a composition including a cell described herein, for example, a cell of any of the above aspects and modalities mentioned cell: or a cell population described herein, for example, a cell population of any of the above mentioned aspects and modalities of the cell population. In aspects, hemoglobinopathy is beta thalassemia. In aspects, hemoglobinopathy is the sickle cell disease.
[0113] [0113] In one aspect, the invention provides a method for increasing fetal hemoglobin expression in a human patient, including administering to said human patient a composition including a cell described herein, for example, a cell of any aspect and above-mentioned cell modalities: or a cell population described herein, for example, a cell population of any of the above-mentioned aspects and modalities of the cell population. In aspects, human patients have beta thalassemia. In aspects, the human patient has sickle cell disease.
[0114] [0114] In aspects of the method of treating a hemoglobinopathy or the method of increasing fetal hemoglobin expression, the human patient is administered with a composition including at least about 1 and 6 cells (for example, cells as described herein) by kg of human patient's body weight, for example, at least about 1 and 6 CD34 + cells (for example, cells as described herein) per kg of human patient's body weight. In aspects of the method of treating a hemoglobinopathy or the method of increasing fetal hemoglobin expression, the human patient is administered with a composition including at least about 2 and 6 cells (e.g., cells as described herein) per kg of weight human patient's body weight, for example, at least about 2and6 CD34 + cells (for example, cells as described herein) per kg of human patient's body weight. In aspects of the method of treating a hemoglobinopathy or the method of increasing fetal hemoglobin expression, the human patient is administered with a composition including about 2 and 6 cells (e.g., cells as described herein) per kg of human patient's body weight. , for example, about 2 and 6 CD34 + cells (for example, cells as described herein) per kg of human patient body weight. In aspects of the method of treating a hemoglobinopathy or the method of increasing fetal hemoglobin expression, the human patient is administered with a composition including at least about 3 and 6 cells (for example, cells as described herein) per kg of body weight. human patient, for example, at least about 3 and 6 CD34 + cells (for example, cells as described herein) per kg of human patient body weight. In aspects of the method of treating a hemoglobinopathy or the method of increasing fetal hemoglobin expression, the human patient is administered with a composition including about 3 and 6 cells (for example, cells as described herein) per kg of human patient's body weight. , for example, about 3 and 6 CD34 + cells (for example, cells as described herein) per kg of human patient body weight. In aspects of the method of treating a hemoglobinopathy or the method of increasing fetal hemoglobin expression, the human patient is administered with a composition including from about 2e6 to about 10e6 cells (for example, cells as described herein) by Kg of human patient's body weight, for example, from about 2 € 6 to about 10 and 6 CD34 + cells (for example, cells as described herein) per kg of human patient's body weight.
[0115] [0115] In one aspect, the invention provides: a gRNA molecule described herein, for example, a gRNA molecule of any of the above mentioned aspects and modalities of the gRNA molecule; a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities of the composition, a nucleic acid described herein, for example, a nucleic acid of any of the above-mentioned aspects and modalities of nucleic acids; a vector described herein, for example, a vector of any of the above-mentioned aspects and modalities of vectors; a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell; or a cell population described herein, for example, a cell population of any of the above mentioned aspects and modalities of the cell population, for use as a medicament.
[0116] [0116] In one aspect, the invention provides: a gRNA molecule described herein, for example, a gRNA molecule of any of the above mentioned aspects and modalities of the gRNA molecule; a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities of the composition, a nucleic acid described herein, for example, a nucleic acid of any of the above-mentioned aspects and modalities of nucleic acids; a vector described herein, for example, a vector of any of the above-mentioned aspects and modalities of vectors; a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell; or a cell population described herein, for example, a cell population of any of the above mentioned aspects and modalities of the cell population, for use in the manufacture of a medicament.
[0117] [0117] In one aspect, the invention provides; a gRNA molecule described herein, for example, a gRNA molecule of any of the above mentioned aspects and modalities of the gRNA molecule; a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities of the composition, a nucleic acid described herein, for example, a nucleic acid of any of the above-mentioned aspects and modalities of nucleic acids; a vector described herein, for example, a vector of any of the above-mentioned aspects and modalities of vectors; a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell; or a cell population described herein, for example, a cell population of any of the above mentioned aspects and modalities of the cell population, for use in the treatment of a disease.
[0118] [0118] In one aspect, the invention provides: a gRNA molecule described herein, for example, a gRNA molecule of any of the above mentioned aspects and modalities of the gRNA molecule; a composition described herein, for example, a composition of any of the above-mentioned aspects and modalities of the composition, a nucleic acid described herein, for example, a nucleic acid of any of the above-mentioned aspects and modalities of nucleic acids; a vector described herein, for example, a vector of any of the above-mentioned aspects and modalities of vectors; a cell described herein, for example, a cell of any of the above-mentioned aspects and modalities of the cell; or a cell population described herein, for example, a cell population of any of the above mentioned aspects and modalities of the cell population, for use in the treatment of a disease, in which the disease is a hemoglobinopathy, for example, beta thalassemia or sickle cell disease. BRIEF DESCRIPTION OF THE DRAWINGS
[0119] [0119] Fig 1: HbF induction 7 days after editing. For each target gRNA sequence tested the percentage of cells with induced expression of HbF corrected to baseline levels based on the simulated transfection, is shown as the mean with error bars indicating the standard deviation. The G8 gRNA against exon 2 of BCLI1A serves as a positive control. A dotted line at 17% indicates the threshold level chosen for the analysis. Several gray shading, as indicated in the legend, relate the degree of HbF induction to the degree of editing at target loci HBG1 or HBG 2.
[0120] [0120] Fig 2: Editing efficiency at the target locus of HBG1. For each gRNA tested, the percentage of indels detected by NGS is shown as mean with an error bar indicating standard deviation. The G8 gRNA against exon 2 of BCL11A serves as a positive control. Two guides for which no NGS data has been obtained are indicated by arrowheads.
[0121] [0121] Fig 3: Editing efficiency at the HBG2 target locus. For each gRNA tested, the percentage of indels detected by NGS is shown as mean with an error bar indicating standard deviation. The G8 gRNA against exon 2 of BCL11A serves as a positive control. Sixteen guides for which no NGS data was obtained are indicated by arrowheads.
[0122] [0122] Fig 4: Summary of the location of high-performance gRNA target sequences (for example,> 17% HbF up-regulation on Day 7), known non-deletion HPFH polymorphisms and transcription factor binding sites in the HBG1 promoter. The figure discloses SEQ ID NOS 293-312, respectively, in order of appearance.
[0123] [0123] Fig 5: Summary of the location of high-performance gRNA target sequences (eg,> 17% HbF up-regulation on Day 7), known non-deletion HPFH polymorphisms and transcription factor binding sites in the HBG2 promoter. The figure discloses SEQ ID NOS 313-332, respectively, in order of appearance.
[0124] [0124] Fig. 6: Editing efficiency in B2M locus directed in CD34 + HSPCs by different Cas9 variants, as assessed by NGS and flow cytometry. NLS = SV40 NLS; His6 (SEQ ID NO: 247) or His8 (SEQ ID NO: 248) refers to 6 (SEQ ID NO: 247) or 8 (SEQ ID NO: 248) histidine residues, respectively; VTE = tobacco etch virus cleavage site; Cas9 = Cas9 of S. pyogenes wild type - mutations or variants are as indicated).
[0125] [0125] Fig. 7: Detection and quantification of HbF positive cells by flow cytometry at 7 (black bars), 14 (light gray bars) or 21 (dark gray bars) days after erythroid differentiation followed by electroporation of HSPCs with RNP's containing saRNA from the indicated target domain. The percentage of HbF + cells for control cultures not treated with sgRNA at each time point was subtracted. Mean + standard deviation is indicated (n = 2 technical replicates).
[0126] [0126] Fig. 8: Detection and quantification of HbF positive cells by flow cytometry at 7 (open black bars), 14 (open light gray bars) or 21 (open dark gray bars) days after erythroid differentiation followed by electroporation of HSPCs with RNPs containing saRNA from the indicated target domain. The percentage of HbF + cells for control cultures not treated with saRNA at each time point was subtracted. The mean (bar) of two independent cell donors is shown, along with the value for each donor (circle = first donor, triangle = second donor).
[0127] [0127] Fig. 9: Visualization of PCR products of the indicated reaction: P1, P2 or P3, as described in the Examples, from cells after electroporation with RNP's containing saRNA from the indicated target domain or control cells not treated with sgaRNA . The expected products are as follows. P1: 7.7 kb for the indelible wild-type / small allele or inversion allele of 4.9 kb, 2.8 kb for the allele with 4.9 kb deletion. P2: 3.8 kb for the indelible wild / small type allele, no product for 4.9 kb deletion or 4.9 kb inversion allele. P3: 1.8 kb for the 4.9 kb inversion allele, no product for the indelible wild / small allele or 4.9 kb deletion allele. L = DNA reference ladder. * = DNA from that band was isolated and subjected to next generation sequencing.
[0128] [0128] Fig. 10: Schematic indicating the genomic location of the primer and probe binding sites for the digital drop PCR assay to quantify 4.9 kb of deletions. The primers (5.2 kb Fwd and 5.2 kb Rev) and probe (FAM probe) bind to an intergenic site downstream of HBG2 and upstream of HBG1. The probe has a second binding site upstream of HBG2, but the region is not bound by the primers. The areas in which the target domains in the HBG1 and HBG2 promoters are located are indicated. If the intervening sequence with the two regions of the target domain is deleted, the primer / probe binding site between HBG1 and HBG2 will be lost.
[0129] [0129] Fig. 11: Separation scheme for HSPC subpopulations for the cell sample followed by electroporation with RNPs containing sgRNA from the target domain GCR-0067. Cell fluorescence plots are shown after immunostaining directed to the indicated cell surface marker. The following populations were separated as shown: P5 = CMP (CD34 + CD45RA-CD38 +), P9 = MPP (CD34 + CD45RA-CD38-CD90-CD49f-), P10 = ST-HSC (CD34 + CD45RA-CD38-CD90-CD49f + ), P1H1 = LT-HSC (CD34 + CD45RA-CD38-CD90 + CD49f +). Total CD34 + cells were also separated (not shown).
[0130] [0130] Fig. 12: Percentage of editing of HSPC subpopulations separated following electroporation with RNPs containing saRNA from the target domain GCR-0067. Indel HBG1 and indel HBG2 indicate the percentage of small insertions and deletions identified by next generation sequencing of PCR amplicons at or near the target domain of the HBG1 or HBG 2 promoter region, respectively (note that the alleles with deletion or inversion 4.9 kb previously described are not amplified). HBG1-HBG2 deletion indicates the percentage of alleles with the 4.9 kb deletion, as determined by the digital drop PCR assay described in the Examples. The total edition is an approximation calculated by the percentage of HBG1-HBG2 deletion added to the percentage without the HBG1-HBG deletion - 2 times the percentage of HBG2 indel.
[0131] [0131] Fig. 13: Figure 13A shows the sum of all indices observed at the HBG1 locus in the indicated cell type after introduction of SAgRNA comprising the target domain of GCR-0067. Indels are arranged with indels most often seen at the top of each bar. Not quantified in this assay is the cell fraction comprising the large 4.9 kb deletion between the HBG1 and HBG2 loci. The number within the bar of each of the most prevalent indels indicates the number of nucleotide differences in the wild-type genomic sequence (- indicates deletion; + indicates insertion). Figure 13B shows the sum of all indices observed at the HBG2 locus in the indicated cell type after introduction of saRNA comprising the target domain of GCR-0067. Indels are arranged with indels most often seen at the top of each bar. Not quantified in this assay is the cell fraction comprising the large 4.9 kb deletion between the HBG1 and HBG2 loci. The number within the bar of each of the most prevalent indels indicates the number of nucleotide differences in the wild-type genomic sequence (- indicates deletion; + indicates insertion). CMP = CD34 + CD45RA-CD38 + cells; MPP = CD34 + CD45RA-CD38-CD90-CD49f-cells; ST-HSC = CD34 + CD45RA-CD38-CD90-CD49f + cells; and LTHSC = CD34 + CD45RA-CD38-CD90 + CD49f + cells.
[0132] [0132] Fig. 14: Percentage of colonies corresponding to the indicated subtype, CFU-GEMM (dark gray bars), CFU-G / M / GM (medium gray bars) or BFU-E / CFU-E (light gray bars), after electroporation with RNPs containing saRNA from the indicated target domain or control cultures not treated with saRNA. Mean +/- standard deviation is indicated (n = 2 independent donors).
[0133] [0133] Fig. 15: Number of proliferation times of total nucleated cells (TNC), CD34 positive cells (CD34 +) and CD34 and CD90 positive double cells (CD34 + CD90 +), as indicated, in medium containing compound 4, after electroporation with RNPs containing the RCR of the target domain GCR-0067 or control cultures not treated with SARNA. Mean +/- standard deviation is indicated (n = 2 independent donors) The mean (bar) of three independent cell donors is shown, along with the value for each donor (square = donor A, triangle = donor B, circle = donor Ç). The differences between the edited and control cultures were not significant (ns) by the unpaired t test (GraphPad Prism).
[0134] [0134] Fig. 16: Representative classifications of cell populations by flow cytometry. Cell fluorescence dot plots are shown after immunostaining directed to the indicated cell surface markers, or control isotype (isotype). The classifications indicated with the bold boxes were used to quantify the percentage of the indicated population and were configured to exclude the cells marked with the isotype control. Only viable cells pre-classified as negative for DAPI and within the direct scatter and lateral scatter cell classifications are shown and are derived from the electroporated donor C with only Cas9.
[0135] [0135] Fig. 17: Percentage of cells with the indicated cell surface phenotype, as assessed by flow cytometry, after electroporation with RNPs containing saRNA from the target domain GCR-0067 (black bars) or control cultures not treated with sa RNA (gray bars) The cells were expanded 2 days after electroporation of RNP in medium comprising Compound 4 and evaluated by flow cytometry, as described in Figure 16. Mean + standard deviation is indicated (n = 3 independent donors). There were no significant differences between edited and unedited cells for a given population by unpaired t test (GraphPad Prism).
[0136] [0136] Fig. 18A. EC-MS quantification of globin subunits in simulated edited HSCs derived from a patient with simulated sickle cell disease (SCD1), edited with Cas9 and without saRNA. After the cells are differentiated into an erythroid lineage, the cells showed a normal level of a-aglobin, no normal b-globin due to sickle cell homozygosity, a high level of b-globin sickle subunit and a low level of fetal g-globin.
[0137] [0137] Fig. 18B. EC-MS quantification of globin subunits in HSPCs edited in genome derived from patients with sickle cell disease (SCD1). After editing the HSCs, the erythroid cells derived from the patient in the sample demonstrated 40% positive regulation of fetal g-globin and 50% negative regulation of the sickle-b-globin subunit.
[0138] [0138] Fig. 19: Schematic protocol for studying the graft of cells edited by genes. Transplantation of HSCs edited by gene with Cas9 and sagRNA comprising the target domain of CRO001128 (sg91128). Five hundred thousand human CD34 + cells were either edited or simulated with gRNA or edited by gene with sg1128, followed by injection into NOD.Cg-Prkdec * “I / 2rg" receptors! Wi / SzJ (NSG) irradiated with 2 Gy. The mice were bled at 4.8, 12, 16 weeks and bone marrow cells were harvested at 16 weeks after transplantation.
[0139] [0139] Fig. 20: Bone marrow reconstitution at 16 weeks post-transplant using the experimental protocol shown in Figure
[0140] [0140] Fig. 21: Reconstitution of myeloid lymphoid cells, Be T in peripheral blood and bone marrow at multiple times using the experimental protocol shown in Figure 19. N = 5 / group, the data show min to max, 4 independent experiments.
[0141] [0141] Fig. 22: Schematic diagram of the transplant study to assess the function of HSCs stem cells edited with saRNAs from the gamma globin promoter region (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg -G0067) compared to the erythroid-specific enhancer region gRNA of the BCL11A gene (sg-G1128; also referred to as sg1128). The cells were left in culture 24 hours after electroporation. The culture conditions before and after electroporation were StemSpan SFEM + IL6, SCF, TPO, FIt3àL; 750 nM of Compound 4.
[0142] [0142] Fig. 23: Human graft and lineage analysis over weeks in NSG mice. Figure 23A) Chimerism of peripheral blood over 18 weeks; Figure 23B) Distribution of the lineage in peripheral blood at 18 weeks. Bone marrow analysis: Figure 23C) Bone marrow analysis of human cells at week 9; Figure 23D) Graft of human CD45 + and distribution of human cell line in the bone marrow at week 9; Figure 23E) Graft of human CD45 + in the bone marrow at week 20 after the graft; Figure 23F) Distribution of the cell line grafted into the bone marrow in 20 weeks.
[0143] [0143] Fig 24A. Graft efficiency of HSCs edited with SARNAs with homology to the gamma globin promoter region (sg-GO0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) compared to the SAgRNA of the specific erythroid enhancer region of the BCL11A gene (s91128) Shows the graft of human cells in NSG mice at 8 weeks after transplantation. N = 10 / group, 3 independent experiments. The graph shows grouped data.
[0144] [0144] Fig. 24B. Graft efficiency of HSCs edited with SAgRNAs homologous to the gamma globin promoter region (sg-GO0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067) compared to the SAgRNA of the specific erythroid enhancer region of the gene
[0145] [0145] Fig. 25: Reconstitution of multiple lineage of NSG mice transplanted with CD34 + cells edited by genes. N = 10 / group, data from a representative experiment.
[0146] [0146] Fig. 26: High editing efficiency was maintained before and after transplantation. "Pre-Xpt": editing efficiency and indelible pattern of individual saRNA in human CD34 + cells, as measured by NGS after editing, but before transplantation. "8 weeks post-Xpt": editing efficiency and indelible pattern in human CD34 + cells 8 weeks after bone marrow transplantation in mice as measured by NGS. "20 weeks post-Xpt": editing efficiency and indelible pattern in human CD34 + cells 20 weeks after bone marrow transplantation in mice as measured by NGS. Electroporation performed in triplicate per group, data from a representative experience. As used in connection with this Figure, "indel" refers to the sum of all indels less than 200 nt; "large deletion" refers to the deletion of the sequence between the predicted binding sites of HBG1 and HBG2 for each gRrRNA.
[0147] [0147] Fig. 27: NGS analysis of CD34 + cells post-editing with RNPs. The saRNA that targets specific regions is indicated on the x axis. Figure 27A: NGS analysis of CD34 + cells on day 2 post-electroporation of RNPs. Figure 27B: NGS analysis of whole bone marrow of NSG mice transplanted with CD34 + bone marrow cells edited at (27B) week 9 post-transplant. Figure 27C: NGS analysis of whole bone marrow of NSG mice transplanted with CD34 + bone marrow cells edited at week 20 post-transplant. Insertion indels are indicated in black, deletion indels (excluding large deletions comprising an excision of the region between HBG1 binding and HBG2 target sequences for each of sg-G51, sg-G48 and sg-G67 gRNA) are indicated in Grey. % of total editing is represented by the height of the bars. N = 10, data are presented as mean + SEM from an independent experiment.
[0148] [0148] Fig. 28: Human HSC grafted, long-term, edited by genes were able to produce increased levels of HbF after differentiation of erythroid cells. Fifty thousand human CD34 + cells were separated from the bone marrow of NSG mice transplanted at 8 or 20 weeks. Separate cells were seeded in erythroid differentiation medium for 14 to 21 days. Cultured mature red blood cells were assayed for HbF expression and to enumerate the number of HbF + cells by flow cytometry. The simulated control represents CD34 + cells edited with Cas9 without gRNA and transplanted into NSG mice in the same way as the control group edited by genes. N = 10 / group, 3 independent experiments.
[0149] [0149] Fig 29. Is the off-target activity for HBG1 and / or HBG guide RNAs was evaluated using a dsDNA oligo-insertion method in HEK-293 cells with Cas9 overexpression. The target site (open circle) and the potential off-target sites (closed circles) detected are indicated; the y-axis indicates the detection frequency. All gRNAs were tested in the dgRNA format with the target domain indicated by the identifier CRXXXxXxxx.
[0150] [0150] Fig. 30: Off-target activity for HBG1 and / or HBG 2 guide RNAs was evaluated using a dsDNA oligo-insertion method in HEK-293 cells with Cas9 overexpression. The target site (open circle) and the potential off-target sites (closed circles) detected are indicated; the y-axis indicates the detection frequency. The gRNAs were tested in the dgRNA format with the target domain indicated by the identifier CROXxXx or in the saRNA format with the target domain indicated by the identifier GXXxxxx.
[0151] [0151] Figure 31A. Count of CD34 + cells derived from peripheral blood mobilized from healthy individuals after genetic editing. The cells were thawed on day 0, cultured for 3 days before electroporation on day 3. Enumeration of the number of CD34 + cells by ISHAGE over 10 days after electroporation. Two independent experiments were performed in duplicate. Total N = 5. The graphs show data from 1 experiment with mean + SEM.
[0152] [0152] Figure 31B. Expansion of CD34 + cells derived from mobilized peripheral blood from healthy individuals after genetic editing. The cells were thawed on day 0, cultured for 3 days before electroporation on day 3. Expansion of total mononuclear cells on day 3, 7 and 10 after editing. Two independent experiments were performed in duplicate. Total N =
[0153] [0153] Figure 31C. Viability of CD34 + cells derived from peripheral blood mobilized from healthy individuals after genetic editing. The cells were thawed on day 0, cultured for 3 days before electroporation on day 3. The viability of mononuclear cells at the same time points to post-editing. Two independent experiments were performed in duplicate. Total N =
[0154] [0154] Figure 32A. Editing efficiency of sg1128 and sgo0067 in CD34 + cells mobilized from the peripheral blood of healthy individuals. The percentage of INDELs captured by NGS after editing using sg1128 (targeting the BCL11A +58 region of ESH) and sg0067 (targeting the HDbG-1 and HbG-2 gene cluster) is shown. This chart also indicates the total editing efficiency of the sg1128, as only small INDELs were generated by this sa RNA. More than five independent experiments were performed in duplicate or triplicate, n = 2-3 / experiment.
[0155] [0155] Figure 32B. Editing efficiency of sg1128 and sg0067 in CD34 + cells mobilized from the peripheral blood of healthy individuals. A. The percentage of INDELs captured by NGS after editing using sg0067 (targeting the HDG-1 and HbG-2 gene cluster). The total editing efficiency and the editing standard of sgo0067 are shown. The sg0067 editing pattern consists of a large 5kb deletion (denoted by black bars) and smaller INDELs (denoted by gray bars). More than five independent experiments were performed in duplicate or triplicate, n = 2-3 / experiment.
[0156] [0156] Figure 33. Number of times of alteration of the g-globin transcript in the erythroid cells of patient samples after CRISPR silencing of BCL11A or indelible / deletion formation in the HBG1 / 2 region. CD34 + cells derived from peripheral blood mobilized from healthy donors were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in previous procedures. On day 11 of erythroid differentiation, cells were harvested from the culture and subjected to qPCR to measure g-globin and b-globin transcripts, normalizing GAPDH. The experiment was performed independently twice, each in duplicate, n = 2-3 / experiment. Data show mean + SEM of donors grouped in one study.
[0157] [0157] Figure 34. Enumeration of HbF + cells from healthy individuals after genetic editing. CD34 + cells derived from peripheral blood mobilized from healthy donors were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in previous procedures. On day 10, of erythroid differentiation, the cells were stained with anti-HbF-FITC antibody to enumerate HbF + cells by flow cytometry. The experiment was performed independently twice, each in duplicate, n = 2-3 / experiment. The data shows mean + SD of a study.
[0158] [0158] Figure 35A. Expansion and viability of CD34 + cells derived from peripheral blood of individuals with sickle cell disease after gene editing. The cells were cultured for 6-10 days before electroporation. DO refers to the day of electroporation. The absolute count of CD34 + cells is shown by ISHAGE over 10 days after electroporation. N = 4, the data show mean + SEM. 4 independent experiments were performed in duplicate. The bars are, from left to right at each time point, simulated, sg1128, sgoo67.
[0159] [0159] Figure 35B. Expansion and viability of CD34 + cells derived from peripheral blood of individuals with sickle cell disease after gene editing. The cells were cultured for 6-10 days before electroporation. DO refers to the day of electroporation. The percentage of CD34 + cells is shown by ISHAGE over 10 days after electroporation. N = 4, the data show mean + SEM. 4 independent experiments were performed in duplicate. The bars are, from left to right at each time point, simulated, sg1128, sgoo67.
[0160] [0160] Figure 35C. Expansion and viability of CD34 + cells derived from peripheral blood of individuals with sickle cell disease after gene editing. The cells were cultured for 6-10 days before electroporation. DO refers to the day of electroporation. The expansion of the total mononuclear cells on days 3, 7 and 10 after editing is shown. N = 4, the data show mean + SEM. 4 independent experiments were performed in duplicate. The bars are, from left to right at each time point, simulated, sg1128, sgoo67.
[0161] [0161] Figure 35D. Expansion and viability of CD34 + cells derived from peripheral blood of individuals with sickle cell disease after gene editing. The cells were cultured for 6-10 days before electroporation. DO refers to the day of electroporation. Mononuclear cell viability is shown on days 3, 7 and 10 after editing. N = 4, the data show mean + SEM. 4 independent experiments were performed in duplicate. The bars are, from left to right at each time point, simulated, sg1128, sg0067.
[0162] [0162] Figure 36. Editing efficiency of sg1128 and sg0067 in CD34 + cells from samples from patients with sickle cell disease. The sg0067 editing pattern was denoted by great exclusion (gray) and sum of small indels (black). The data shows mean + SEM of four independent editing experiments performed in duplicate with CD34 + cells from four patients with different sickle cell disease (SCD1-4).
[0163] [0163] Figure 37. Capacity of multi-lineage differentiation in vitro of HSPCs measured by unitary colony formation assay. BFU-E = erythroid rupture forming unit; CFU-GM = colony forming unit, granulocyte, monocyte; CFU-GEMM = colony-forming unit, granulocyte, erythroid, monocyte, megakaryocyte. The graph shows three independent CD34 + cell experiments from three different sickle cell disease patients (SCD1-3). The experiments were carried out in triplicate. Data represent mean + SEM.
[0164] [0164] Figure 38. Number of times of alteration of the g-globin transcript in the erythroid cells of the patient samples after CRISPR silencing of BCL11A or the indelible formation / deletion in the g-globin gene. CD34 + cells derived from 3 patients with sickle cell disease (SCD1-3) were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the Examples. On day 11 of erythroid differentiation, cells were harvested from the culture and subjected to qQPCR to measure g-globin and b-globin transcripts, normalizing GAPDH. The experiment was performed in duplicate. Data show mean + SEM of grouped donors.
[0165] [0165] Figure 39. Enumeration of HDF + cells in samples from patients with sickle cell disease after gene editing. CD34 + cells derived from 3 patients with sickle cell disease (SCD1-3) were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the Examples. On day 11, 14 and 21 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody to enumerate HbF + cells by flow cytometry. The experiment was performed in duplicate. Data show mean + SEM. Shown on day 11 are the results of SCD1-3; shown on day 14 are the results of SCD-1 and SCD-2; shown on day 21 are the results of SCD-2 and SCD-3.
[0166] [0166] Figure 40. Measurement of fetal hemoglobin expression in samples from patients with sickle cell disease, edited by genes, by flow cytometry. CD34 + cells derived from 3 patients with sickle cell disease (SCD1-3) were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in the Examples. On day 11, 14 and 21 of erythroid differentiation, cells were stained with anti-HbF-FITC antibody to measure the intensity of HbF expression in each cell by flow cytometry. The experiment was performed in duplicate. Data show mean + SEM. MFl = average fluorescent intensity. Shown on day 11 are the results of SCD1-3; shown on day 14 are the results of SCD-1 and SCD-2; shown on day 21 are the results of SCD-2 and SCD-3.
[0167] [0167] Figure 41. Enumeration of the number of sickle cells versus normal cells after editing patient samples by CRISPR. CD34 + cells derived from 3 patients with sickle cell disease (SCD1-3) were edited by CRISPR and differentiated into the erythroid lineage in vitro as described in previous procedures. On day 21 of erythroid differentiation, cells were subjected to a% hypoxia chamber for 4 days, fixed and followed by single cell imaging flow cytometry. Figure 41A (left panel) shows the change in the number of sickle cells in patient samples edited by genes, as listed by single cell imaging. Figure 41B (right panel) shows the change in the number of normal cells after editing by gene in patient samples, as enumerated by single cell imaging. Three independent experiments were carried out, with each experiment in duplicate. Forty thousand images of individual cells were enumerated from each patient. Graph shows mean + SEM of the grouped data. DEFINITIONS
[0168] [0168] The terms "CRISPR system", "Cas system" or "CRISPR / Cas' system" refer to a set of molecules comprising an RNA-guided nuclease or other effector molecule and a gRNA molecule that together are needed and enough to target and effect nucleic acid modifications in a target sequence by RNA-guided nuclease or other effector molecules. In one embodiment, a CRISPR system comprises a gRNA and a Cas protein, for example, a Cas9 protein. Such systems comprising a Cas9 or a modified Cas9 molecule are referred to herein as "Cas9 systems" or "CRISPR / Cas9 systems". In one example, the gRNA molecule and the Cas molecule can be complexed to form a ribonuclear protein (RNP) complex.
[0169] [0169] The terms "guide RNA", "guide RNA molecule", "gRNA molecule" or "gRNA" are used interchangeably and refer to a set of nucleic acid molecules that promote the specific targeting of a guided nuclease by RNA or another effector molecule (typically in complex with the gRNA molecule) for a target sequence. In some embodiments, said targeting is accomplished by hybridizing a portion of the gRNA to DNA (for example, through the target domain gRNA), and by binding a portion of the gRNA molecule to the nuclease guided by RNA or another effector molecule (for example, through at least gRNA tracing). In embodiments, a gRNA molecule consists of a single contiguous polynucleotide molecule, referred to herein as a "single guide RNA" or "sagRNA" and the like. In other embodiments, a gRNA molecule consists of a plurality, usually two polynucleotide molecules, which are themselves capable of association, usually through hybridization, referred to herein as a "double guide RNA" or "dgRNA" and the like. The gRNA molecules are described in more detail below, but they usually include a target domain and a tracr. In modalities, the target domain and tracr are arranged on a single polynucleotide. In other embodiments, the target domain and tracr are arranged in separate polynucleotides.
[0170] [0170] The term "target domain", as the term is used in connection with a gRNA, is the portion of the gRNA molecule that recognizes, for example, it is complementary to a target sequence, for example, a target sequence inside the nucleic acid of a cell, for example, inside a gene.
[0171] [0171] The term "crRNA" as the term is used in connection with a gRNA molecule, is a portion of the gRNA molecule that comprises a target domain and a region that interacts with a tracr to form a mast region.
[0172] [0172] The term "target sequence" refers to a sequence of nucleic acids - complementary, for example, fully complementary, to a target domain of gRNA. In modalities, the target sequence is arranged on the genomic DNA. In one embodiment, the target sequence is adjacent to (either in the same or complementary DNA strand) a sequence of the adjacent protospace motif (PAM) recognized by a protein having a nuclease or other effector activity, for example, a PAM sequence recognized by Cas9. In embodiments, the target sequence is a target sequence within a gene or locus that affects the expression of a globin gene, for example, that affects the expression of fetal beta globin or hemoglobin (HbF). In embodiments, the target sequence is a target sequence within a non-deletion HPFH region, for example, it is within an HBG1 and / or HBG2 promoter region.
[0173] [0173] The term "mast" as used here in connection with a gRNA molecule, refers to the portion of the gRNA where the crRNA and tracr bind or hybridize to each other.
[0174] [0174] The term "tracr" as used here in connection with a gRNA molecule, refers to the portion of the gRNA that binds to a nuclease or other effector molecule. In embodiments, tracr comprises a nucleic acid sequence that specifically binds to Cas9. In modalities, tracr comprises the nucleic acid sequence that is part of the mast.
[0175] [0175] The terms "Cas9" or "Cas9 molecule" refer to an enzyme of the bacterial CRISPR / Cas Type Il system responsible for DNA cleavage. Cas9 also includes the wild-type protein, as well as its functional and non-functional mutants. In modalities, Cas9 is a Cas9 of S. pyogenes.
[0176] [0176] The term "complementary" as used in connection with nucleic acid, refers to the base pair, A with T or U, and G with C. The complementary term refers to nucleic acid molecules that are completely complementary, that is, they form base pairs of À with T or U and G pairs with C throughout the reference sequence, as well as molecules that are at least 80%, 85%, 90%, 95%, 99% complementary.
[0177] [0177] "Nucleic Acid Mold", as used in connection with homology-directed repair or homologous recombination, refers to nucleic acid to be inserted into the modification site by the donor sequence of the CRISPR system for gene repair (insertion) at the site cutting.
[0178] [0178] An "indel", as the term is used herein, refers to a nucleic acid comprising one or more nucleotide insertions, one or more nucleotide deletions, or a combination of nucleotide insertions and deletions, relative to a reference nucleic acid, which results after being exposed to a composition comprising a gRNA molecule, for example, a CRISPR system. Indels can be determined by nucleic acid sequencing after being exposed to a composition comprising a gRNA molecule, for example, by NGS. With respect to the site of an indel, an indel is considered "at or near" a reference site (for example, a site complementary to a target domain of a gRNA molecule) if it comprises at least one insertion or deletion within approximately 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide (s) of the reference site, or is superimposed on part or all of said reference site (for example, comprises at least at least one insertion or deletion superimposed with, or within 10, 9, 8, 7, 6, 5, 4, 3, 20U 1 nucleotides from a site complementary to the target domain of a gRNA molecule, for example, a molecule of gRNA described here). In modalities, the indel is a large deletion, for example, comprising more than about 1 kb, more than about 2 kb, more than about 3 kb, more than about 4 kb, more than about 5 kb, more than about 6 kb or more than about 10 kb of nucleic acid. In embodiments, the 5 'end, the 3' end or both the 5 'and 3' ends of the large deletion are arranged at or near a target sequence of a gRNA molecule described herein. In modalities, the large deletion comprises about 4.9 kb of DNA, arranged between a target sequence of a gRNA molecule, for example, described here, arranged within the region of the HBG1 promoter and a target sequence of a molecule of gRNA, for example, described herein, disposed within the HBG2 promoter region.
[0179] [0179] An "indel pattern", as the term is used here, refers to a set of indels that results after exposure to a composition comprising a gRNA molecule. In one embodiment, the indel pattern consists of the first three indels, by the frequency of appearance. In one embodiment, the indel pattern consists of the first five indels, by the frequency of appearance. In one embodiment, the indel pattern consists of indels that are present with a frequency greater than about 1% in relation to all sequencing readings. In one embodiment, the indel pattern consists of indels that are present with a frequency greater than about 5% in relation to all sequencing readings. In one embodiment, the indel pattern consists of indels that are present more than about 10% of the total number of indel sequencing readings (that is, those readings that do not consist of the reference nucleic acid sequence unmodified). In one embodiment, the indel pattern includes any of the first three most frequently observed indels. The pattern of indel can be determined, for example, by methods described herein, for example, by sequencing cells from a population of cells that have been exposed to the gRNA molecule.
[0180] [0180] An "indel off-target", as the term is used here, refers to
[0181] [0181] The terms "one" and "one" relate to one or more than one (that is, at least one) of the grammatical object of the article. By way of example, "an element" means an element or more than an element.
[0182] [0182] The term "about", when related to a measurable value, such as a quantity, a time duration, and the like, is intended to cover variations of + 20% or, in some cases, + 10%, or in some cases + 5%, or in some cases + 1%, or in some cases + 0.1% of the specified value, as such variations are appropriate to carry out the revealed methods.
[0183] [0183] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent cells, or both. The person skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, the antigens can be derived from recombinant or genomic DNA. One skilled in the art will understand that any DNA, which comprises nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response, therefore encodes an "antigen" as the term is used herein. In addition, one skilled in the art will understand that an antigen need not be encoded by a complete nucleotide sequence of a gene alone. It is clear that the present invention includes, but is not limited to, use of partial nucleotide sequences from more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. In addition, one skilled in the art will understand that an antigen does not need to be encoded by a "gene" at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or can be a macromolecule in addition to a polypeptide. Such a biological sample may include, but is not limited to, a tissue sample, a cell or a fluid with other biological components.
[0184] [0184] The term "autologous" refers to any material derived from the same subject in whom it will later be reintroduced.
[0185] [0185] The term "allogeneic" refers to any material derived from an animal other than the same species as the individual into which the material is introduced. Two or more subjects are considered to be allogeneic to each other when the genes in one or more / oci are not identical. In some respects, allogeneic material from subjects of the same species can be genetically quite different to interact antigenically.
[0186] [0186] The term "xenogeneic" refers to a graft derived from an animal of a different species.
[0187] [0187] "Derived from" as the term is used here, indicates a relationship between a first and a second molecule. It generally refers to the structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule.
[0188] [0188] The term "encoding" relates to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA or an mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having a defined sequence of nucleotides (for example, rRNA, tRNA and mMRNA) or a defined sequence of amino acids and the resulting biological properties. Thus, a gene, cDNA, or RNA, encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the MRNA sequence, as is usually provided in sequence lists, and the non-coding strand, used as a template for the transcription of a gene or cDNA, can be referred to as encoding the protein or another product of that gene or cDNA.
[0189] [0189] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one another and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or an RNA can also include introns in that the nucleotide sequence encoding the protein may, in some versions, contain an intron (s).
[0190] [0190] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein, and relate to an amount of a compound, formulation, material or composition, as described herein, effective in achieving a particular biological result.
[0191] [0191] The term "endogenous" refers to any material from or produced within an organism, cell, tissue or system.
[0192] [0192] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.
[0193] [0193] The term "expression" relates to the transcription and / or translation of a particular nucleotide sequence directed by a promoter.
[0194] [0194] The term "transfer vector" refers to a composition of matter that comprises an isolated nucleic acid and that can be used to distribute the isolated nucleic acid within a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "transfer vector" includes a plasmid or an autonomously replicating virus. The term should also be understood to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid to cells, such as, for example, a polylysine compound, liposome and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors and the like.
[0195] [0195] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-action elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (for example, naked or contained in liposomes) and viruses (for example, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that incorporate the recombinant polynucleotide.
[0196] [0196] The term "homologous" or "identity" relates to the identity of the subunit sequence between two polymeric molecules, for example, between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit in both molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical in that position. The homology between two sequences is a direct function of the number of corresponding or homologous positions; for example, if half (for example, five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (for example, 9 out of 10) are corresponding or homologous, the two strings are 90% homologous.
[0197] [0197] The term "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in substantially purified form, or it can exist in a non-native environment such as, for example, a host cell.
[0198] [0198] The term "operably linked" or "transcriptional control" refers to the functional link between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operationally linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operatively linked DNA sequences can be contiguous with each other and, for example, when necessary to unite two protein coding regions, are in the same reading frame.
[0199] [0199] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal, intratumor or infusion injection techniques.
[0200] [0200] The term "nucleic acid" or "polynucleotide" relates to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and their polymers in the form of single or double strands. Unless specifically limited, the term encompasses nucleic acids containing known natural nucleotide analogs that have binding properties similar to the reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants of these (for example, degenerate codon substitutions), alleles, orthologists, SNPs and complementary sequences, as well as the sequence explicitly indicated. Specifically, substitutions of degenerate codes can be obtained by generating sequences in which the third position of one or more selected codons (or all) is replaced with mixed base residues and / or deoxyinosine (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).
[0201] [0201] The terms "peptide", "polypeptide" and "protein" are used interchangeably and relate to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that a protein or peptide sequence can comprise. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which are generally referred to in the art as proteins, of which there are many kinds. "Polypeptides" include, for example, biologically active fragments, polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others, substantially homologous. A polypeptide includes a natural peptide, a recombinant peptide or a combination thereof.
[0202] [0202] The term "promoter" relates to a DNA sequence recognized by the synthetic machinery of the cell or synthetic machinery introduced, required to initiate the specific transcription of a polynucleotide sequence.
[0203] [0203] The term "promoter / regulatory sequence" relates to a nucleic acid sequence that is necessary for expression of a genetic product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the main sequence of the promoter and in other cases, this sequence may also include an enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. The promoter / regulatory sequence can, for example, be one that expresses the gene product in a tissue-specific manner.
[0204] [0204] The term "constitutive" promoter refers to a nucleotide sequence that, when operatively linked to a polynucleotide that encodes or specifics a gene product, causes the gene product to be produced in a cell under most or all the physiological conditions of the cell.
[0205] [0205] The term "inducible" promoter refers to a nucleotide sequence that, when operatively linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer that corresponds to the promoter is present in the cell.
[0206] [0206] The term "tissue specific" promoter refers to a nucleotide sequence that, when operatively linked to a polynucleotide encodes or specifies a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
[0207] [0207] As used here in connection with a messenger RNA (mMRNA), a 5 'cap (also called an RNA cap, 7-RNA methylguanosine cap or RNA m7G cap) is a modified guanine nucleotide that has been added to " front "or at the 5 'end of a eukaryotic messenger RNA right after the start of transcription. Cap 5 'consists of a terminal group that is attached to the first transcribed nucleotide. Its presence is crucial for ribosome recognition and protection of RNases. The addition of cap is coupled to the transcription and occurs transcriptionally, in such a way that each influences the other. Shortly after the start of transcription, the 5 'end of the mRNA being synthesized is linked by a cap synthesizer complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions that are necessary for MRNA capping. The synthesis proceeds as a multi-step biochemical reaction. The capping fraction can be modified to modulate mMRNA functionality, such as its stability or translation efficiency.
[0208] [0208] As used here, "RNA transcribed in vitro" relates to RNA, preferably mRNA, which has been synthesized in vitro. Generally, RNA transcribed in vitro is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the RNA transcribed in vitro.
[0209] [0209] As used here, a "poly (A)" is a series of adenosines linked by polyadenylation to MRNA. In the preferred embodiment of a construct for transient expression, polyA is between 50 and 5000 (SEQ ID NO: 190), preferably greater than 64, more preferably greater than 100, more preferably greater than 300 or 400. The poly (A ) can be modified chemically or enzymatically to modulate MRNA functionality, such as localization, stability or translation efficiency.
[0210] [0210] As used here, "polyadenylation" relates to the covalent attachment of a polyadenyl fraction, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (MRNA) molecules are polyadenylated at the 3 'end. The poly (A) 3rd tail is a long sequence of adenine nucleotides (often hundreds) added to the pre-mRNA through the action of a polyadenylate polymerase enzyme. In higher eukaryotes, the poly (A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly (A) tail and the protein attached to it help protect MRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, core mMRNA export and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but it can also occur later in the cytoplasm. After transcription termination, the mMRNA chain is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is generally characterized by the presence of the AAUAAA base sequence close to the cleavage site. After mMRNA cleavage, adenosine residues are added to the free 3 'end at the cleavage site.
[0211] [0211] As used here, "transient" relates to the expression of a non-integrated transgene over a period of hours, days or weeks, in which the period of time for expression is less than the time for expression of the gene whether integrated into the genome or contained within a stable plasmid replicon in the host cell.
[0212] [0212] As used here, the terms "treat", "treatment" and "treating" refer to reducing or improving the progression, severity and / or duration of a disorder, for example, a hemoglobinopathy, or the improvement of a or more symptoms (preferably, one or more discernible symptoms) of a disorder, for example, hemoglobinopathy, resulting from the administration of one or more therapies (for example, one or more therapeutic agents, such as a gRNA molecule, CRISPR system or modified cell of the invention). In specific modalities, the terms "treat", "treatment" and "treating" refer to the improvement of at least one measurable physical parameter of a hemoglobinopathy disorder, not discernible by the patient. In other modalities, the terms "treat", "treatment" and "treating" refer to inhibiting the progression of a disorder, whether physically, for example, stabilizing a discernible symptom, physiologically by, for example, stabilizing a parameter physical, or both. In other embodiments, the terms "treat", "treatment" and "treating"
[0213] [0213] The term "signal transduction pathway" relates to the biochemical relationship between a variety of signal transduction molecules that play a role in transmitting a signal from one portion of a cell to another portion of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting the signal across a cell's membrane.
[0214] [0214] The term "subject" is intended to include living organisms in which an immune response can be triggered (for example, mammals, human).
[0215] [0215] The term a "substantially purified" cell refers to a cell that is essentially devoid of other types of cells. A substantially purified cell also refers to a cell that has been separated from other types of cells with which it is normally associated in its natural state. In some cases, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, this term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some ways, cells are grown in vitro. In other respects, the cells are not cultured in vitro.
[0216] [0216] The term "therapeutic" as used here means a treatment. A therapeutic effect is obtained by reducing, suppressing, remitting or eradicating a disease state.
[0217] [0217] The term "prophylaxis" as used herein means the prevention or protective treatment for an illness or disease state.
[0218] [0218] The term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid and / or protein is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid and / or protein. The cell includes the main cell and its descendants.
[0219] [0219] The term "specifically binds" refers to a molecule that recognizes and binds with a binding partner (for example, a protein or nucleic acid) present in a sample, but whose molecule does not substantially recognize or binds to other molecules in the sample.
[0220] [0220] The term "bioequivalent" refers to an amount of an agent other than the reference compound, necessary to produce an effect equivalent to the effect produced by the reference dose or reference quantity of the reference compound.
[0221] [0221] "Refractory", as used herein, refers to a disease, for example, hemoglobinopathy, which does not respond to treatment. In modalities, a refractory hemoglobinopathy may be resistant to treatment before or at the beginning of treatment. In other modalities, refractory hemoglobinopathy can become resistant during treatment. A refractory hemoglobinopathy is also called resistant hemoglobinopathy.
[0222] [0222] "Recurrence" as used herein refers to the return of a disease (eg, hemoglobinopathy) or the signs and symptoms of a disease, such as a hemoglobinopathy after a period of improvement, for example, after previous treatment of a therapy, for example, hemoglobinopathy therapy.
[0223] [0223] Intervals: throughout this disclosure, various aspects of the invention can be presented in an interval format. It should be understood that the description in interval format is merely for convenience and brevity and should not be interpreted as an inflexible limitation within the scope of the invention. Therefore, the description of an interval should be considered as having specifically disclosed all possible sub-intervals, as well as individual numerical values within that interval. For example, the description of a range from 1 to 6 should be considered to have specifically disclosed subintervals, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range, such as 95-99% identity, includes something with 95 %, 96%, 97%, 98% or 99% identity and includes subintervals such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity . This applies regardless of the span of the range.
[0224] [0224] The term "BCL11a" refers to B cell lymphoma / leukemia 11A, a DNA-binding protein specific to the sequence of the proximal region of the major RNA polymerase promoter | and the gene that encodes that protein, along with all the introns and exons. This gene encodes a C2H2 type zinc finger protein. BCL11A was found to play a role in suppressing fetal hemoglobin production. BCL11a is also known as B Cell CLL / Lymphoma 11A (Zinc Finger Protein), CTIP1, EVI9, Ecotropic Viral Integration Site 9 Protein Homolog, COUP-TF Interactive Protein 1, Zinc Finger Protein 856, KIAA1809 , BCL-11A, ZNF856, EVI-9 and B Cell CLL / Lymphoma 11A. The term covers all BLC11a isoforms and splice variants. The human gene encoding BCL11a is mapped to the chromosomal location 2p16.1 (by Ensembl). The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot., And the human BCL11a genomic sequence can be found in GenBank at NC 000002.12. The BCL11a gene refers to this genomic location, including all introns and exons. There are several known isotypes of BCL11a.
[0225] [0225] The MRNA sequence encoding human BCL11a isoform 1 can be found in NM 022893.
[0226] [0226] The peptide sequence of human BCL11a isoform 1 is: 40 50
[0227] [0227] The sequences of other BCL11a protein isoforms are provided in: Isoform 2: Q9H165-2 Isoform 3: Q9H165-3 Isoform 4: Q9H165-4 Isoform 5: Q9H165-5 Isoform 6: Q9H165-6
[0228] [0228] As used herein, a human BCL11a protein also includes proteins that have over their total length, at least about 70%, 71%, 72%, 73%, T4%, T5%, 16%, 17% , 18%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 294 %, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with BCL11A isoform 1-6, wherein such proteins still have at least one of the functions of BCL11a.
[0229] [0229] The term "globin locus" as used herein refers to the region of human chromosome 11 comprising genes for embryonic (e), fetal (G (y) and A (y)) globin genes, adults (y B ), locus control regions and DNase hypersensitivity sites |
[0230] [0230] The term "complementary" as used in connection with nucleic acid, refers to the base pair, A with T or U, and G with C. The complementary term refers to nucleic acid molecules that are completely complementary, that is, they form base pairs of À with T or U and G pairs with C throughout the reference sequence, as well as molecules that are at least 80%, 85%, 90%, 95%, 99% complementary.
[0231] [0231] The term "HPFH non-deletion" refers to a mutation that does not comprise an insertion or deletion of one or more nucleotides, which results in hereditary persistence of fetal hemoglobin and is characterized by an increase in fetal hemoglobin in adult red blood cells. In exemplary modalities, non-deletion HPFH is a mutation described in Nathan and Oski Hematology and Oncology of Infancy and Childhood, 8th Ed., 2015, Orkin SH, Fisher DE, Look T, Lux SE, Ginsburg D, Nathan DG, Eds. Elsevier Saunders, whose total content is incorporated by reference, for example, the non-deletion HPFH mutations described in Table 21-5. The term "non-deletion HPFH region" refers to a genomic site that comprises or is close to a non-deletion HPFH. In exemplary embodiments, the non-deletion HPFH region is the nucleic acid sequence of the HBG1 promoter region (Chr11: 5,249,833 to Chr11: 5,250,237, hg38; - strand), the nucleic acid sequence of the HBG2 promoter region (Chr11: 5,254,738 to Chr11: 5,255,164, hg38; - chain) or combinations thereof. In exemplary embodiments, the non-deletion HPFH region includes one or more of the non-deletion HPFH described in Nathan and Oski Hematology and Oncology of Infancy and Childhood, 8th Ed., 2015, Orkin SH, Fisher DE, Look T, Lux SE, Ginsburg D , Nathan DG, Eds. Elsevier Saunders (for example, described in Table 21-5). In exemplary embodiments, the non-deletion HPFH region is the nucleic acid sequence in chr11: 5,250,094-5,250,237, - strand, hg38; or the nucleic acid sequence in chr11: 5,255,022-5,255,164, - strand, hg38; or the chr11 nucleic acid sequence: 5,249,833-5,249,927, - strand, hg38; or the nucleic acid sequence in chr11: 5,254,738-5,254,851, - strand, hg38; or the nucleic acid sequence in chr11: 5,250,139-5,250,237, - strand, hg38; or their combinations.
[0232] [0232] "BCL11a enhancer" as the term used herein, refers to the nucleic acid sequence that affects, for example, enhances, the expression or function of BCL11a. See, for example, Bauer et al., Science, vol. 342, 2013, pp. 253-257. The BCL11a enhancer may, for example, be operative only on certain types of cells, for example, cells of the erythroid lineage. An example of a BCL11a enhancer is the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene (for example, the nucleic acid at or corresponding to positions +55: Chr2: 60497676- 60498941; +58: Chr2: 60494251 - 60495546; +62: Chr2: 60490409- 60491734 as recorded in hg38). In one embodiment, the BCL11a Enhancer is the +62 region of the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene. In one embodiment, the BCL11a Enhancer is the +58 region of the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene. In one embodiment, the BCL11a Enhancer is the +55 region of the nucleic acid sequence between exon 2 and exon 3 of the BCL11a gene.
[0233] [0233] The terms "hematopoietic stem cells and progenitors" or "HSPC" are used interchangeably and refer to a population of cells comprising hematopoietic stem cells ("HSCs") and hematopoietic progenitor cells ("HPCs") . Such cells are characterized, for example, as CD34 +. In exemplary modalities, HSPCs are isolated from bone marrow. In other exemplary modalities, HSPCs are isolated from peripheral blood. In other exemplary modalities, HSPCs are isolated from umbilical cord blood. In one embodiment, HSPCs are characterized as CD34 + / CD38- / CD90 + / CD45RA-. In modalities, HSPCs are characterized as CD34 + / CD90 + / CD49f + cells. In modalities, HSPCs are characterized as CD34 + cells. In modalities, HSPCs are characterized as CD34 + / CD90 + cells. In modalities, HSPCs are characterized as CD34 + / CD90 + / CD45RA- cells.
[0234] [0234] "Stem cell expander" as used herein refers to a compound that causes cells, for example, HSPCs, HSCs and / or HPCs to proliferate, for example, to increase in number, at a more rapid rate fast for the same cell types absent from said agent. In an exemplary aspect, the stem cell expander is an inhibitor of the aryl hydrocarbon receptor pathway. Additional examples of stem cell expanders are provided below. In modalities, proliferation, for example, increase in number, is carried out ex vivo.
[0235] [0235] "Graft" refers to the incorporation of a cell or tissue, for example, a population of HSPCs, into the body of a recipient, for example, a mammal or human subject. In one example, the graft includes the growth, expansion and / or differentiation of the grafted cells in the recipient. In one example, the HSPC graft includes the differentiation and growth of said HSPCs in erythroid cells within the recipient's body.
[0236] [0236] The term "hematopoietic progenitor cells" (HPCs) as used here refers to primitive hematopoietic cells that have a limited capacity for self-renewal and the potential for multi-lineage differentiation (eg myeloid, lymphoid), differentiation of single lineage (eg, myeloid or lymphoid) or restricted cell type differentiation (eg, erythroid parent) depending on placement within the hematopoietic hierarchy (Doulatov et al., Cell Stem Cell 2012).
[0237] [0237] "Hematopoietic stem cells" (HSCs) as used herein refer to immature blood cells capable of self-renewing and differentiating into more mature blood cells comprising granulocytes (eg, promyelocytes, neutrophils, eosinophils, basophils ), erythrocytes (for example, reticulocytes, erythrocytes), thrombocytes “(for example, megakaryoblasts, platelet-producing megakaryocytes, platelets) and monocytes (for example, monocytes, macrophages). HSCs are interchangeably described as stem cells throughout the specification.
[0238] [0238] "Expansion" or "Expanding" in the context of cells refers to an increase in the number of a characteristic cell type, or cell types, of an initial cell population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells generated from the expansion.
[0239] [0239] "Cell population" refers to eukaryotic mammalian cells, preferably human, isolated from biological sources, for example, blood products or tissues and derivatives of more than one cell.
[0240] [0240] "Enriched" when used in the context of the cell population refers to a cell population selected based on the presence of one or more markers, for example, CD34 +.
[0241] [0241] The term "CD34 + cells" refers to cells that express the CD34 marker on their surface. CD34 + cells can be detected and counted using, for example, fluorescently labeled flow cytometry and anti-CD34 antibodies.
[0242] [0242] "Enriched in CD34 + cells" means that a cell population was selected based on the presence of the CD34 marker. Therefore, the percentage of CD34 + cells in the cell population after the selection method is higher than the percentage of CD34 + cells in the initial cell population before selecting the step based on the CD34 markers. For example, CD34 + cells can represent at least 50%, 60%, 70%, 80% or at least 90% of the cells in a cell population enriched in CD34 + cells.
[0243] [0243] The terms "F cell" and "F cell" refer to cells, usually erythrocytes (for example, red blood cells) that contain and / or produce (for example, express) fetal hemoglobin. For example, an F cell is a cell that contains or produces detectable levels of fetal hemoglobin. For example, an F cell is a cell that contains or produces at least 5 picograms of fetal hemoglobin. In another example, an F cell is a cell that contains or produces at least 6 picograms of fetal hemoglobin. In another example, an F cell is a cell that contains or produces at least 7 picograms of fetal hemoglobin. In another example, an F cell is a cell that contains or produces at least 8 picograms of fetal hemoglobin. In another example, an F cell is a cell that contains or produces at least 9 picograms of fetal hemoglobin. In another example, an F cell is a cell that contains or produces at least 10 picograms of fetal hemoglobin. Fetal hemoglobin levels can be measured using an assay described herein or by another method known in the art, for example, flow cytometry using an anti-fetal hemoglobin detection reagent, high performance liquid chromatography, mass spectrometry or an assay enzyme-linked immunosorbent.
[0244] [0244] Unless otherwise specified, all coordinates of the genome or chromosome are in accordance with hg38. DETAILED DESCRIPTION
[0245] [0245] The gRNA molecules, compositions and methods described here refer to genome editing in eukaryotic cells using the CRISPR / Cas9 system. In particular, the gRNA molecules, compositions and methods described herein refer to the regulation of globin levels and are useful, for example, in the regulation of the expression and production of globin genes and proteins. The gRNA molecules, compositions and methods can be useful in the treatment of hemoglobinopathies. |. GRNA molecules
[0246] [0246] A gRNA molecule can have multiple domains, as more fully described below, however, a gRNA molecule typically comprises at least one cr 'RNA domain (comprising a target domain) and a tracr. The gRNA molecules of the invention, used as a component of a CRISPR system, are useful for modifying DNA (for example, modifying the sequence) at or near the target site. Such modifications include deletions and or insertions that result, for example, in the reduced or eliminated expression of a functional product of the gene comprising the target site. These uses, and additional uses, are described in more detail below.
[0247] [0247] In one embodiment, a unimolecular or sgRNA preferably comprises 5 'to 3 ": a crRNA (containing a target domain complementary to a target sequence and a region that is part of a mast (ie a crRNA mast region)), a loop, and a tracr (which contains a complementary domain to the crRNA mast region and a domain that additionally binds a nuclease or other effector molecule, for example, a Cas molecule, for example, a molecule aCas9), and can have the following format (from 5 'to 3'): [target domain] - [crRNA mast region] - [optional first mast extension] - [loop] - [first optional tracr extension ] - [tracr mast region] - [tracr nuclease binding domain].
[0248] [0248] In embodiments, the tracr nuclease binding domain binds to a Cas protein, for example, a Cas9 protein.
[0249] [0249] In one embodiment, a bimolecular or sa RNA comprises two polynucleotides; the first, preferably from 5 'to 3: a cr »RNA (which contains a target domain complementary to a target sequence and a region that is part of a mast; and the second, preferably from 5' to 3; and a tracr (which contains a domain complementary to the mast region of the crRNA and a domain that additionally binds a nuclease or other effector molecule, for example, a Cas molecule, for example, a Cas9 molecule), and can have the following format (of 5 'to 3):
[0250] [0250] Polynucleotide 1 (cr »RNA): [target domain] - [crRNA mast region] - [optional first mast extension] - [optional second mast extension]
[0251] [0251] Polynucleotide 2 (tracr): [first optional tracr extension] - [tracr mast region] - [tracr nuclease binding domain]
[0252] [0252] In embodiments, the tracr nuclease binding domain binds to a Cas protein, for example, a Cas9 protein.
[0253] [0253] In some respects, the target domain comprises or consists of a target domain sequence described herein, for example, a target domain described in Table 1, or a target domain comprising or consisting of 17, 18, 19 or 20 (preferably,
[0254] [0254] In some respects, the mast, for example, the mast region of the crRNA, comprises from 5 'to 3: GUUUUAGAGCUA (SEQ ID NO: 182).
[0255] [0255] In some respects, the mast, for example, the mast region of the crRNA, comprises from 5 'to 3. GUUUAAGAGCUA (SEQ ID NO: 183).
[0256] [0256] In some respects, the loop comprises from 5 'to 3: GAAA (SEQ ID NO: 186).
[0257] [0257] In some respects, the tracr comprises from 5 'to 3 ":
[0258] [0258] In some respects, the tracr comprises from 5 'to 3º
[0259] [0259] In some respects, the gRNA may also comprise, at the 3 'end, additional U nucleic acids. For example, the gRNA can comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U nucleic acids (SEQ ID NO: 249) at the 3 'end. In one embodiment, the gRNA comprises an additional 4 U nucleic acids at the 3 'end. In the case of dgRNA, one or more of the polynucleotides of the dgRNA (for example, the polynucleotide comprising the target domain and the polynucleotide comprising the tracr) may comprise, na 3 'end, additional U nucleic acids. For example, the case of AgRNA, one or more of the polynucleotides of the dgRNA (for example, the polynucleotide comprising the target domain and the polynucleotide comprising the tracr) may comprise an additional 1, 2,3,4,5,6 ,
[0260] [0260] In some respects, the gRNA may also comprise, at the 3 'end, additional A nucleic acids. For example, the gRNA may comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 90Uu 10 A nucleic acids A (SEQ ID NO: 250) at the 3 'end. In one embodiment, the gRNA comprises an additional 4 nucleic acids A at the 3 'end. In the case of dgRNA, one or more of the polynucleotides of the dgRNA (for example, the polynucleotide comprising the target domain and the polynucleotide comprising the tracr) may comprise , at the 3 'end, additional nucleic acids A. For example, the case of dgRNA, one or more of the polynucleotides of the dgRNA (for example, the polynucleotide comprising the target domain and the polynucleotide comprising the tracr) may comprise an additional 1,2,3,4,5,6 , 7.8, 9uU 10 A nucleic acids A (SEQ ID NO: 250) at the 3 'end. In one embodiment, in the case of dogRNA, one or more of the polynucleotides in the doRNA (for example, the polynucleotide comprising the target domain and the polynucleotide comprising the tracr) comprise an additional 4 nucleic acids A at the end 3. In one embodiment of a doRNA, only the polynucleotide comprising the tracr comprises the additional A nucleic acid (s), for example, 4 nucleic acids A. In a dgRNA embodiment, only the polynucleotide comprising the domain Target comprises the additional nucleic acid (s) A. In a dgRNA embodiment, both the polynucleotide comprising the target domain and the polynucleotide comprising the tracr comprise additional U nucleic acids, for example, 4 A nucleic acids.
[0261] [0261] In embodiments, one or more of the polynucleotides of the gRNA molecule may comprise a cap at the 5 "end.
[0262] [0262] In one embodiment, a unimolecular or gRNA preferably comprises from 5 'to 3 a crRNA (which contains a target domain complementary to a target sequence; a crRNA mast region; first mast extension; a a first tacr extension (which contains a domain complementary to at least a portion of the first mast extension) and a tracr (which contains a domain complementary to the crRNA mast region, and a domain that additionally binds a molecule In some respects, the target domain comprises or consists of a target domain sequence described herein, for example, a target domain described in Table 1, or a target domain comprising or consisting of 17, 18, 19 or (preferably 20) consecutive nucleotides from a sequence of the target domain described in Table 1, for example, 3 '17, 18, 19 or 20 (preferably, 20) consecutive nucleotides from a sequence of the target domain described in Table 1.
[0263] [0263] In aspects comprising a first mast extension and / or a first tracr extension, the mast, loop and tracr sequences can be as described above. In general, any first mast extension and first tracer extension can be used, as long as they are complementary. In modalities, the first mast extension and first tracr extension consist of 3, 4, 5, 6, 7, 8, 9, 10 or more complementary nucleotides.
[0264] [0264] In some respects, the first mast extension comprises from 5 'to 3: UGCUG (SEQ ID NO: 184). In some respects, the first extension of the mast consists of SEQ ID NO: 184.
[0265] [0265] In some respects, the first extension of the tracr comprises from 5 'to 3: CAGCA (SEQ ID NO: 189). In some respects, the first extension of tracr consists of SEQ ID NO: 189.
[0266] [0266] In one embodiment, a dgRNA comprises two nucleic acid molecules. In some aspects, the dgRNA comprises a first nucleic acid that contains, preferably from 5 'to 3: a target domain complementary to a target sequence; a crRNA mast region; optionally, a first mast extension; and, optionally, a second mast extension; and a second nucleic acid (which may be referred to herein as a tracr) and comprising at least one domain that binds to a Cas molecule, for example, a Cas9 molecule) preferably comprising 5 'to 3': optionally a first tracr extension; and a tracr (which contains a domain complementary to the CR »RNA mast region and a domain that additionally binds a Cas molecule, for example, Cas9). The second nucleic acid may additionally comprise, at the 3 'end (e.g., 3' for tracr), additional U nucleic acids. For example, the tracr can comprise an additional 1, 2, 3,4,5,6, 7, 8, 9 or 10 U nucleic acids (SEQ ID NO: 249) at the 3 'end (for example, 3' for the tracr ). The second nucleic acid may additionally or alternately comprise, at the 3 'end (e.g., 3' for tracr), additional A nucleic acids. For example, the tracr can comprise an additional 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 A nucleic acids (SEQ ID NO: 250) at the 3 'end (for example, 3' for the tracr ). In some respects, the target domain comprises a target domain sequence described herein, for example, a target domain described in Table 1, or a target domain comprising or consisting of 17, 18, 19 or 20 (preferably, 20 ) consecutive nucleotides of a target domain sequence described in Table 1.
[0267] [0267] In aspects involving a dgRNA, the crRNA mast region, the optional first mast extension, the optional first tracr extension and the tracr sequences can be as described above.
[0268] [0268] In some respects, the optional second mast extension comprises 5 'to 3 ": UUUUG (SEQ ID NO: 185).
[0269] [0269] In embodiments, nucleotides 3 '1, 2, 3, 4 or 5, nucleotides 5' 1, 2, 3, 4 or 5, or both nucleotides 3 'and 5' 1,2,3, 4 or 5 of the gRNA molecule (and in the case of a dgRNA molecule, the polynucleotide comprising the target domain and / or the polynucleotide comprising the tracr) are modified nucleic acids, as described more fully in section XIII, below.
[0270] [0270] The domains are discussed briefly below: 1) The target domain:
[0271] [0271] Guidance on selecting target domains can be found, for example, in Fu Y el al. NAT BIOTECHNOL 2014 (doi:
[0272] [0272] The target domain comprises a nucleotide sequence that is complementary, for example, at least 80, 85, 90, 95, or 99% complementary, for example, completely complementary, with the target sequence in the target nucleic acid. The target domain is part of an RNA molecule and therefore comprises the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). Although not intended to be limited by theory, it is believed that the complementarity of the target domain with the target sequence contributes to the specificity of the interaction of the gRNA / Cas9 molecule complex with a target nucleic acid. It is understood that in a target domain and a pair of target sequences, the uracil bases in the target domain will pair with the adenine bases in the target sequence.
[0273] [0273] In one embodiment, the target domain is 5 to 50, for example, 10 to 40, for example, 10 to 30, for example, 15 to 30, for example, 15 to 25 nucleotides in length. In one embodiment, the target domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In one embodiment, the target domain is 16 nucleotides in length. In one embodiment, the target domain is 17 nucleotides in length. In one embodiment, the target domain is 18 nucleotides in length. In one embodiment, the target domain is 19 nucleotides in length. In one embodiment, the target domain is 20 nucleotides in length. In embodiments, the 16, 17, 18, 19 or 20 nucleotides mentioned above comprise the 5'-16, 17, 18, 19 or 20 nucleotides of a target domain described in Table 1. In embodiments, the 16, 17, 18 , 19 or 20 nucleotides mentioned above comprise the 3'-16, 17, 18, 19 or nucleotides of a target domain described in Table 1.
[0274] [0274] Without being bound by theory, it is believed that the 8, 9, 10, 11 or 12 nucleic acids of the target domain arranged at the 3 'end of the target domain are important for targeting the target sequence, and can thus be referred to as the "central" region of the target domain. In one embodiment, the central domain is completely complementary to the target sequence.
[0275] [0275] The target nucleic acid strand with which the target domain is complementary is referred to here as the target sequence. In some ways, the target sequence is arranged on a chromosome, for example, it is a target within a gene. In some ways, the target sequence is arranged within an exon of a gene. In some ways, the target sequence is arranged within an intron of a gene. In some respects, the target sequence comprises, or is proximal (for example, within 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1000 nucleic acids) to a binding site of a regulatory element, for example, a promoter or transcription factor binding site, of a gene of interest. Some or all of the nucleotides in the domain may have a modification, for example, a modification found here in Section XIII. 2) crRNA mast region:
[0276] [0276] The mast contains parts of the crRNA and the tracr. The crRNA mast region is complementary with a portion of the tracr and, in one embodiment, has sufficient complementarity with a portion of the tracr to form a duplex region under at least some physiological conditions, for example, normal physiological conditions. In one embodiment, the crRNA mast region is 5 to 30 nucleotides in length. In one embodiment, the mast region of the cr »RNA is 5 to 25 nucleotides in length. The CR »RNA mast region can share homology with, or be derived from, a naturally occurring portion of the repeat sequence from a bacterial CRISPR array. In one embodiment, it has at least 50% homology to a crRNA mast region disclosed herein, for example, a S. pyogenes or S. thermophilus RNA mast region.
[0277] [0277] In one embodiment, the mast, for example, the mast region of RNA, comprises SEQ ID NO: 182. In one embodiment, the mast, for example, the mast region of RNA, comprises a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with SEQ ID NO: 182. In one embodiment, the mast, for example, the region crRNA mast, comprises at least 5, 6, 7, 8, 9, 10 or 11 nucleotides of SEQ ID NO: 182. In one embodiment, the mast, for example, the crRNA mast region, comprises SEQ ID NO: 183. In one embodiment, the mast comprises a sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with SEQ ID NO: 183. In one embodiment, the mast, for example, the mast region of crRNA, comprises at least 5, 6, 7, 8, 9, 10 or 11 nucleotides of SEQ ID NO: 183.
[0278] [0278] Some or all nucleotides in the domain may have a modification, for example, modification described here in Section XIII. 3) First mast extension
[0279] [0279] When a tracr comprising a first tracr extension is used, the crRNA may comprise a first mast extension. In general, any first mast extension and first tracer extension can be used, as long as they are complementary. In modalities, the first mast extension and first tracr extension consist of 3, 4, 5, 6, 7, 8, 9, 10 or more complementary nucleotides.
[0280] [0280] The first mast extension may comprise nucleotides that are complementary, for example, 80%, 85%, 90%, 95% or 99%, for example, fully complementary, with nucleotides from the first tracr extension. In some respects, the first nucleotides of the mast extension that hybridize with complementary nucleotides of the first tracr extension are contiguous. In some aspects, the first nucleotides of the mast extension that hybridize with complementary nucleotides of the first tracr extension are discontinuous, for example, comprise two or more hybridization regions separated by nucleotides that do not form base pairs with nucleotides of the first tracr extension. In some respects, the first mast extension comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some respects, the first mast extension comprises 5 'to 3: UGCUG (SEQ ID NO: 184). In some respects, the first mast extension consists of SEQ ID NO: 184. In some aspects, the first mast extension comprises nucleic acid that is at least 80%, 85%, 90%, 95% or 99% homology with SEQ ID NO: 184.
[0281] [0281] Some or all of the nucleotides in the first tracr extension may have a modification, for example, a modification found here in Section XIII. 3) The handle
[0282] [0282] A loop serves to connect the crRNA mast region (or, optionally, the first mast extension, when present) with the tracr (or optionally the first tracr extension, when present) of a sgRNA. The loop can connect the RNA mast region and tracr covalently or non-covalently. In one embodiment, the bond is covalent. In one embodiment, the loop covalently engages the crRNA and tracr mast region. In one embodiment, the handle covalently engages the first extension of the mast and the first extension tracr. In one embodiment, the loop is, or comprises, a covalent bond interposed between the cr 'RNA mast region and the tracr domain that hybridizes to the crRNA mast region. Typically, the loop comprises one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
[0283] [0283] In dgRNA molecules, the two molecules can be associated by virtue of hybridization between at least a portion of the crRNA (for example, the mast region of crRNA) and at least a portion of the tracr (for example , the tracr domain that is complementary to the crRNA mast region).
[0284] [0284] A wide variety of handles are suitable for use in SARNAs. The loops may consist of a covalent bond, or be as short as one or a few nucleotides, for example, 1, 2, 3,4 or 5 nucleotides in length. In one embodiment, a loop is 2, 3, 4,5,6,7, 8,9, 10, 15, 20, or 25 or more nucleotides in length. In one embodiment, a loop is 2 to 50, 2 to 40, 2a 30.2 to 20.2 to 10, or 2 to 5 nucleotides in length. In one embodiment, a loop shares homology with, or is derived from, a naturally occurring sequence. In one embodiment, the loop has at least 50% homology with a loop shown here. In one embodiment, the handle comprises SEQ ID NO: 186.
[0285] [0285] Some or all nucleotides in the domain may have a modification, for example, modification described here in Section XIII. 4) The second extension of the mast
[0286] [0286] In one embodiment, a dgRNA may comprise an additional sequence, 3 'to the crRNA mast region or, when present, the first mast extension, referred to herein as the second mast extension. In one embodiment, the second extension of the mast is 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length. In one embodiment, the second extension of the mast is 2,3,4,5,6,7,8,9 or 10 or more nucleotides in length. In one embodiment, the second extension of the mast comprises SEQ ID NO: 185. 5) The Tracr:
[0287] [0287] Tracr is the nucleic acid sequence necessary for nuclease binding, for example, Cas9. Without being limited by theory, it is believed that each Cas9 species is associated with a particular tracr sequence. Tracr sequences are used in the SgRNA and dgRNA systems. In one embodiment, the tracr comprises a sequence of, or derived from, a S. pyogenes tracr. In some respects, the tracr has a portion that hybridizes to the mast portion, for example, it has sufficient complementarity to the crRNA mast region to form a duplex region under at least some physiological conditions (sometimes referred to here as the region tracr mast or a tracr domain complementary to the crRNA mast region). In modalities, the domain of the tracr that hybridizes with the mast region of cr »RNA comprises at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that hybridize to complementary nucleotides from the mast region of crRNA. In some respects, the tracr nucleotides that hybridize to complementary nucleotides from the crRNA mast region are contiguous. In some respects, the tracr nucleotides that hybridize to complementary nucleotides from the crRNA mast region are discontinuous, for example, comprise two or more hybridization regions separated by nucleotides that do not form base pairs with nucleotides from the CR »RNA region. In some respects, the tracr portion that hybridizes to the mast region of the crRNA, comprises 5 'to 3: UAGCAAGUUAAAA (SEQ ID NO: 191). In some respects, the tracr portion that hybridizes to the mast region of the CcrRNA, comprises 5 'to 3: UAGCAAGUUUAAA (SEQ ID NO: 192). In embodiments, the sequence that hybridizes to the mast region of crRNA is arranged in tracr 5'- for the tracr sequence that additionally binds a nuclease, for example, a Cas molecule, for example, a Cas9 molecule.
[0288] [0288] Tracr further comprises a domain that additionally binds to a nuclease, for example, a Cas molecule, for example, a Cas9 molecule. Without being limited by theory, it is believed that Cas9 of different species bind to different tracr sequences. In some ways, tracr comprises the sequence that binds to a Cas9 molecule of S. pyogenes. In some respects, tracr comprises the sequence that binds to a Cas9 molecule disclosed herein. In some respects, the domain that additionally binds to a Cas9 molecule comprises 5 to 3 "
[0289] [0289] In some modalities, tracr comprises SEQ ID NO:
[0290] [0290] Some or all of the tracr nucleotides may have a modification, for example, a modification found here in Section XIII. In embodiments, the gRNA (for example, the saRNA or the tracr and / or crRNA of a dgRNA), for example, any of the gRNA or gRNA components described above, comprises an inverted abasic residue at the 5 'end, at the 3' end or at both the 5 'and 3' ends of the gRNA. In embodiments, the gRNA (for example, the saRNA or the tracr and / or crRNA of a daoRNA), for example, any of the gRNA or gRNA components described above, comprises one or more phosphorothioate bonds between the residues at the 5 'end of the polynucleotide, for example, a phosphorothioate bond between the first two residues 5 ', between each of the first three residues 5', between each of the first four residues 5 ', or between each of the first five residues 5'. In embodiments, the gRNA or gRNA component may alternatively or additionally comprise one or more phosphorothioate bonds between the residues at the 3 'end of the polynucleotide, for example, a phosphorothioate bond between the first two 3' residues, between each of the first three residues 3 ', between each of the first four residues 3', or between each of the first five residues 3 '. In one embodiment, gRNA (for example,
[0291] [0291] In one embodiment, the gRNA (for example, the saRNA or the tracr and / or crRNA of a dagRNA), for example, any of the gRNA or gRNA components described above, comprises, for example, consists of a link phosphorothioate between each of the first four 5 'residues (for example, comprises, for example, consists of three, phosphorothioate bonds at the 5' end (s) of the polynucleotide (s)) and a phosphorothicoate bond between each of the first four 3 'residues (for example, comprises, for example, consists of three phosphorothioate bonds at the 5' ends of the polynucleotide (s)), 2 'O-methyl modification in each of the first three residues in 5 ', 2' O-methyl modification in each of the first three residues in 3 'and an additional inverted abasic residue in each of the 5' and 3 "ends.
[0292] [0292] In one embodiment, the gRNA (for example, the saRNA or the tracr and / or cr »RNA of a dagRNA), for example, any of the gRNA or gRNA components described above, comprises, for example, consists of, a phosphorothioate bond between each of the first four 5 'residues (for example, comprises, for example, consists of three phosphorothioate bonds at the 5' end (s) of the polynucleotide (s)) and a phosphoroticate bond between each of the first four 3 'residues (for example, comprises, for example, consists of three phosphorothioate bonds at the 5' ends of the polynucleotide (s)), a 2 'O-methyl modification in each of the first three residues 5 'modification, and 2' O-methyl in each of residues 4º to the terminal, 3º to the terminal and 2º to the terminal in 3 'and an additional inverted abasic residue at each of the 5' and
[0293] [0293] In one embodiment, gRNA is a dgRNA and comprises, for example, consists of: crRNA: MN * MN * mMN * NNNNNNNNNNNNNNNNNGUUUUVUAGAGCUAU * mG * mC * mU (SEQ ID NO: 251), where m indicates a base with 2'-O- modification
[0294] [0294] In one embodiment, gRNA is a dgRNA and comprises, for example, consists of: crRNA: MN * MN * MN * NNNNNNNNNNNNNNNNNGUUUUAGAGCUAU * mG * mC * mU (SEQ ID NO: 251), where m indicates a base with modification 2'-O-methyl, * indicates a phosphorothioate bond, and N's indicate residues of the target domain, for example, as described herein, (optionally with an abasic residue inverted at the 5 'and / or 3' terminal); and tracr: MA * MA * MC * AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU * mMU * mU * mU (SEQ ID NO: 246), where m indicates a base with 2'-O-methyl modification, * indicates the residues and indicates the residue, * indicates of the target domain, for example, as described herein, (optionally with an abasic residue inverted at the 5 'and / or 3' terminal).
[0295] [0295] In one embodiment, gRNA is a dgRNA and comprises, for example, consists of: crRNA: MN * mMN * mMN * NNNNNNNNNNNNNNNNNGUUUVAGAGCUAUGCUGU U * mMU * mU * mG (SEQ ID NO: 252), where m indicates with 2'-O-methyl modification, * indicates a phosphoroticate bond, and N's indicate residues of the target domain, for example, as described herein,
[0296] [0296] In one embodiment, gRNA is a dgRNA and comprises, for example, consists of: crRNA: MN * MN * MN * NNNNNNNNNNNNNNNNNGUUUUVAGAGCUAUGCUGU U * mMU * mU * mG (SEQ ID NO: 252), where m indicates a base with 2'-O-methyl modification, * indicates a phosphoroticate bond, and N's indicate residues of the target domain, for example, as described herein, (optionally with an abasic residue inverted at the 5 'and / or 3' end); and tracr: MA * MA * MC * AGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA ACUUGAAAAAGUGGCACCGAGUCGGUGCUUU * mMU * mU * mU (SEQ ID NO: 246), where m indicates a base with a 2'-O-methyl modification (a link is optional) inverted abasic terminal 5 'and / or 3').
[0297] [0297] In one embodiment, gRNA is a dgRNA and comprises, for example, consists of: crRNA:
[0298] [0298] In one embodiment, gRNA is a sgRNA and comprises, for example, consists of: NNNNNNNNNNNNNNNNNNNNGUUUUVAGAGCUAGAAAUAGCAAGU
[0299] [0299] In one embodiment, gRNA is a sgRNA and comprises, for example, consists of: MN * MN * MN * NNNNNNNNNNNNNNNNNGUUUUVAGAGCUAGAAAUA
[0300] [0300] In one embodiment, gRNA is a sgRNA and comprises, for example, consists of: MN * MN * MN * NNNNNNNNNNNNNNNNNGUUUUVAGAGCUAGAAAUA
[0301] [0301] When the gRNA comprises a first mast extension, the tracr can comprise a first tracr extension. The first tracr extension can comprise nucleotides that are complementary, for example, 80%, 85%, 90%, 95% or 99%, for example, fully complementary, with nucleotides from the first mast extension. In some respects, the first nucleotides of the tracr extension that hybridize to complementary nucleotides of the first mast extension are contiguous. In some respects, the first nucleotides of the tracr extension that hybridize to complementary nucleotides of the first mast extension are discontinuous, for example, they comprise two or more hybridization regions separated by nucleotides that do not form base pairs with nucleotides of the first mast extension. In some respects, the first tracer extension comprises at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some respects, the first tracer extension comprises SEQ ID NO: 189. In some respects, the first tracr extension comprises nucleic acid that is at least 80%, 85%, 90%, 95% or 99% homology with SEQ ID NO: 189.
[0302] [0302] Some or all of the nucleotides in the first tracr extension may have a modification, for example, a modification found here in Section XIII.
[0303] [0303] In some modalities, the saRNA can comprise, from 5 'to 3', arranged in 3 'to the target domain:
[0304] [0304] In one embodiment, a sgRNA of the invention comprises, for example, consists of, from 5 'to 3: [target domain] -
[0305] [0305] In one embodiment, a sgRNA of the invention comprises, for example, consists of, from 5 'to 3: [target domain] - GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG
[0306] [0306] In some modalities, dgRNA can comprise:
[0307] [0307] A crRNA comprising, from 5 'to 3', preferably directly arranged in 3 'for the target domain: a) GUUUUAGAGCUA (SEQ ID NO: 182); b) GUUUAAGAGCUA (SEQ ID NO: 183); c) GUUUUAGAGCUAUGCUG (SEQ ID NO: 199); d) GUUUAAGAGCUAUGCUG (SEQ ID NO: 200); e) GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 201); fN) GUUUAAGAGCUAUGCUGUUUUG (SEQ ID NO: 202); or 9) GSUUUUAGAGCUAUGCU (SEQ ID NO: 226): and a tracer comprising from 5 'to 3': a)
[0308] [0308] In one embodiment, the k) sequence, above, comprises the 3 'UUUUUU sequence, for example, if a U6 promoter is used for transcription. In one embodiment, the k) sequence above comprises the 3 'UUÚU sequence, for example, if an HI promoter is used for transcription. In one embodiment, the sequence of k) above comprises variable numbers of Us in 3 ', depending, for example, on the termination signal of the policy promoter.
[0309] [0309] In one embodiment, the cr »RNA comprises, for example, consists of a target domain and, arranged 3 'in relation to the target domain (for example, arranged directly in 3' in relation to the target domain) , a sequence comprising, for example, consisting of, SEQ ID NO: 201, and the tracr comprising, for example, consists of
[0310] [0310] In one embodiment, the cr »RNA comprises, for example, consists of a target domain and, arranged 3 'in relation to the target domain (for example, arranged directly in 3' in relation to the target domain) , a sequence comprising, for example, consisting of, SEQ ID NO: 202, and the tracr comprising, for example, consists of
[0311] [0311] In one embodiment, the cr »RNA comprises, for example, consists of a target domain and, arranged in 3 'in relation to the target domain (for example, arranged directly in 3' in relation to the target domain) , a sequence comprising, for example, consisting of, GUUUUAGAGCUAUGCU (SEQ ID NO: 226, and the tracr comprises, for example, consists of GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGG
[0312] [0312] In one embodiment, the cr »RNA comprises, for example, consists of a target domain and, arranged 3 'in relation to the target domain (for example, arranged directly in 3' in relation to the target domain) , a sequence comprising, for example, consisting of, GUUUUAGAGCUAUGCU (SEQ ID NO: 226, and the tracr comprises, for example, consists of
[0313] [0313] In one embodiment, the cr »RNA comprises, for example, consists of a target domain and, arranged 3 'in relation to the target domain (for example, arranged directly in 3' in relation to the target domain) , a sequence comprising, for example, consisting of, GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 201, and the tracr comprises, for example, consists of GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAG
[0314] [0314] In the table below are provided target domains targeting non-deletion HPFH regions, gRNA molecules of the present invention, and for use in the various aspects of the present invention, for example, in altering the expression of globin genes, for example , a fetal hemoglobin gene or a beta hemoglobin gene. Table 1: GRNA target domains targeting non-deletion HPFH regions. SEQ ID NO: s refers to the sequence of the gRNA target domain.
[0315] [0315] Table 2, below, shows those target domains that, when included in a gRNA molecule, result in at least 17% increase in fetal hemoglobin (for example, in erythroid cells differentiated from modified HSPCs) in 7 days according to the methods described in the Examples. GRNA molecules comprising any of these target domains are collectively referred to here as Level 2 gRNA molecules. Table 2 - Target domains for Level 2 gRNA molecules Target Domain ID GCR-0001 GCR-0006 GCR-0008 GCR -0009 GCR-0010 GCR-0011 GCR-0012 GCR-0028 GCR-0034 GCR-0045 GCR-0046 GCR-0047 GCR-0048 GCR-0050 GCR-0051
[0316] [0316] Table 3a and Table 3b, below, show those target domains that, when included in a gRNA molecule, result in the largest increase in fetal hemoglobin (for example, in erythroid cells differentiated from modified HSPCs) in 7 days of according to the methods described in the Examples. The gRNA molecules comprising these target domains are collectively referred to herein as Level 1 gRNA molecules (for example, Level 1a or Level 1b).
[0317] [0317] Methods for creating gRNAs are described here, including methods for selecting, creating and validating target sequences. Exemplary target domains are also provided here. Target domains discussed here can be incorporated into the gRNAs described here.
[0318] [0318] Methods for selecting and validating target sequences, as well as off-target analyzes are described, for example, in Mali et al. , 2013 SCIENCE 339 (6121): 823-826; Hsu et al 2013 NAT BIOTECHNOL, 31 (9): 827-32; Fu et al, 2014 NAT BIOTECHNOL, doi:
[0319] [0319] For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, for example, to minimize total off-target activity across the genome. Off-target activity can be different from cleavage. For each possible choice of gRNA, for example, using Cas9 of S. bpyogenes, the tool can identify all off-target sequences (for example, preceding NAG or NGG PAMs) across the genome that contains up to a certain number (for example, 1, 2, 3, 4, 5, 6,7, 8, 92 or 10) of unpaired base pairs. The cleavage efficiency in each off-target sequence can be predicted, for example, using an experimentally derived weighting scheme. Each possible gRNA is then classified according to its cleavage outside the predicted total target; the best classified gRNAs represent those that are likely to have the highest cleavage on the target and the least off-target. Other functions, for example, creating an automated reagent for building CRISPR, creating primers for the assay
[0320] [0320] Although software algorithms can be used to generate an initial list of potential gRNA molecules, cutting efficiency and specificity will not necessarily reflect predicted values, and gRNA molecules typically require screening on specific cell lines, for example , primary human cell lines, for example, human HSPCs, for example, human CD34 + cells, to determine, for example, cutting efficiency, indel formation, cutting specificity and alteration in the desired phenotype. These properties can be tested by the methods described here.
[0321] [0321] In aspects of the invention, a gRNA comprising the target domain of GCR-0001 (SEQ ID NO: 1, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: sSaRNA GCR-0001 1: AGUCCUGGUAUCCUCUAUGAGUUUUAGAGCUAGAAAUAGCAAGU
[0322] [0322] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0323] [0323] In aspects of the invention, a gRNA comprising the target domain of GCR-0006 (SEQ ID NO: 6, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SaRNA GCR-0006 * 1: AAAAACUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGU
[0324] [0324] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "MN" (where N = A, G, C or U) denotes a 2- modified nucleotide. OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0325] [0325] In aspects of the invention, a gRNA comprising the target domain of GCR-0008 (SEQ ID NO: 8, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: sSaRNA GCR-0008 * 1: GGAGAAGGAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGU
[0326] [0326] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0327] [0327] In aspects of the invention, a gRNA comprising the target domain of GCR-009 (SEQ ID NO: 9, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: sSaRNA GCR-0009 * 1: GUUUCCUUCUCCCAUCAUAGGUUUUAGAGCUAGAAAUAGCAAGU
[0328] [0328] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein.
[0329] [0329] In aspects of the invention, a gRNA comprising the target domain of GCR-0010 (SEQ ID NO: 10, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgaRNA GCR-0010 * & 1: GGGAGAAGGAAACUAGCUAAGUUUUAGAGCUAGAAAUAGCAAGU
[0330] [0330] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "MN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein.
[0331] [0331] In aspects of the invention, a gRNA comprising the target domain of GCR-0011 (SEQ ID NO: 11, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgRNA GCR-0011 * & 1: CACUGGAGCUAGAGACAAGAGUUUUAGAGCUAGAAAUAGCAAGU
[0332] [0332] In each of the gRNA molecules described above, a
[0333] [0333] In aspects of the invention, a gRNA comprising the target domain of GCR-0012 (SEQ ID NO: 12, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SaRNA GCR-0012 H1: AGAGACAAGAAGGUAAAAAAGUUUUAGAGCUAGAAAUAGCAAGU
[0334] [0334] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein.
[0335] [0335] In aspects of the invention, a gRNA comprising the target domain of GCR-0028 (SEQ ID NO: 28, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgaRNA GCR-0028 * 1: GGCUAGGGAUGAAGAAUAAAGUUUUAGAGCUAGAAAUAGCAAGU
[0336] [0336] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2'- modified nucleotide OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0337] [0337] In aspects of the invention, a gRNA comprising the target domain of GCR-0034 (SEQ ID NO: 34, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgRNA GCR-0034 * 1: AAAAAUUGGAAUGACUGAAUGUUUUAGAGCUAGAAAUAGCAAGU
[0338] [0338] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2 'modified nucleotide -MOM. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0339] [0339] In aspects of the invention, a gRNA comprising the target domain of GCR-0045 (SEQ ID NO: 45, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgRNA GCR-0045 * 1: UGGUCAAGUUUGCCUUGUCAGUUUUAGAGCUAGAAAUAGCAAG
[0340] [0340] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2- modified nucleotide. OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein.
[0341] [0341] In aspects of the invention, a gRNA comprising the target domain of GCR-0046 (SEQ ID NO: 46, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here:
[0342] [0342] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2'- modified nucleotide OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein.
[0343] [0343] In aspects of the invention, a gRNA comprising the target domain of GCR-0047 (SEQ ID NO: 47, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgRNA GCR-0047 H1: GGCAAGGCUGGCCAACCCAUGUUUUAGAGCUAGAAAUAGCAAG
[0344] [0344] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2 'modified nucleotide -MOM. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0345] [0345] In aspects of the invention, a gRNA comprising the target domain of GCR-0048 (SEQ ID NO: 48, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgRNA GCR-0048 * 1: ACGGCUGACAAAAGAAGUCCGUUUUVUAGAGCUAGAAAUAGCAAGU
[0346] [0346] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2- modified nucleotide. OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0347] [0347] In aspects of the invention, a gRNA comprising the target domain of GCR-0050 (SEQ ID NO: 50, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: sSaRNA GCR-0050 1: CCEUGGCUAAMACUCCACCCAUGUUUUAGAGCUAGAAAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA
[0348] [0348] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0349] [0349] In aspects of the invention, a gRNA comprising the target domain of GCR-0051 (SEQ ID NO: 51, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SaRNA GCR-0051 1: GGAGAAGAAAACUAGCUAAAGUUUUAGAGCUAGAAAUAGCAAGU
[0350] [0350] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2- modified nucleotide. OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0351] [0351] In aspects of the invention, a gRNA comprising the target domain of GCR-0053 (SEQ ID NO: 53, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgRNA GCR-0053 * 1: CUUGUCAAGGCUAUUGGUCAGUUUUAGAGCUAGAAAUAGCAAG
[0352] [0352] In each of the gRNA molecules described above, an n "*" denotes a phosphorothioate bond between adjacent nucleotides, and "MN" (where N = A, G, C or U) denotes a 2- modified nucleotide. OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0353] [0353] In aspects of the invention, a gRNA comprising the target domain of GCR-0054 (SEQ ID NO: 54, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SaRNA GCR-0054 1: AGUCCUGGUAUCUUCUAUGGGUUUUAGAGCUAGAAAUAGCAAG
[0354] [0354] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides,
[0355] [0355] In aspects of the invention, a gRNA comprising the target domain of GCR-0058 (SEQ ID NO: 58, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SaRNA GCR-0058 * 1: GUCCUGEGUAUCUUCUAUGGUGUUUUAGAGCUAGAAAUAGCAAG
[0356] [0356] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "MN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein.
[0357] [0357] In aspects of the invention, a gRNA comprising the target domain of GCR-0062 (SEQ ID NO: 62, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SgaRNA GCR-0062 * 1: CUUGACCAAUAGCCUUGACAGUUUUAGAGCUAGAAAUAGCAAGU
[0358] [0358] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2'- modified nucleotide OMe. In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0359] [0359] In aspects of the invention, a gRNA comprising the target domain of GCR-0063 (SEQ ID NO: 63, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: sSaRNA GCR-0063 * 1: CAAGGCUAUUGGUCAAGGCAGUUUUAGAGCUAGAAAUAGCAAGU
[0360] [0360] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described herein, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in the methods, cells and other aspects and modalities of the invention, for example, described herein.
[0361] [0361] In aspects of the invention, a gRNA comprising the target domain of GCR-0067 (SEQ ID NO: 67, unmodified sequence underlined below), for example, one of the gRNA molecules described below, is useful in CRISPR systems, methods, cells and other aspects and modalities of the invention, including aspects involving more than one gRNA molecule, for example, described here: SaRNA GCR-0067 * 1: ACUGAAUCGGAACAAGGCAAGUUUUAGAGCUAGAAAUAGCAAGU
[0362] [0362] In each of the gRNA molecules described above, an "*" denotes a phosphorothioate bond between adjacent nucleotides, and "mN" (where N = A, G, C or U) denotes a 2-OMe modified nucleotide . In embodiments, any of the gRNA molecules described herein, for example, described above, are complexed with a Cas9 molecule, for example, as described here, to form a ribonuclear protein (RNP) complex. Such RNPs are particularly useful in methods, cells and other aspects and modalities of the invention, for example, described herein. IV. Mol Molecules Cas Molecules Cas9
[0363] [0363] In preferred embodiments, the Cas molecule is a Cas9 molecule. Cas9 molecules of a variety of species can be used in the methods and compositions described herein. Although the Cas9 molecule of S. pyogenes is the subject of many of the disclosures presented here, Cas9 molecules of,
[0364] [0364] A Cas9 molecule, as the term is used here, refers to a molecule that can interact with a gRNA molecule (for example, a tracr domain sequence) and, in conjunction with the gRNA molecule, locate (for example, target or house) for a site comprising a target sequence and a PAM sequence.
[0365] [0365] In one embodiment, the cas9 molecule is able to cleave a target nucleic acid molecule, which can be referred to here as an active Cas9 molecule. In one embodiment, an active Cas9 molecule comprises one or more of the following activities: nickase activity, that is, the ability to cleave a single strand, for example, the non-complementary strand or the complementary strand of a nucleic acid molecule; a double-stranded nuclease activity, that is, the ability to cleave both strands of a double-stranded nucleic acid and create a double-stranded break, which in one embodiment is the presence of two nickase activities; an endonuclease activity; an exonuclease activity; and a helicase activity, that is, the ability to unwind the helical structure of a double-stranded nucleic acid.
[0366] [0366] In one embodiment, an enzymatically active Cas9 molecule cleaves both strands of DNA and results in a double strand break. In one embodiment, a Cas9 molecule cleaves only one strand, for example, the strand to which the gRNA hybridizes, or the strand complementary to the strand with which gRNA hybridizes. In one embodiment, an active Cas9 molecule comprises cleavage activity associated with an HNH-like domain. In one embodiment, an active Cas9 molecule comprises cleavage activity associated with an N-terminal RuvC-like domain. In one embodiment, an active Cas9 molecule comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an RuvC-like domain in N-terminal. In one embodiment, an active Cas9 molecule comprises an active or competent HNH-like domain for cleavage and a RuvC-like domain in an inactive or incompetent N-terminal for cleavage. In one embodiment, an active Cas9 molecule comprises an inactive or incompetent HNH-like domain for cleavage and an RuvC-like domain in active or competent N-terminal for cleavage.
[0367] [0367] In one embodiment, the ability of an active Cas9 molecule to interact with and cleave a target nucleic acid is dependent on the PAM sequence. A PAM sequence is a sequence in the target nucleic acid. In one embodiment, cleavage of the target nucleic acid occurs upstream of the PAM sequence. Active Cas9 molecules from different bacterial species can recognize different sequence motifs (for example, PAM sequences). In one embodiment, an active Cas9 molecule from S. pyogenes recognizes the NGG sequence motif and directs the cleavage of a target nucleic acid sequence from 1 to 10, for example, 3 to 5, base pairs upstream of that sequence. See, for example, Mali el al, SCIENCE 2013; 339 (6121): 823-266. In one embodiment, an active Cas9 molecule from S. thermophilus recognizes the NGGNG and NNAG AAW sequence motif (W = A or T) and directs the cleavage of a major target nucleic acid sequence from 1 to 10, for example, 3 to 5, base pairs upstream of these sequences. See, for example, Horvath et al., SCIENCE 2010; 327 (5962): 167-170 and Deveau et al, J BACTERIOL 2008; 190 (4): 1390-
[0368] [0368] In one embodiment, an active Cas9 molecule from S. aureus recognizes the NNGRR sequence motif (R = A or G) and directs the cleavage of a target nucleic acid sequence from 1 to 10, for example, 3 to 5 , base pairs upstream of that sequence. See, for example, Ran F. et al., NATURE, vol. 520, 2015, pp. 186-191. In one embodiment, an active Cas9 molecule of N. meningitidis recognizes the NNNNGATT sequence motif and directs the cleavage of a target nucleic acid sequence from 1 to 10, for example, 3 to 5, base pairs upstream of that sequence. See, for example, Hou et al., PNAS EARLY EDITION 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, for example, using a transformation assay described in Jinek et al, SCIENCE 2012, 337: 816.
[0369] [0369] Some Cas9 molecules have the ability to interact with a gRNA molecule and in conjunction with the gRNA molecule are addressed (for example, targeted or localized) to a major target domain, but are unable to cleave the target nucleic acid, or unable to cleave at efficient rates. Cas9 molecules that have no cleavage activity or have non-substantial cleavage activity can be referred to here as an inactive Cas9 (an enzymatically inactive Cas9), a dead Cas9 or a dCas9 molecule. For example, an inactive Cas9 molecule may lack cleavage activity or have substantially less, for example, less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, as measured by an essay described here.
[0370] [0370] Examples of naturally occurring Cas9 molecules are described in Chylinski et al, RNA Biology 2013; 10: 5, 727-737. Such Cas9 molecules include the Cas9 molecules of a bacterial family cluster 1, bacterial family cluster 2, bacterial family cluster 3, bacterial family cluster 4, bacterial family cluster 5, bacterial family cluster 6, bacterial family cluster 7, bacterial family cluster 8, family bacterial cluster 9, bacterial family cluster 10, bacterial family cluster 11, bacterial family cluster 12, bacterial family cluster 13,
[0371] [0371] Examples of naturally occurring Cas9 molecules include a Cas9 molecule from a cluster 1 bacterial family.
[0372] [0372] In one embodiment, a Cas9 molecule, for example, an active Cas9 molecule or an inactive Cas9 molecule, comprises a sequence of amino acids: having 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, 96%, 97%, 98%, or 99% homology with; differs to no more than 1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the amino acid residues when compared to; differs by at least 1,2, 5, or 20 amino acids, but not more than 100, 80, 70, 60, 50, 40 or amino acids from; or is identical to; any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence, for example, a Cas9 molecule of the species listed here or described in Chylinski et al., RNA Biology 2013, 10: 5, 121-T, 1 Hou et al. PNAS Early Edition 2013, 1-6.
[0373] [0373] In one embodiment, a Cas9 molecule comprises a sequence of amino acids having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with; differs to no more than 1%, 2%, 5%, 10%, 15%, 20%, 30%, or 40% of the amino acid residues when compared to; differs by at least 1, 2, 5, or 20 amino acids, but not more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or is identical to; S. pyogenes cas9: Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
[0374] [0374] In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 that includes one or more mutations for positively charged amino acids (eg, lysine, arginine or histidine) that introduce a non-polar amino acid or not loaded, for example, alanine, in said position. In modalities, the mutation is for one or more positively charged amino acids in the Cas9 n-sulcus. In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 that includes a mutation at position 855 of SEQ ID NO: 205, for example, a mutation for an uncharged amino acid, for example, alanine , at position 855 of SEQ ID NO: 205. In embodiments, the Cas9 molecule has a mutation only at position 855 of SEQ ID NO: 205, in relation to SEQ ID NO: 205, for example, for an uncharged amino acid, for example example, alanine. In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 that includes a mutation at position 810, a mutation at position 1003 and / or a mutation at position 1060 of SEQ ID NO: 205, for example example a mutation for alanine at position 810, position 1003 and / or position 1060 of SEQ ID NO: 205. In embodiments, the Cas9 molecule has a mutation only at position 810, position 1003 and position 1060 of SEQ ID NO: 205, in relation to SEQ ID NO: 205, for example, where each mutation is for an uncharged amino acid, for example, alanine. In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 that includes a mutation at position 848, a mutation at position 1003 and / or a mutation at position 1060 of SEQ ID NO: 205, for example example a mutation for alanine at position 848, position 1003 and / or position 1060 of SEQ ID NO: 205. In embodiments, the Cas9 molecule has a mutation only at position 848, position 1003 and position 1060 of SEQ ID NO: 205, in relation to SEQ ID NO: 205, for example, where each mutation is for an uncharged amino acid, for example, alanine. In modalities, the Cas9 molecule is a Cas9 molecule as described in Slaymaker et al., Science Express, available online, December 1, 2015 in Science DOI:
[0375] [0375] In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 includes one or more mutations. In embodiments, the Cas9 variant comprises a mutation at position 80 of SEQ ID NO: 205, for example, includes a leucine at position 80 of SEQ ID NO: 205 (ie comprises, for example, consists of SEQ ID NO: 205 with a C80L mutation). In embodiments, the Cas9 variant comprises a mutation at position 574 of SEQ ID NO: 205, for example, includes a glutamic acid at position 574 of SEQ ID NO: 205 (ie comprises, for example, consists of SEQ ID NO : 205 with a C574E mutation). In embodiments, the Cas9 variant comprises a mutation at position 80 and a mutation at position 574 of SEQ ID NO: 205, for example, includes a leucine at position 80 of SEQ ID NO: 205, and a glutamic acid at position 574 of SEQ ID NO: 205 (i.e., comprises, for example, consists of SEQ ID NO: 205 with a C80L mutation and a C574E mutation). Without being limited by theory, it is believed that such mutations improve the solution properties of the Cas9 molecule.
[0376] [0376] In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 includes one or more mutations. In embodiments, the Cas9 variant comprises a mutation at position 147 of SEQ ID NO: 205, for example, includes a tyrosine at position 147 of SEQ ID NO: 205 (ie comprises, for example, consists of SEQ ID NO: 205 with a D147Y mutation). In embodiments, the Cas9 variant comprises a mutation at position 411 of SEQID NO: 205, for example, which includes a threonine at position 411 of SEQ ID NO: 205 (ie, comprises, for example, consists of SEQ ID NO: 205 with a P411T mutation). In embodiments, the Cas9 variant comprises a mutation at position 147 and a mutation at position 411 of SEQ ID NO: 205, for example, includes tyrosine at position 147 of SEQ ID NO: 205, and a threonine at position 411 of
[0377] [0377] In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 includes one or more mutations. In embodiments, the Cas9 variant comprises a mutation at position 1135 of SEQ ID NO: 205, for example, includes a glutamic acid at position 1135 of SEQ ID NO: 205 (ie comprises, for example, consists of SEQ ID NO : 205 with a D1135E mutation). Without being limited by theory, it is believed that such mutations improve the selectivity of the Cas9 molecule for the NGG PAM sequence versus the NAG PAM sequence.
[0378] [0378] In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 that includes one or more mutations that introduce a non-polar or uncharged amino acid, for example, alanine, at certain positions. In embodiments, the Cas9 molecule is a Cas9 variant of S. pyogenes of SEQ ID NO: 205 that includes a mutation at position 497, a mutation at position 661, a mutation at position 695 and / or a mutation at position 926 of SEQ ID NO: 205, for example a mutation for alanine at position 497, position 661, position 695 and / or position 926 of SEQ ID NO: 205. In embodiments, the Cas9 molecule has a mutation only at position 497, position 661, position 695 and position 926 of SEQ ID NO: 205, in relation to SEQ ID NO: 205, for example, where each mutation is for an uncharged amino acid, for example, alanine. Without being limited by theory, it is believed that such mutations reduce the cut by the Cas9 molecule at sites outside the target.
[0379] [0379] It should be understood that the mutations described here for the Cas9 molecule can be combined and can be combined with any of the fusions or other modifications described here, and the Cas9 molecule tested in the assays described here.
[0380] [0380] Various types of Cas molecules can be used to practice the inventions disclosed here. In some embodiments, Cas molecules from Type | 1 Cas systems are used. In other embodiments, Cas molecules from other Cas systems are used. For example, Cas molecules of Type | or Type Ill. Examples of Cas molecules (and Cas systems) are described, for example, in Haft et al, PLoOS COMPUTATIONAL BIOLOGY 2005, 1 (6): e60 and Makarova et al, NATURE REVIEW MICROBIOLOGY 201 1, 9: 467 -477, the contents of both references are hereby incorporated by reference in their entirety.
[0381] [0381] In one embodiment, the Cas9 molecule comprises one or more of the following activities: a nickase activity; a double-stranded cleavage activity (for example, an endonuclease and / or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to locate a target nucleic acid. Changed Cas9 Molecules
[0382] [0382] The naturally occurring Cas9 molecules have a number of properties, including: nickase activity, nuclease activity (for example, endonuclease and / or exonuclease activity); helicase activity; the ability to functionally associate with a gRNA molecule; and the ability to target (or locate) a site on a nucleic acid (for example, PAM recognition and specificity). In one embodiment, Cas9 molecules can include all or a subset of these properties. In typical embodiments, Cas9 molecules have the ability to interact with a gRNA molecule and, in conjunction with the gRNA molecule,
[0383] [0383] Cas9 molecules with desired properties can be made in a number of ways, for example, by altering one of the parental Cas9 molecules, for example, naturally occurring, to provide an altered Cas9 molecule having a desired property. For example, one or more mutations or differences relating to a parent Cas9 molecule can be introduced. Such mutations and differences include: substitutions (for example, conservative substitutions or substitutions of non-essential amino acids); insertions; or exclusions. In one embodiment, a Cas9 molecule can comprise one or more mutations or differences, for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations, but less than 200, 100 or 80 mutations in relation to a reference Cas9 molecule.
[0384] [0384] In one embodiment, a mutation or mutations does not have a substantial effect on a Cas9 activity, for example, a Cas9 activity described here. In one embodiment, a mutation or mutations has a substantial effect on a Cas9 activity, for example, a Cas9 activity described here. In one embodiment, the activities - exemplary - comprise one or more of specificity of MAP, cleavage activity and helicase activity. A mutation (s) can be present, for example, in: one or more RuvC-like domain, for example, an RuvC-like domain in N-terminal; a domain similar to HNH; a region outside the RuvC-like domains and the HNH-like domain. In some embodiments, a mutation (s) is present in a domain similar to RuvC at N-terminal. In some embodiments, a mutation (s) is present in a domain similar to HNH. In some embodiments, the mutations are present in both a domain similar to RuvC in N-terminal and in a domain similar to HNH.
[0385] [0385] Whether a specific sequence, for example, a substitution, can affect one or more activities, such as targeting activity, cleavage activity, etc., can be assessed or predicted, for example, by assessing whether the mutation is conservative or by the method described in Section Ml. In one embodiment, a "non-essential" amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be changed from the wild-type sequence of a Cas9 molecule, for example, a Cas9 molecule that occurs naturally, for example, an active Cas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (for example, cleavage activity), while altering an "essential" amino acid residue results in a substantial loss of activity (e.g., cleavage activity). Cas9 molecules with altered PAM recognition or without PAM recognition
[0386] [0386] Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.
[0387] [0387] In one embodiment, a Cas9 molecule has the same specificities as PAM as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with a naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, for example, to alter PAM recognition, for example, to alter the PAM sequence that the Cas9 molecule recognizes, to decrease off-target sites and / or improve specificity; or eliminate a PAM recognition requirement. In one embodiment, a Cas9 molecule can be altered, for example, to increase the length of the PAM recognition sequence and / or improve the specificity of Cas9 to a high level of identity to decrease off-target sites and increase specificity. In one embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules that recognize different PAM sequences and / or that reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for the directed evolution of Cas9 molecules are described, for example, in Esvelt el al, Nature 2011, 472 (7344): 499-503. Candidate Cas9 molecules can be evaluated, for example, by the methods described here.
[0388] [0388] In one embodiment, a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, for example, that differs from the naturally occurring Cas9 molecule that has the closest homology. For example, a Cas9 molecule may differ from naturally occurring Cas9 molecules, for example, a Cas9 molecule from S. pyogenes, as follows: its ability to modulate the cleavage, for example, decrease or increase, from a double chain break (endonuclease and / or exonuclease activity), for example, compared to a naturally occurring Cas9 molecule (for example, a Cas9 molecule of S. byogenes); its ability to modulate the cleavage, for example, decrease or increase, of a single strand of a nucleic acid,
[0389] [0389] In one embodiment, an active Cas9 molecule comprises one or more of the following activities: cleavage activity associated with an RuvC-like domain at N-terminal; cleavage activity associated with an HNH domain; cleavage activity associated with a domain similar to HNH and cleavage activity associated with a domain similar to RuvC in N-terminal.
[0390] [0390] In one embodiment, the Cas9 molecule is a Cas9 nickase, for example, it cleaves only a single strand of DNA. In one embodiment, the Cas9 nickase includes a mutation at position 10 and / or a mutation at position 840 of SEQ ID NO: 205, for example, comprises a D10A and / or H840A mutation for SEQ ID NO: 205. Inactive Cas9 molecules No -Clivantes
[0391] [0391] In one embodiment, the altered Cas9 molecule is an inactive Cas9 molecule that does not cleave a nucleic acid molecule (double-stranded or single-stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, for example example, less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, for example, as measured by an assay described herein. The reference Cas9 molecule can be an unmodified, naturally occurring Cas9 molecule, for example, a naturally occurring Cas9 molecule, such as a Cas9 molecule from S. pyogenes, S. thermophilus, S.
[0392] [0392] In one embodiment, the Cas9 molecule is dCasº9. Tsai et al. (2014), Nat. Biotech. 32: 569-577.
[0393] [0393] A catalytically inactive Cas9 molecule can be fused with a transcription repressor. An inactive Cas9 fusion protein complex with a gRNA and locates in a DNA sequence specified by the target domain of the gRNA, but, unlike an active Cas9, it will not cleave the target DNA. Merging an effective domain, such as a transcriptional repression domain, into an inactive Cas9 allows recruitment of the effector to any DNA site specified by the gRNA. Site-specific targeting of a Cas9 fusion protein to a promoter region of a gene can block or affect polymerase binding to the promoter region, for example, a Cas9 fusion with a transcription factor (for example, a transcription activator) and / or a transcriptional enhancer that binds nucleic acid to increase or inhibit activation of transcription. Alternatively, the site specific targeting of a Cas9 fusion to a transcriptional repressor for a promoter region of a gene can be used to decrease transcriptional activation.
[0394] [0394] Transcription repressors or transcription repressor domains that can be fused to an inactive Cas9 molecule may include ruppel associated box (KRAB or SKD), the Mad mSIN3 interaction domain (SID) or the ERF repressor domain (ERD).
[0395] [0395] In another embodiment, an inactive Cas9 molecule can be fused with a protein that modifies chromatin. For example, an inactive Cas9 molecule can be fused to heterochromatin protein 1 (HP1), a histone lysine methyltransferase (for example, SUV39H 1, SUV39H2, G9A, ESET / SETDB 1, Pr-SET7 / 8, SUV4-20H 1 , RIZ1), a demethylated lysine histone (e.g. LSD1 / BHC1 10, SpLsdl / Sw, ISafl 10, Su (var) 3-3, JMJID2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, Rph 1, JARID 1 A / RBP2, JARI DIB / PLU-I, JARID 1C / SMCX, JARID1 D / SMCY, Lid, Jhn2, Jmj2), a lysine histone deacetylase (e.g. HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hos 1, Cir6 , HDACA4, HDAC5, HDAC7, HDAC9, Hdal, Cir3, SIRT 1, SIRT2, Sir2, Hst 1, Hst2, Hst3, Hst4, HDAC 11) and a DNA methylase (DNMT1, DNMT2a / DMNT3b, MET1). An inactive Cas9-chromatin-modifying molecule fusion protein can be used to alter the state of the chromatin to reduce the expression of a target gene.
[0396] [0396] The heterologous sequence (for example, the transcriptional repressor domain) can be fused with the N- or C-terminal of the inactive Cas9 protein. In an alternative embodiment, the heterologous sequence (e.g., the transcriptional repressor domain) can be fused to an inner portion (i.e., a portion other than the N-terminal or C-terminal) of the inactive Cas9 protein.
[0397] [0397] The ability of a Cas9 molecule / gRNA molecule complex to bind and cleave a target nucleic acid can be assessed, for example, by the methods described here in Section Ml. The activity of a Cas9 molecule, for example, either an active Cas9 or inactive Cas9, alone or in a complex with a gRNA molecule can also be assessed by methods well known in the art, including gene expression assays and chromatin-based assays, for example, chromatin immunoprecipitation (ChiP) and in vivo chromatin assay (CiA). Other Molecules Cas9 of Fusions
[0398] [0398] In modalities, the Cas9 molecule, for example, a Cas9 of S. pyogenes, can additionally comprise one or more amino acid sequences that confer additional activity.
[0399] [0399] In some respects, the Cas9 molecule may comprise one or more nuclear localization sequences (NLSs), such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the Cas9 molecule comprises at least 1,2,3,4,5, 6, 7, 8, 9, 10, or more NLSs at or near the amino terminus, at least 1, 2,3, 4,5,6,7,8,9,10 or more NLSs at or near the carboxy terminus, or a combination thereof (for example, one or more NLSs at the amino terminus and one or more NLSs at the carboxy terminus). When more than one NLS is present, each can be selected independently from the others, so that a single NLS can be present in more than one copy and / or in combination with one or more other NLS present in one or more copies. In some embodiments, an NLS is considered close to the N or C terminal when the amino acid closest to the NLS is within about 1, 2,3,4,5,10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N or C terminus. Typically, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the surface of the protein, but other types of NLS are known. Non-limiting examples of NLS include an NLS sequence comprising or derived from: the SVL virus large T antigen NLS, having the amino acid sequence PKKKRKV (SEQ ID NO: 206); the nucleoplasmin NLS (for example, the split nucleleoplasmin NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 207); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 208) or RQRRNELKRSP (SEQ ID NO: 209 ), the NLS hRNPA1 M9 having the sequence
[0400] [0400] In one embodiment, the Cas9 molecule, for example, the Cas9 molecule of S. pyogenes, comprises an NLS sequence of SVA40, for example, arranged at the N-terminus of the Cas9 molecule. In one embodiment, the Cas9 molecule, for example, the Cas9 molecule of S. bpyogenes, comprises an SVL NLS sequence arranged at the N-terminus of the Cas9 molecule and an SV40 NLS sequence disposed at the C-terminus of the Cas9 molecule. In one embodiment, the Cas9 molecule, for example, the Cas9 molecule of S. pyogenes, comprises an SVL NLS sequence disposed at the N-terminus of the Cas9 molecule and a nucleoplasmin NLS sequence disposed at the C-terminus of the Cas9 molecule. In any of the above-mentioned embodiments, the molecule may additionally comprise a marker, for example, a His marker, for example, a His (6) marker (SEQ ID NO: 247) or His (8) marker (SEQ ID NO: 248), for example, at the N or C terminal.
[0401] [0401] In some ways, the Cas9 molecule may comprise one or more amino acid sequences that allow the Cas9 molecule to be specifically recognized, for example, a marker. In one embodiment, the marker is a histidine marker, for example, a histidine marker comprising at least 3, 4, 5, 6, 7, 8, 9, 10 or more histidine amino acids. In embodiments, the histidine marker is a His6 marker (six histidines (SEQ ID NO: 247). In other embodiments, the histidine marker is a His8 marker (SEQ ID NO: 248). The histidine marker can be separated from one or more other portions of the Cas9 molecule by a ligand In embodiments, the ligand is GGS An example of such a fusion is the Cas9 iProt 106520 molecule.
[0402] [0402] In some respects, the Cas9 molecule may comprise one or more amino acid sequences that are recognized by a protease (for example, they comprise a protease cleavage site). In embodiments, the cleavage site is the tobacco etch virus (TEV) cleavage site, for example, it comprises the sequence ENLYFOG (SEQ ID NO: 230). In some respects, the protease cleavage site, for example, the TEV cleavage site is arranged between a marker, for example, a His marker, for example, a His6 marker (SEQ ID NO: 247) or His8 marker ( SEQ ID NO: 248), and the remainder of the Cas9 molecule. Without being limited by theory, it is believed that this introduction will allow the use of the marker for, for example, the purification of the Cas9 molecule and then the subsequent cleavage, so that the marker does not interfere with the function of the Cas9 molecule.
[0403] [0403] In embodiments, the Cas9 molecule (for example, a Cas9 molecule as described herein) comprises an N-terminal NLS, and a C-terminal NLS (for example, comprises, NLS-cas9-NLS from N- to C-terminal), for example, where each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (for example, a Cas9 molecule as described herein) comprises an NLS at the N-terminus, an NLS at the C-terminus, and a His6 marker at the C-terminus (SEQ ID NO: 247) (for example, comprises, from the N-terminal to C NLS-Cas9-NLS-His marker), for example, where each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (for example, a Cas9 molecule as described herein) comprises an N-terminal His marker (for example, His6 marker (SEQ ID NO: 247)), an N-terminal NLS, and a C- NLS terminal (for example, comprises, from the N terminal to the C terminal tag tag-NLS-Cas9-NLS), for example, where each NLS is an SV40 NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an NLS at the N-terminus and a His marker at the C-terminus (e.g., His6 marker (SEQ ID NO: 247)) (for example, comprises, from the N-terminal to C marker His-Cas9-NLS), for example, where the NLS is an SVL NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (e.g., a Cas9 molecule as described herein) comprises an NLS at the N-terminus and a His marker at the C-terminus (e.g., His6 marker (SEQ ID NO: 247)) (for example, comprises, from the N-terminal to C NLS-Cas9-His marker), for example, where the NLS is an SVL NLS (PKKKRKV (SEQ ID NO: 206)). In embodiments, the Cas9 molecule (for example, a Cas9 molecule as described herein) comprises an N-terminal His tag (eg, His8 tag (SEQ ID NO: 248)), an N-terminal cleavage domain (eg, tobacco etch virus (TEV) cleavage domain (for example, comprises the sequence ENLYFQG (SEQ ID NO: 230)), an N-terminal at N-terminal (for example, an NLS of SV40; SEQ ID NO: 206) , and a C-terminal NLS (e.g., an NLS in SV40; SEQ ID NO: 206) (for example, it comprises His-TEV-NLS-Cas9-NLS marker from N- to C-terminal). In any of the aforementioned modalities, Cas9 has a sequence of SEQ | D NO: 205. Alternatively, in any of the aforementioned modalities, Cas9 has a sequence of the Cas9 variant of SEQ ID NO: 205, for example, as above described. In any of the aforementioned embodiments, the Cas9 molecule comprises a linker between the His marker and another portion of the molecule, for example, a GGS linker. The amino acid sequences of exemplary Cas9 molecules “described above are provided below. The "iProt" identifiers correspond to those in Figure 60.
[0404] [0404] iProt105026 (also referred to as iProt106154, iProt106331, iProt 106545 and PID426303, depending on protein preparation) (SEQ ID NO: 233): MAPKKKRKVD —KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL —QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVD KLFIQLVATY NQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA - KLQLSKDTYD DDLDNLLAQ! GDQYADLFLA AKNLSDAILL SDILRVNTEI TKAPLSASMI! KRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQOSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNREDLLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV - GPLARGNSRF - AWMTRKSEET ITPWNFEEVWV - DKGASAQSFI ERMTNFDKNL —PNEKVLPKHS LLYEYFTVYN ELTKVKYVTE GMRKPAFLSG EQKKAIVDLL FKTNRKVTIVK - QLKEDYFKKI ECFDSVEISG - VEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHL - FDDKVMKQLK RRRYTGWGRL -SRKLINGIRD KQSGKTILDF LKSDGFANRN FMQLIHDDSL - TFKEDIQKAQ VSGQGDSLHE -HIANLAGSPA IKKGILOTIVK VVDELVKVMG RHKPENIVIE - MARENQTTOK -GOKNSRERMK RIEEGIKELG SQILKEHPVE - NTQLONEKLY LYYLOANGRDM -YVDQELDINR LSDYDVDHIV - PQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY - WRQLLNAKLI TQORKFDNLTK -AERGGLSELD KAGFIKRQLV ETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL - VSDFRKDFOQF - YKVREINNYH HAHDAYLNAV VGTALIKKYP KLESEFVYGD YKVYDVRKMI AKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKRPL IETNGETGEI VWDKGRDFAT —VRKVLSMPQV NIVKKTEVOT GGFSKESILP KRNSDKLIAR —KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY - EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL ADANLDKVLS AYNKHRDKPI REQAENIIHL FTLTNLGAPA AFKYFDTTID RKRYTSTKEV LDATLIHOS! TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH
[0405] [0405] iProt 106518 (SEQ ID NO: 234): MAPKKKRKVD —KKYSIGLDIG TNSVGWAVIT —DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRILYL QEIFSNEMAK - VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVD KLFIQLVATY NQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA - KLQLSKDTYD DDLDNLLAQ! - GDOYADLFLA - AKNLSDAILL - SDILRVNTEI TKAPLSASMI - KRYDEHHOQDL TLLKALVRQQ - LPEKYKEIFF DOSKNGYAGY IDGGASQEEF. YKFIKPILEK MDGTEELLVK LNREDLLRKQ - RTFDNGSIPH - QIHLGELHAI -LRRQEDFYPF LKDNREKIEK ILTFRIPYYVY -GPLARGNSRF - AWMTRKSEET ITPWNFEEVWV - DKGASAQSFI ERMTNFDKNL - PNEKVLPKHS LIVYVEYFIVYN - ELTKVKYVTE. GMRKPAFLSG - EQKKAIVDLL FKTNRKVTIVK QLKEDYFKKI EEFDSVEISGS VEDRFNASLG TYHDLLKIK .- DKDFLDONEEN - EDILEDIVLT - LTLFEDREMI EERLKTYAHL - FDDKVMKQLK RRRYTGWGRL - SRKLINGIRD KOSGKTILDF - LKSDGFANRN - FMQLIHDDSL - TFKEDIQKAQ VSGOGDSLHE -HIANLAGSPA IKKGILOTVK VVDELVKVMG RHKPENIVIE - MARENOTTOK .GOKNSRERMK RIEEGIKELG SOILKEHPVE - NTQLONEKLY LYYLONGRDM - YVDOQELDINR LSDYDVDHIV - "POSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY - WROQLLNAKLI TOQORKFDNLTK AERGGLSELD KAGFIKRQLV - ETROITKHVA - QILDSRMNTK - YDENDKLIRE VKVITLKSKL - VSDFRKDFOF - YKVREINNYH - HAHDAYLNAV VGTALIKKYP - KLESEFVYGD - YKVYDVRKMI -AKSEQEIGKA TAKYFFYSNI - MNFFKTEITL - ANGEIRKRPL - IETNGETGEI VWDKGRDFAT is VRKVLSMPQV NIVKKTEVOT GGFSKESILP KRNSDKLIAR -KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE .- LLGITIMERS - SFEKNPIDEL - EAKGYKEVKK DLIIKLPKYS - LFELENGRKR - MLASAGELOK - GNELALPSKY VNFLYLASHY .EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL - ADANLDKVLS - AYNKHRDKPI - REQAENIIHL FTLINLGAPA - AFKYFDTTID - RKRYTSTKEV - LDATLIHOS! TGLYETRIDL SOLGGDSRAD PKKKRKVHHH HHH
[0406] [0406] - iProt106519 (SEQ ID NO: 235): MGSSHHHHHH - HHENLYFOGS MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR - RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA - QIGDQYADLF LAAKNLSDA! LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQALPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL - VKLNREDLLR - KORTFDNGSI PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQOS FIERMTNFDK NLPNEKVLPK —HSLLYEYFIV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE —MIEERLKTYA HLFDDKVMKQ —LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILQOT VKVVDELVKV - MGRHKPENIV IEMARENQTT -QKGQKNSRER MKRIEEGIKE LGSQILKEHP - VENTOQLONEK - LYLYYLONGR DMYVDQELDI NRLSDYDVDH IVPOSFLKDD - SIDNKVLTRS DKNRGKSDNV - PSEEVVKKMK NYWROQOLLNAK LITARKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH - VAQILDSRMN TKYDENDKLI! REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN - AVVGTALIKK YPKLESEFVY GDYKVYDVRK
[0407] [0407] - iProt106520 (SEQ ID NO: 236): MAHHHHHHGG. SPKKKRKVDK KYSIGLDIGT NSVGWAVITD EYKVPSKKFK “VLGNTDRHSI KKNLIGALLFE DSGETAEATR LKRTARRRYT RRKNRICYLA EIFSNEMAKV DDSFFHRLEE SFLVEEDKKH - ERHPIFGNIV -DEVAYHEKYP - TIYHLRKKLV DSTDKADLRL - IVLALAHMIK "FRGHFLIEGD - LNPDNSDVDK LFIOLVOTYN - QLFEENPINA - SGVDAKAILS - ARLSKSRRLE NLIAQLPGEK - KNGLFGNLIA -LSLGLTPNFK - SNFDLAEDAK LOLSKDTYDD - DLDNLLAQIG - DOYADLFLAR - KNLSDAILLS DILRVNTEIT KAPLSASMIK RYDEHHODLT - LLKALVRQQOL PEKYKEIFED QSKNGYAGYI DGGASQEEFY KFIKPILEKM DGTEELLVKL - NREDLLRKOR - TFDNGSIPHQ - IHLGELHAIL RROEDFYPFL - KDNREKIEKI LTFRIPYYVG - PLARGNSRFA WMTRKSEET! TPWNFEEVVD KGASAQSFIE RMTNFDKNLP NEKVLPKHSL -LYEYFTVYNE LTKVKYVTEG MRKPAFLSGE QKKAIVDLLE - KTNRKVIVKQ - LKEDYFKKIE - CFDSVEISGV EDRFNASLGT - YHDLLKIKD -KDFLDNEENE - DILEDIVLTL TLFEDREMIE -ERLKTYAHLF -DDKVMKOQLKR RRYTGWGRLS RKLINGIRDK - QSGKTILDFL KSDGFANRNF - MQLIHDDSLT FKEDIOKAQVY - SGOGDSLHEH -IANLAGSPAI - KKGILOTVKV VDELVKVMGR - HKPENIVIEM ARENOTTOKG OKNSRERMKR IEEGIKELGS - QILKEHPVEN - TQLONEKLYL - YYLONGRDMY VDOQELDINRL - SDYDVDHIVP - QSFLKDDSID - NKVLTRSDKN RGKSDNVPSE - EVVKKMKNYW - ROLLNAKLIT QRKFDNLTKA ERGGLSELDK AGFIKRQLL TKITFIKRQLVE TROITKR
[0408] [0408] - iProt106521 (SEQ ID NO: 237): MAPKKKRKVD - KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS - IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL - QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI -VDEVAYHEKY -PTIVHLRKKL - VDSTDKADLR LIVLALAHMI- KFRGHFLIEG - DLNPDNSDVD - KLFIQLVATY NQLFEENPIN - ASGVDAKAIL - SARLSKSRRL - ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQ! - GDQYADLFLA - AKNLSDAILL - SDILRVNTEI TKAPLSASMI - KRYDEHHOQDL TLLKALVRQQ - LPEKYKEIFF DQSKNGYAGY IDGGASQEEF. YKFIKPILEK MDGTEELLVK LNREDLLRKQ - RTFDNGSIPH - QIHLGELHAI - LRRQEDFYPF LKDNREKIEK ILTFRIPYYVY -GPLARGNSRF - AWMTRKSEET ITPWNFEEVWV - DKGASAQSFI ERMTNFDKNL PNEKVLPKHS LLIYVEYFIVYN - ELTKVKYVTE - GMRKPAFLSG - EQKKAIVDLL FKTNRKVIVK -QLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIK .- DKDFLDNEEN - EDILEDIVLT - LTLFEDREMI EERLKTYAHL - FDDKVMKQLK RRRYTGWGRL - SRKLINGIRD KOSGKTILDF - LKSDGFANRN - FMQLIHDDSL - TFKEDIQKAQ VSGQGDSLHE —HIANLAGSPA IKKGILOTIVK VVDELVKVMG RHKPENIVIE - MARENOTTOK GOKNSRERMK RIEEGIKELG SQILKEHPVE - NTQLONEKLY LYYLONGRDM —YVDQELDINR LSDYDVDHIV - POSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY WROLLNAKLI TQRKFDNLTK AERGGLSELD KAGFIKRQLV - ETRQITKHVA - QILDSRMNTK - YDENDKLIRE VKVITLKSKL - VSDFRKDFOF - YKVREINNYH - HAHDAYLNAV VGTALIKKYP - KLESEFVYGD YKVYDVRKMI AKSEQEIGKA TAKYFFYSNI - MNFFKTEITL - ANGEIRKRPL - IETNGETGEI VWDKGRDFAT IS VRKVLSMPQV NIVKKTEVOT GGFSKESILP KRNSDKLIAR KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE. LLGITIMERS —SFEKNPIDEL - EAKGYKEVKK DLIKLPKYS “LFELENGRKR MLASAGELOK GNELALPSKY VNFLYLASHY EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL - ADANLDKVLS - AYNKHRDKPI - REQAENIIHL FTLTINLGAPA - AFKYFDTTID —RKRYISTKEV - LDATLIHOS! TGLYETRIDL SQLGGDSRAD HHHHHH
[0409] [0409] - iProt106522 (SEQ ID NO: 238): MAHHHHHHGG. SDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR - HSIKKNLIGA LLFDSGETAE ATRLKRTARR RYTRRKNRIC. YLQEIFSNEM -AKVDDSFFHR - LEESFLVEED KKHERHPIFG - NIVDEVAYHE KYPTIVHLRK - KLVDSTDKAD LRLIVLALAH - MIKFRGHFLI EGDLNPDNSD - VDKLFIQLVQ TYNQLFEENP - INASGVDAKA -ILSARLSKSR - RLENLIAQLP GEKKNGLFGN -LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA - QIGDOYADLF - LAAKNLSDAI - LLSDILRVNT EITKAPLSAS - MIKRYDEHHOQ - DLTLLKALVR - QQLPEKYKEI FFDOSKNGYA. GYIDGGASQE .EFYKFIKPIL EKMDGTEELL VKLNREDLLR - KORTFDNGSI PHOQIHLGELH - AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS - RFAWMTRKSE ETITPWNFAS - VVKKN HSLLYEYFTIV - YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA - HLFDDKVMKQ -LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD - SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILOAT - VKVVDELVKV MGRHKPENIV IEMARENQTT -QKGQOKNSRER MKRIEEGIKE LGSQILKEHP - VENTOQLONEK -LYLYYLOANGR -DMYVDQELDI NRLSDYDVDH -IVPQSFLKDD -SIDNKVLTRS -DKNRGKSDNV PSEEVVKKMK - NYWROQLLNAK LITAORKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS KLVSDFRKDF - QFYKVREINN - YHHAHDAYLN AVVGTALIKK —YPKLESEFVY GDYKVYDVRK MIAKSEQE KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF - ATVRKVLSMP - QVNIVKKTEV —QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTIVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGDSR ADPKKKRKV
[0410] [0410] iProt 106658 (SEQ ID NO: 239): MGSSHHHHHH HHENLYFOGS MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE ATRLKRTARR - RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA - QIGDQYADLF LAAKNLSDA! LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA GYIDGGASQE EFYKFIKPIL EKMDGTEELL - VKLNREDLLR - KORTFDNGS! I PHQIHLGELH AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE VVDKGASAQOS FIERMTNFDK NLPNEKVLPK —HSLLYEYFIV YNELTKVKYV TEGMRKPAFL SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQO —LKRRRYTGWG RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL HEHIANLAGS PAIKKGILAT VKVVDELVKV - MGRHKPENIV IEMARENQOTT —QKGQKNSRER MKRIEEGIKE LGSQILKEHP - VENTQLONEK - LYLYYLOQNGR DMYVDQELDI NRLSDYDVDH IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV —PSEEVVKKMK NYWROQLLNAK LITARKFDNL TKAERGGLSE LDKAGFIKRQ LVETRQITKH - VAQILDSRMN TKYDENDLL REVKVITLKS KLVSDFRKDF QFYKVREINN YHHAHDAYLN - AVVGTALIKK YPKLESEFVY GDYKVYDVRK MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF - ATVRKVLSMP - QVNIVKKTEV QTGGFSKES! LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK YSLFELENGR KRMLASAGEL QKGNELALPS - KYVNFLYLAS —HYEKLKGSPE DNEQKQLFVE QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA PAAFKYFDTT IDRKRYTSTK EVLDATLIHO SITGLYETRI DLSQLGGDGG GSPKKKRKV
[0411] [0411] iProt106745 (SEQ ID NO: 240): MAPKKKRKVD —KKYSIGLDIG TNSVGWAVIT —DEYKVPSKKF KVLGNTDRHS IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL - QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI VDEVAYHEKY PTIYHLRKKL VDSTDKADLR LIYLALAHMI KFRGHFLIEG DLNPDNSDVD KLFIQLVATY NQLFEENPIN ASGVDAKAIL SARLSKSRRL ENLIAQLPGE KKNGLFGNLI] ALSLGLTPNF KSNFDLAEDA - KLQLSKDTYD DDLDNLLAQ! GDQYADLFLA AKNLSDAILL SDILRVNTEI TKAPLSASMI! KRYDEHHQDL TLLKALVRQQ LPEKYKEIFF DQOSKNGYAGY IDGGASQEEF YKFIKPILEK MDGTEELLVK LNREDLLRKQ RTFDNGSIPH QIHLGELHAI LRRQEDFYPF LKDNREKIEK ILTFRIPYYV - GPLARGNSRF - AWMTRKSEET ITPWNFEEVW —DKGASAQSFI ERMTNFDKNL —PNEKVLPKHS LLYEYFTVYN ELTKVKYVTE GMRKPAFLSG EQKKAIVDLL FKTNRKVIVK - QLKEDYFKKI ECFDSVEISG - VEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHL - FDDKVMKQLK "RRRYTGWGRL -SRKLINGIRD KQOSGKTILDF LKSDGFANRN FMQLIHDDSL - TFKEDIQKAQ VSGQGDSLHE -HIANLAGSPA IKKGILOAIVK VVDELVKVMG RHKPENIVIE - MARENQTTOK - GQOKNSRERMK RIEEGIKELG SQILKEHPVE - NTOQLONEKLY - LYYLONGRDM - YVDQELDINR LSDYDVDHIV - POQSFLKDDSI DNAVLTRSDK NRGKSDNVPS EEVVKKMKNY - WRQLLNAKLI TORKFDNLTK -AERGGLSELD KAGFIKRQLV ETRQITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL - VSDFRKDFOQF —YKVREINNYH HAHDAYLNAV VGTALIKKYP KLESEFVYGD YKVYDVRKMI! AKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKRPL IETNGETGEI VWDKGRDFAT —VRKVLSMPQV NIVKKTEVAT GGFSKESILP KRNSDKLIAR - “KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY. EKLKGSPEDN - EQKOQLFVEQH - KHYLDEIIEQ ISEFSKRVIL - ADANLDKVLS - AYNKHRDKPI - REQAENIIHL FTLINLGAPA - AFKYFDTTID - RKRYTSTKEV - LDATLIHOS! TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH
[0412] [0412] - iProt106746 (SEQ ID NO: 241): MAPKKKRKVD - KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS - IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL - QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI -VDEVAYHEKY -PTIVHLRKKL - VDSTDKADLR LIVLALAHMI- KFRGHFLIEG - DLNPDNSDVD - KLFIQLVOTY NOLFEENPIN - ASGVDAKAIL - SARLSKSRRL - ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQ! - GDQYADLFLA - AKNLSDAILL - SDILRVNTEI TKAPLSASMI - KRYDEHHODL —TLLKALVROQ LPEKYKEIFF DOSKNGYAGY IDGGASQEEF. YKFIKPILEK MDGTEELLVK LNREDLLRKQ - RTFDNGSIPH - QIHLGELHAI -LRRQEDFYPF LKDNREKIEK ILTFRIPYYVY -GPLARGNSRF - AWMTRKSEET ITPWNFEEVV - DKGASAQOSFI ERMTNFDKNL - PNEKVLPKHS LIVYVEYFIVYN - ELTKVKYVTE - GMRKPAFLSG - EQKKAIVDLL FKTNRKVTIVK -QLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIK-DKDFLDNEEN -. EDILEDIVLT - LTLFEDREMI EERLKTYAHL - FDDKVMKOLK RRRYTGWGRL - SRKLINGIRD KOSGKTILDF - LKSDGFANRN - FMQLIHDDSL - TFKEDIQKAQ VSGOGDSLHE -HIANLAGSPA IKKGILOTVK VVDELVKVMG RHKPENIVIE - MARENOTTOK .GOKNSRERMK RIEEGIKELG SQILKEHPVE - NTQLONEALY LYYLONGRDM - YVDQELDINR LSDYDVDHIV -POSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY - WROLLNAKLI TORKFDNLTK AERGGLSELD KAGFIKRQLV - ETROITKHVA - OILDSRMNTK - YDENDKLIRE VKVITLKSKL - VSDFRKDFOF - YKVREINNYH - HAHDAYLNAV VGTALIKKYP - ALESEFVYGD - YKVYDVRKMI —AKSEQEIGKA TAKYFFYSNI - MNFFKTEITL - ANGEIRKAPL - IETNGETGEI VWDKGRDFAT is VRKVLSMPQV NIVKKTEVOT GGFSKESILP KRNSDKLIAR -KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE - LLGITIMERS - SFEKNPIDEL - EAKGYKEVKK DLIIKLPKYS - LFELENGRKR - MLASAGELOK - GNELALPSKY VNFLYLASHY. EKLKGSPEDN —EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL - ADANLDKVLS - AYNKHRDKPI - REQAENIIHL FTLINLGAPA - AFKYFDTTID - RKRYTSTKEV - LDATLIHOS! TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH
[0413] [0413] - iProt106747 (SEQ ID NO: 242): MAPKKKRKVD - KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS - IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL - QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI -VDEVAYHEKY -PTIVHLRKKL - VDSTDKADLR LIVLALAHMI- KFRGHFLIEG - DLNPDNSDVD - KLFIQLVATY NQLFEENPIN - ASGVDAKAIL - SARLSKSRRL - ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQ! - GDQYADLFLA - AKNLSDAILL - SDILRVNTEI TKAPLSASMI - KRYDEHHOQDL TLLKALVRQQ - LPEKYKEIFF DQSKNGYAGY IDGGASQEEF. YKFIKPILEK MDGTEELLVK LNREDLLRKQ - RTFDNGSIPH - QIHLGELHAI - LRRQEDFYPF LKDNREKIEK ILTFRIPYYVY -GPLARGNSRF - AWMTRKSEET ITPWNFEEVWV - DKGASAQSFI ERMTNFDKNL PNEKVLPKHS LLIYVEYFIVYN - ELTKVKYVTE - GMRKPAFLSG - EQKKAIVDLL FKTNRKVIVK -QLKEDYFKKI ECFDSVEISG VEDRFNASLG TYHDLLKIK .- DKDFLDNEEN - EDILEDIVLT - LTLFEDREMI EERLKTYAHL - FDDKVMKQLK RRRYTGWGRL -SRKLINGIRD KOSGKTILDF - LKSDGFANRN - FMQLIHDDSL - TFKEDIQKAQ VSGQGDSLHE —HIANLAGSPA IKKGILOTIVK VVDELVKVMG RHKPENIVIE - MARENQOTTOK GOKNSRERMK RIEEGIKELG SQILKEHPVE - NTOQLONEKLY LYYLONGRDM - YVDQELDINR LSDYDVDHIV -POSFLADDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY - WROLLNAKLI TQRKFDNLTK AERGGLSELD KAGFIKRQLV - ETRQITKHVA - QILDSRMNTK - YDENDKLIRE VKVITLKSKL - VSDFRKDFOF - YKVREINNYH - HAHDAYLNAV VGTALIKKYP - ALESEFVYGD - YKVYDVRKMI -AKSEQEIGKA TAKYFFYSNI - MNFFKTEITL - ANGEIRKAPL - IETNGETGEI VWDKGRDFAT is VRKVLSMPQV NIVKKTEVOT GGFSKESILP KRNSDKLIAR -KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE .- LLGITIMERS - SFEKNPIDEL - EAKGYKEVKK DLIIKLPKYS - LFELENGRKR - MLASAGELOK - GNELALPSKY VNFLYLASHY .EKLKGSPEDN -EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL - ADANLDKVLS - AYNKHRDKPI - REQAENIIHL FTLINLGAPA - AFKYFDTTID - RKRYTSTKEV - LDATLIHOS! TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH
[0414] [0414] - iProt106884 (SEQ ID NO: 243): MAPKKKRKVD - KKYSIGLDIG TNSVGWAVIT DEYKVPSKKF KVLGNTDRHS - IKKNLIGALL FDSGETAEAT RLKRTARRRY TRRKNRICYL - QEIFSNEMAK VDDSFFHRLE ESFLVEEDKK HERHPIFGNI -VDEVAYHEKY -PTIVHLRKKL - VDSTDKADLR LIVLALAHMI- KFRGHFLIEG - DLNPDNSDVD - KLFIQLVATY NQLFEENPIN - ASGVDAKAIL - SARLSKSRRL - ENLIAQLPGE KKNGLFGNLI ALSLGLTPNF KSNFDLAEDA KLQLSKDTYD DDLDNLLAQ! - GDQYADLFLA - AKNLSDAILL - SDILRVNTEI TKAPLSASMI - KRYDEHHQDL TLLKALVROQQ - LPEKYKEIFF DOSKNGYAGY IDGGASQEEF. YKFIKPILEK MDGTEELLVK LNREDLLRKQ - RTFDNGSIPH - QIHLGELHAI - LRRQEDFYPF LKDNREKIEK ILTFRIPYYVY —GPLARGNSRF - AWMTRKSEET ITPWNFEEVV - DKGASAQSL ERM LLYEYFTVYN ELTKVKYVTE GMRKPAFLSG EQKKAIVDLL FKTNRKVTIVK - QLKEDYFKKI ECFDSVEISG - VEDRFNASLG TYHDLLKIIK DKDFLDNEEN EDILEDIVLT LTLFEDREMI EERLKTYAHL - FDDKVMKQLK RRRYTGWGAL —SRKLINGIRD KQSGKTILDF LKSDGFANRN FMALIHDDSL TFKEDIQKAQ VSGQGDSLHE -HIANLAGSPA IKKGILOIVK VVDELVKVMG RHKPENIVIE - MARENQTTOK -GOKNSRERMK RIEEGIKELG SQILKEHPVE - NTQLONEKLY LYYLANGRDM -YVDQELDINR LSDYDVDHIV - PQSFLKDDSI DNKVLTRSDK NRGKSDNVPS EEVVKKMKNY - WRQLLNAKLI TQORKFDNLTK -AERGGLSELD KAGFIKRQLV ETRAITKHVA QILDSRMNTK YDENDKLIRE VKVITLKSKL - VSDFRKDFOQF - YKVREINNYH HAHDAYLNAV VGTALIKKYP KLESEFVYGD YKVYDVRKMI AKSEQEIGKA TAKYFFYSNI MNFFKTEITL ANGEIRKRPL IETNGETGEI VWDKGRDFAT —VRKVLSMPQV NIVKKTEVOT GGFSKESILP KRNSDKLIAR —KKDWDPKKYG GFDSPTVAYS VLVVAKVEKG KSKKLKSVKE LLGITIMERS SFEKNPIDFL EAKGYKEVKK DLIIKLPKYS LFELENGRKR MLASAGELQK GNELALPSKY VNFLYLASHY - EKLKGSPEDN EQKQLFVEQH KHYLDEIIEQ ISEFSKRVIL ADANLDKVLS AYNKHRDKPI REQAENIIHL FTLTNLGAPA AFKYFDTTID RKRYTSTKEV LDATLIHOS! TGLYETRIDL SQLGGDSRAD PKKKRKVHHH HHH
[0415] [0415] iProt 20109496 (SEQ ID NO: 244) MAPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTD RHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRILYLQEIFSN EMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIY HLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF IQLVATYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAOQ! GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD LTLLKALVRQQLPEKYKEIFFDAOSKNGYAGYIDGGASQEEFYKFIKPIL EKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQED FYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPW
[0416] [0416] Nucleic acids encoding Cas9 molecules, for example, an active Cas9 molecule or an inactive Cas9 molecule are provided herein.
[0417] [0417] Examples of nucleic acids encoding Cas9 molecules are described in Cong et al, SCIENCE 2013, 399 (6121): 819-823; Wang et al, CELL 2013, 153 (4): 910-918; Mali et al., SCIENCE 2013, 399 (6121): 823-826; Jinek et al, SCIENCE 2012, 337 (6096): 816-821.
[0418] [0418] In one embodiment, a nucleic acid encoding a Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, for example, as described in Section XII. In one embodiment, MRNA Cas9 has one or more of, for example, all of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and / or pseudouridine.
[0419] [0419] In addition or alternatively, the synthetic nucleic acid sequence can be optimized codons, for example, at least one non-common codon or less common codon that has been replaced by a common codon. For example, synthetic nucleic acid can direct the synthesis of an optimized messenger mMRNA, for example, optimized for expression in a mammalian expression system, for example, described herein.
[0420] [0420] An exemplary and optimized nucleic acid sequence encoding a Cas9 molecule of S. pyogenes is provided below. atggataaaa agtacagcat cgggctggac atcggtacaa actcagtagg gtagacegta 60 attacggacg agtacaaggt accctecaaa aaatttaaag tactgggtaa cacggacaga - 120 cactctataa agaaaaatct tattggagcc ttactatteg acteaggega gacagecgaa - 180 gccacaaggt tgaageggac cgccaggagg cggtatacca ggagaaagaa ccgcatatge 240 tacctgcaag aaatcttcag taacgagatg gcaaagattg acgatagctt tttecatege - 300 ctggaagaat cctttettgt têaggaagac aagaagcacg aacggcaccc catetttagge - 360 aatattgtcg acgaagtggc atatcacgaa aagtaccega ctatetacca ceteaggaag - 420 aagctagtgg actctaccega taaggcggac ctcagactta tttatttage actegeccac - 480 atgattaaat ttagaggaca tttcttgate gagggegacc tgaaccegga caacagtgac - 540 gtcgataagc tattcatcca acttgtgcag acctacaatc aactgttega agaaaacect - 600 ataaatgctt caggagtcga cgctaaagca atccetgtecg cgegectete aaaatetaga - 660 agactigaga atctgattgc teagttgcec ggggaaaaga aaaatgagatt gtttggcaac - 720 ctgatcgcecec teagtetegg actgacececa aattteaaaa gtaacttega cctggecgaa - 780 gacgctaagc tecagctgte caaggacaca tacgatgacg acctegacaa tetgcta GEC - 840 cagattigggg atcagtacgc cgatctettt ttggcagcaa agaaccetgte cgacgecate - 900 ctattgageg atatcttgag agtgaacacc gaaattacta aagcaccect tagegcatet - 960 atgatcaagc ggtacgacga gcatcatcag gatctgacee tactagaagac tettatáaga - 1020 caacagctcc cegaaaaata caaggaaatc ttetttyacc agagcaaaaa cagcetacget 1080 ggctatatag atagtggggc cagtcaggag gaattctata aattcatcaa gcecattete - 1140 gagaaaatgg acggcacaga ggagttgcta gtcaaactta acagggagga cctgctgcag 1200 aagcagcgga ccetttgacaa caggtetate ccecaccaga tteatetagg cgaactacac - 1260 gcaatcctga ggaggcagga ggatttttat ccttttetta aagataaceg cgagaaaata 1320 gaaaagattc ttacattcag gatccegtac tacgtgggac ctetegeceg gggcaattrca - 1380 cggtttacct ggatgacaag gaagtcagag gagactatta caccttggaa cttegaagaa - 1440 gtggtggaca agggtgcatc taccecagtet tteategage ggatgacaaa ttttgacaag - 1500 aacctceccta atgagaaggt getgcccaaa cattetetgc tetacgagta ctttaccgte - 1560 tacaatgaac tgactaaagt caagtacgtc accgagggaa tagaggaagec ggcattectt “1620 agtggagaac agaagaaggc gattgtagac ctgttatt ca agaccaacag gaaggatgact 1680 gtgaagcaac ttaaagaaga ctactttaag aagatcgaat gttttgacag tatagaaatt 1740 tcaggggttg aagaccegctt caatgcegtea ttggggactt accatgatct teteaagate - 1800 ataaaggaca aagacttcct ggacaacgaa gaaaatgagg atattetega agacategte - 1860 ctcacccetga ceccetattega agacagggaa atgatagaag agcgcttgaa aacctatace - 1920 cacctcttog acgataaagt tatgaagcag ctgaagegcea ggagatacac aggataggga 1980 agattgtcaa ggaagctgat caatggaatt agggataaac agagtggcaa gaccatacta 2040 gatttcctca aatctgatgg cttegccaat aggaacttca tgcaactgat ttcacgatgac - 2100 tctettacct teaaggagga cattrcaaaag gcteaggtga gegggcagag agactecett 2160 catgaacaca tcegcegaattt ggcagattec ceegcetatta aaaagggcat cctteaaact 2220 gtcaaggtag tagatgaatt ggtcaaggta atgggcagac ataagccaga aaatattatgag 2280 atcgagatgg ccogegaaaa ccagaccaca cagaagggec agaaaaatag tagagagegg 2340 atgaagagga tcgaggagag catcraaagag ctgggatete agattctecaa agaacaccec 2400 gtagaaaaca cacagctgca gaacgaaaaa ttgtacttgt actatctgca gaacggcaga - 2460 gacatgtacg tegaccaaga acttgatatt aatagactgt ccgactatga cgtagaccat 2520 atcgtgecec agtecttect gaaggacgac tecattgata acaaagtctt gacaagaage - 2580 gacaagaaca ggggtaaaag tgataatatga cctagcgagg aggtagigaa aaaaatgaag aactactggc gacagctgct taatgcaaag cteattacac aacggaagtt cgataateta - 2700 acgaaagcag agagaggtgg cttgtctgag ttagacaagg cagggtttat taageggecag 2760 ctggtggaaa ctaggcagat cacaaagcac gtggcgcaga ttttggacag ceggatgaac 2820 acaaaatacg acgaaaatga taaactgata cgagagytca aagttatcac gctgaaaage 2880 aagctagtat ccgatttteg gaaagacttc cagttetaca aagttegega gattaataac - 2940 taccatcatg ctcacgatgc gtacctgaac gcetattateg ggacegcectt gataaagaag - 3000 tacccaaagc tagaatcega gttegtatac ggggattaca aagtgtacga tagtgaggaaa - 3060 atgatagcca agtcegagca ggagattaga aaggccacag ctaagtactt cttttattict 3120 aacatcatga atttttttaa gacggaaatt accctggcca acggagagat cagaaagegg 3180 ccceccttatag agacaaatgg tgaaacaggt gaaatcgtct gggataaggg cagggattte - 3240 gctactgtga ggaaggtgact gagtatgcca caggtaaata tegtgaaaaa aaccgaagta - 3300 cagaccgga g gatttteccaa ggaaagcatt ttgcctaaaa gaaactcaga caagcteate- 3360 gccegceaaga aagattggga ccctaagaaa tacgggggat ttgacteace cacegtagec 3420 tattctatac tagtggtage taaggtagaa aaaggaaagt ctaagaagct gaagtcegta - 3480 aaggaactct tagggaatcac tatcatggaa agatcatcect ttygaaaagaa ccctategat - 3540 ttcctagagg ctaagggtta caaggaggtc aagaaagacc teatcattaa actgccaaaa - 3600 tactctctet tegagetgga aaatggcagg aagagaatgt taggccagege cagagageta - 3660 caaaagggaa acgagcttgc tetgeccetec aaatatgtta atttteteta tetegettec 3720 cactatgaaa agctgaaagg gtctccegaa gataacgagec agaagcagcet gttegtegaa - 3780 cagcacaagc actatctgga tgaaataatc gaacaaataa gcgagttcag caaaagagtt 3840 atcctggcgg atactaattt ggacaaagta ctgtctgctt ataacaageca cegggataag - 3900 cctattaggg aacaagcega gaatataatt cacctcettta cacteacgaa teteggagec - 3960 cecegecgcect traaatactt tgatacgact ategaccegga aacggtatac cagtaccaaa - 4020 gaggtcecteg atgecaccecet catceaceag teaattactg gectatacga aacacggate - 4080 gacctctetc aactaggegg cgactag 4107 (SEQ ID NO: 222 )
[0421] [0421] An exemplary and optimized nucleic acid sequence encoding a Cas9 molecule including SEQ ID NO: 244 is provided below: ATGGCTCCGAAGAAAAAGCGTAAAGTGGATAAAAAATACAGCATT GGTCTGGACATTGGCACGAACTCAGTGGGTTGGGCGGTCATCAC GGATGAATATAAGGTCCCGTCAAAAAAGTTCAAAGTGCTGGGCAA CACCGATCGCCATTCGATTAAAAAGAATCTGATCGGCGCGCTGCT GTTTGATAGCGGTGAAACCGCGGAAGCAACGCGTCTGAAACGTA CCGCACGTCGCCGTTACACGCGCCGTAAAAATCGTATTCTGTATC TGCAGGAAATCTTTAGCAACGAAATGGCGAAAGTTGATGACTCATT TTTCCACCGCCTGGAAGAATCGTTTCTGGTCGAAGAAGACAAAAA GCATGAACGTCACCCGATTTTCGGTAATATCGTTGATGAAGTCGC GTACCATGAAAAATATCCGACGATTTACCATCTGCGTAAAAAACTG GTGGATTCAACCGACAAAGCCGATCTGCGCCTGATTTACCTGGCA CTGGCTCATATGATCAAATTTCGTGGCCACTTCCTGATTGAAGGTG ACCTGAACCCGGATAACTCTGACGTTGATAAGCTGTTCATCCAGC TGGTCCAAACCTATAATCAGCTGTTCGAAGAAAACCCGATCAATG CAAGTGGCGTTGATGCGAAGGCCATTCTGTCCGCTCGCCTGAGTA AATCCCGCCGTCTGGAAAACCTGATTGCACAACTGCCGGGCGAAA AGAAAAACGGCCTGTTTGGTAATCTGATCGCTCTGTCACTGGGTC TGACGCCGAACTTTAAATCGAATTTCGACCTGGCAGAAGATGCTA AGCTGCAGCTGAGCAAAGATACCTACGATGACGATCTGGACAACC TGCTGGCGCAAATTGGTGACCAGTATGCCGACCTGTTTCTGGCGG CCAAAAATCTGTCAGATGCCATTCTGCTGTCGGACATCCTGCGCG TGAACACCGAAATCACGAAAGCGCCGCTGTCAGCCTCGATGATTA AACGCTACGATGAACATCACCAGGACCTGACCCTGCTGAAAGCAC TGGTTCGTCAGCAACTGCCGGAAAAGTACAAGGAAATTTTCTTTGA CCAATCTAAGAACGGCTATGCAGGTTACATCGATGGCGGTGCTAG TCAGGAAGAATTCTACAAGTTCATCAAGCCGATCCTGGAAAAAATG GATGGCACGGAAGAACTGCTGGTGAAACTGAATCGTGAAGATCTG CTGCGTAAACAACGCACCTTTGACAACGGCAGCATTCCGCATCAG ATCCACCTGGGTGAACTGCATGCGATTCTGCGCCGTCAGGAAGAT TTTTATCCGTTCCTGAAAGACAACCGTGAAAAAATTGAAAAGATCC TGACGTTTCGCATCCCGTATTACGTTGGCCCGCTGGCGCGTGGTA ATAGCCGCTTCGCCTGGATGACCCGCAAATCTGAAGAAACCATTA CGCCGTGGAACTTTGAAGAAGTGGTTGATAAAGGTGCAAGCGCTC AGTCTTTTATCGAACGTATGACCAATTTCGATAAAAMACCTGCCGAA TGAAAAGGTCCTGCCGAAACATAGCCTGCTGTATGAATACTTTACC GTGTACAACGAACTGACGAAAGTGAAGTATGTTACCGAAGGCATG CGCAAACCGGCGTTTCTGTCTGGTGAACAGAAAAAAGCCATTGTG GATCTGCTGTTCAAGACCAATCGTAAAGTTACGGTCAAACAGCTG AAGGAAGATTACTTCAAAAAGATCGAAGAATTCGACAGCGTGGAA ATTTCTGGCGTTGAAGATCGTTTCAACGCCAGTCTGGGTACCTAT CATGACCTGCTGAAGATCATCAAGGACAAGGATTTTCTGGATAAC GAAGAAAATGAAGACATTCTGGAAGATATCGTGCTGACCCTGACG CTSGTTCGAAGATCGTGAAATGATTGAAGAACGCCTGAAAACGTAC GCACACCTGTTTGACGATAAAGTTATGAAGCAGCTGAAACGCCGT CGCTATACCGGCTGGGGTCGTCTGTCTCGCAAACTGATTAATGGC ATCCGCGATAAGCAAAGTGGTAAAACGATTCTGGATTTCCTGAAAT CCGACGGCTTTGCCAACCGTAATTTCATGCAGCTGATCCATGACG ATAGTCTGACCTTTAAGGAAGACATTCAGAAAGCACAAGTGTCAG GCCAGGGTGATTCGCTGCATGAACACATTGCGAACCTGGCCGGC TCCCCGGCTATTAAMAAAGGGTATCCTGCAGACCGTCAAAGTCGTG GATGAACTGGTGAAGGTTATGGGCCGTCACAAACCGGAAAACATT GTGATCGAAATGGCGCGCGAAAATCAGACCACGCAAAAGGGTCA GAAAAACTCACGTGAACGCATGAAGCGCATTGAAGAAGGCATCAA AGAACTGGGTTCGCAGATTCTGAAAGAACATCCGGTTGAAAACAC CCAGCTGCAAAATGAAAAACTGTACCTGTATTACCTGCAAAATGGC CGTGACATGTATGTCGATCAGGAACTGGACATCAACCGCCTGAGC GACTATGATGTCGACCACATTGTGCCGCAGAGCTTTCTGAAGGAC GATTCTATCGATAATAAAGTGCTGACCCGTTCTGATAAGAACCGC GGTAAAAGCGACAATGTTCCGTCTGAAGAAGTTGTCAAAAAGATG AAGAACTACTGGCGTCAACTGCTGAATGCGAAGCTGATTACGCAG CGTAAATTCGATAACCTGACCAAGGCGGAACGCGGCGGTCTGAG TGAACTGGATAAGGCCGGCTTTATCAAACGTCAACTGGTGGAAAC CCGCCAGATTACGAAACATGTTGCCCAGATCCTGGATTCCCGCAT GAACACGAAATATGACGAAAATGATAAGCTGATTCGTGAAGTCAAA GTGATCACCCTGAAGAGTAAGCTGGTGTCCGATTTCCGTAAGGAC TTTCAGTTCTACAAAGTTCGCGAAATTAACAATTACCATCACGCAC ACGATGCTTATCTGAATGCAGTGGTTGGCACCGCTCTGATCAAAA AGTATCCGAAACTGGAAAGCGAATTTGTGTATGGTGATTACAAAGT CTATGACGTGCGCAAGATGATTGCGAAAAGTGAACAGGAAATCGG CAAGGCGACCGCCAAGTACTTTTTCTATTCCAACATCATGAACTTT TTCAAGACCGAAATCACGCTGGCAAATGGCGAAATTCGTAAACGC CCGCTGATCGAAACCAACGGCGAAACGGGTGAAATTGTGTGGGA TAAAGGTCGTGACTTCGCGACCGTTCGCAAAGTCCTGTCAATGCC GCAAGTGAATATCGTTAAAAAGACCGAAGTTCAGACGGGCGGTTT TAGTAAAGAATCCATCCTGCCGAAGCGTAACTCGGATAAACTGATT GCGCGCAAAAAGGATTGGGACCCGAAAAAGTACGGCGGTTTTGA TAGTCCGACCGTTGCATATTCCGTCCTGGTCGTGGCTAAAGTTGA AAAAGGCAAGAGTAAAAAGCTGAAGTCCGTCAAAGAACTGCTGGG TATTACCATCATGGAACGTAGCTCTTTTGAAAAGAACCCGATTGAC TTCCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGATCTGATT ATCAAGCTGCCGAAATATTCGCTGTTCGAACTGGAAAACGGTCGT AAACGCATGCTGGCAAGCGCTGGCGAACTGCAGAAGGGTAATGA ACTGGCACTGCCGTCTAAATATGTGAACTTTCTGTACCTGGCTAG CCATTATGAAAAACTGAAGGGTTCTCCGGAAGATAACGAACAGAA GCAACTGTTCGTTGAACAACATAAACACTACCTGGATGAAATCATC GAACAGATCTCAGAATTCTCGAAACGCGTCATTCTGGCGGATGCC AATCTGGACAAAGTGCTGAGCGCGTATAACAAGCATCGTGATAAA CCGATTCEGCGAACAGGCCGAAAATATTATCCACCTGTTTACCCTG ACGAACCTGGGCGCACCGGCAGCTTTTAAATACTTCGATACCACG ATCGACCGTAAGCGCTATACCAGCACGAAAGAAGTTCTGGATGCT ACCCTGATTCATCAGTCAATCACCGGTCTGTATGAAACGCGTATTG
[0422] [0422] If the above Cas9 sequences are fused to a polypeptide or peptide at the C-terminus (for example, an inactive Cas9 fused to a transcriptional repressor at the C-terminus), it is understood that the termination codon will be removed.
[0423] [0423] Nucleic acids, vectors and cells are also provided here for the production of a Cas9 molecule, for example, a Cas9 molecule described here. Recombinant production of polypeptide molecules can be carried out using techniques known to a person skilled in the art. Molecules and methods for recombinant production of polypeptide molecules, such as Cas9 molecules, for example, as described herein are described herein. As used in connection with this, the "recombinant" and production molecules include all polypeptides (for example, Cas9 molecules, for example, as described herein) that are prepared, expressed, created or isolated by recombinant means, such as polypeptides isolated from an animal (for example, a mouse) that is transgenic or transcromosomal to the nucleic acid encoding the molecule of interest, a hybridoma prepared from it, molecules isolated from a host cell transformed to express the molecule, for example, of a transfectome, molecules isolated from a recombinant combinatorial library and molecules prepared, expressed, created or isolated by any other means that involve the splicing of all or a portion of a gene encoding the molecule (or portion thereof) to others DNA sequences. Recombinant production can be from a host cell, for example, a host cell that comprises the nucleic acid encoding a molecule described herein, for example, a Cas9 molecule, for example, a Cas9 molecule described herein.
[0424] [0424] Nucleic acid molecules encoding a molecule (e.g., Cas9 molecule and / or gRNA molecule) are provided herein, for example, as described herein. Nucleic acid molecules are specifically provided comprising the sequence encoding any of SEQ ID NO: 233 to SEQ ID NO: 244, or encoding a fragment of any one of SEQ ID NO: 233 to SEQ ID NO: 244, or encoding a polypeptide comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%% or at least 99% sequence homology with any of SEQ ID NO: 233 to SEQ ID NO: 244.
[0425] [0425] They are provided here, for example, as described herein, comprising any of the nucleic acid molecules described above. In embodiments, said nucleic acid molecules are operably linked to a promoter, for example, a promoter operable in the host cell into which the vector is introduced.
[0426] [0426] Host cells comprising one or more nucleic acid molecules and / or vectors described herein are provided herein. In embodiments, the host cell is a prokaryotic host cell. In embodiments, the host cell is a eukaryotic host cell. In embodiments, the host cell is a yeast or E. coli cell. In embodiments, the host cell is a mammalian cell, for example, a human cell. Such host cells can be used for the production of a recombinant molecule described herein, for example, a Cas9 or gRNA molecule, for example, as described herein. SAW. Functional Analysis of Candidate Molecules
[0427] [0427] Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule / gRNA molecule complexes can be evaluated by methods known in the art or as described herein. For example, exemplary methods for assessing the endonuclease activity of the Cas9 molecule are described, for example, in Jinek el al, SCIENCE 2012; 337 (6096): 8 16-821.
[0428] [0428] The term "template nucleic acid" or "template donor" as used herein refers to a nucleic acid to be inserted into or near the target sequence that has been modified, for example, cleaved, by a CRISPR system from present invention. In one embodiment, the nucleic acid sequence at or near the target sequence is modified to have some or all of the template nucleic acid sequence, generally at or near the cleavage site (s). In one embodiment, the template nucleic acid is single-stranded. In an alternative embodiment, the template nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, for example, double-stranded DNA. In an alternative embodiment, the template nucleic acid is single-stranded DNA.
[0429] [0429] In embodiments, the template nucleic acid comprises the sequence encoding a globin protein, for example, a beta globin, for example, comprising a beta globin gene. In one embodiment, the beta globin encoded by the nucleic acid comprises one or more mutations, for example, antiphallic mutations. In one embodiment, the beta globin encoded by the nucleic acid comprises the T87Q mutation. In one embodiment, the beta globin encoded by the nucleic acid comprises the G16D mutation. In one embodiment, the beta globin encoded by the nucleic acid comprises the E22A mutation. In one embodiment, the beta globin gene comprises the G16D, E22A and T87Q mutations. In embodiments, the template nucleic acid further comprises one or more regulatory elements, for example, a promoter (for example, a human beta globin promoter), a 3 'enhancer, and / or at least a portion of a region control of the globin locus (for example, one or more DNAsel hypersensitivity sites (for example, HS2, HS3 and / or HS4 of the human globin locus)).
[0430] [0430] In other embodiments, the template nucleic acid comprises a sequence encoding a gamma globin, for example, it comprises a gamma globin gene. In embodiments, the template nucleic acid comprises a sequence that encodes more than one copy of a gamma globin protein, for example, comprises two or more, for example, two sequences of the gamma globin gene. In embodiments, the template nucleic acid further comprises one or more regulatory elements, for example, a promoter and / or enhancer.
[0431] [0431] In one embodiment, the template nucleic acid alters the structure of the target position, participating in a homology-directed repair event. In one embodiment, the template nucleic acid changes the target position sequence. In one embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring, base into the target nucleic acid.
[0432] [0432] Mutations in a gene or pathway described here can be corrected using one of the approaches discussed here. In one embodiment, a mutation in a gene or path described herein is corrected by homology-directed repair (HDR) using a template nucleic acid. In one embodiment, a mutation in a gene or pathway described herein is corrected by homologous recombination (HR) using a template nucleic acid. In one embodiment, a mutation in a gene or pathway described here is corrected by non-homologous end-point (NHEJ) repair using non-homologous end joining using a template nucleic acid. In other embodiments, nucleic acids encoding molecules of interest can be inserted into or near a site modified by a CRISPR system of the present invention. In embodiments, the template nucleic acid comprises regulatory elements, for example, one or more promoters and / or enhancers, operably linked to the nucleic acid sequence encoding a molecule of interest, for example, as described herein. Repair by HDR or HR and Nucleic Acids Mold
[0433] [0433] As described herein, nuclease-induced homology-directed repair (HDR) or homologous recombination (HR) can be used to alter a target sequence and correct (for example, repair or edit) a mutation in the genome. While not wanting to be limited by theory, it is believed that alteration of the target sequence occurs by repair based on a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target sequence. It is contemplated that a donor plasmid or linear double-stranded template can be used as a template for homologous recombination. It is further contemplated that a single-stranded donor template can be used as a template for altering the target sequence by alternating homology-directed repair methods (e.g., single-stranded annealing) between the target sequence and the donor template. The change induced by the donor template of a target sequence may depend on cleavage by a Cas9 molecule. Cas9 cleavage can comprise a double strand break, a single strand break or two single strand breaks.
[0434] [0434] In one embodiment, a mutation can be corrected by a single double-strand break or two single-strand breaks. In one embodiment, a mutation can be corrected by providing a mold and a CRISPR / Cas9 system that creates (1) a double strand break, (2) two single strand breaks, (3) two double strand breaks, with one break occurring on each side of the target sequence, (4) a double chain break and two single chain breaks, with the double chain break and two single chain breaks occurring on each side of the target sequence, (5) four breaks single strand with a pair of single strand breaks occurring on each side of the target sequence, or (6) a single strand break. Correction mediated by double chain break
[0435] [0435] In one embodiment, double-stranded cleavage is performed by a Cas9 molecule, having cleavage activity associated with a domain similar to HNH and cleavage activity associated with a domain similar to RuvC, for example, a domain similar to RuvC N-terminal, for example, a wild type Cas9. Such modalities require only a single gRNA. Correction mediated by simple chain break
[0436] [0436] In other modalities, two single chain breaks, or nicknames, are made by a Cas9 molecule, having nickase activity, for example, cleavage activity associated with a domain similar to HNH or cleavage activity associated with a domain similar to N-terminal RuvC. Such modalities require two gRNAs, one for the placement of each single chain break. In one embodiment, the Cas9 molecule having nickase activity cleaves the chain to which the gRNA hybridizes, but not the chain that is complementary to the chain to which the gRNA hybridizes. In one embodiment, the Cas9 molecule that has nickase activity does not cleave the chain to which the gRNA hybridizes, but instead cleaves the chain that is complementary to the chain to which the gRNA hybridizes.
[0437] [0437] In one embodiment, the nickase has HNH activity, for example, a Cas9 molecule having RuvC activity inactivated, for example, a Cas9 molecule having a D10 mutation, for example, the D10A mutation. D10A inactivates RuvC; therefore, Cas9 nickase has (only) HNH activity and will cut in the chain to which the gRNA hybridizes (for example, the complementary chain, which lacks the
[0438] [0438] In one embodiment, where a nickase and two gRNAs are used to position two single-stranded nicknames, a nick is on the + chain and a nick is on the - target nucleic acid chain. PAMs are facing outwards. The gRNAs can be selected in such a way that the gRNAs are separated by about 0-50, 0-100 or 0-200 nucleotides. In one embodiment, there is no overlap between the target sequence that is complementary to the target domains of the two gRNAs. In one embodiment, the gRNAs do not overlap and are separated by as much as 50, 100 or 200 nucleotides. In one embodiment, the use of two gRNAs can increase specificity, for example, by decreasing off-target binding (Ran el al., CELL 2013).
[0439] [0439] In one embodiment, a single nick can be used to induce HDR. It is contemplated here that a single nick can be used to increase the ratio of HDR, HR or NHEJ at a given cleavage site.
[0440] [0440] The placement of the double chain break or a single chain break in relation to the target position
[0441] [0441] The double chain break or single chain break in one of the chains must be close enough to the target position for the correction to occur. In one embodiment, the distance is not greater than 50, 100, 200, 300, 350 or 400 nucleotides. Although not wanting to be limited by theory, it is believed that the break should be close enough to the target position, such that the break is within the region that is subject to exonuclease-mediated removal during the final resection. If the distance between the target position and a break is too great, the mutation may not be included in the final resection and, therefore, may not be corrected, as the donor sequence can only be used to correct the sequence within the final resection region. .
[0442] [0442] In one embodiment, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease induce a double strand break with the objective of inducing correction mediated by HDR- or HR-, the cleavage site is between 0-200 bp (for example, 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175.25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200.75 to 175.75 to 150, 75 to 125.75 to 100 bp) away from the target position. In one embodiment, the cleavage site is between 0-100 bp (for example, 0 to 75.0 to 50, 0 to 25.25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.
[0443] [0443] In a modality in which two gRNAs (independently, unimolecular (or chimeric) or modular gRNA) complexed with Cas9 nickases induce two single chain breaks in order to induce HDR-mediated correction, the closest nick is between 0 -200 bp (for example, 0 to 175, 0 to 150, 0 to 125.0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position and the two nicks are ideally within 25-55 bp of each other (for example, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp) and no more than 100 bp apart (for example, no more than 90, 80, 70, 60,
[0444] [0444] In one embodiment, two gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break on both sides of a target position. In an alternative embodiment, three gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (that is, a complex gRNA with a Cas9 nuclease) and two single strand breaks or paired single strand breaks (ie, two gRNAs complex with Cas9 nickases) on each side of the target position (for example, the first gRNA is used to target upstream (ie, 5 ') of the target position and the second gRNA is used to direct downstream (ie 3 ') from the target position). In another embodiment, four gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single-strand breaks (ie, two pairs of two gRNAs complexed with Cas9 nickase) on both sides from the target position (for example, the first gRNA is used to target upstream (i.e., 5 ') from the target position and the second gRNA is used to target downstream (i.e., 3') from the target position). The double strand break (s) or the closest of the two single strand nicks in a pair, ideally, will be within 0-500 bp of the target position (for example, no more than 450, 400, 350 , 300, 250, 200, 150, 100, 50 or bp from the target position). When nickases are used, the two nicks in a pair are between 25 and 55 bp from each other (for example, between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 at 55, 40 to 55, at 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50.45 to 50, 35 to 45 or 40 to 45 bp) and no more than 100 bp apart from each other (for example, no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).
[0445] [0445] In one embodiment, two gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break on both sides of a target position. In an alternative embodiment, three gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (that is, a complex gRNA with a Cas9 nuclease) and two single strand breaks or paired single-strand breaks (ie, two gRNAs complex with Cas9 nickases) into two target sequences (for example, the first gRNA is used to target an upstream target sequence (ie, 5 ') and the second gRNA is used to target a target sequence downstream (i.e., 3 ') from an insertion site. In another embodiment, four gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single strand breaks (that is, two pairs of two gRNAs complexed with Cas9 nickases) on either side of an insertion site (for example, the first gRNA is used to target an upstream target sequence (i.e., 5 ') described here, and the second the gRNA is used to target a downstream target sequence (i.e., 3 ') described herein). The double strand break (s) or the closest of the two single strand nicks in a pair, ideally, will be within 0-500 bp of the target position (for example, no more than 450, 400, 350 , 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are between 25 and 55 bp from each other (for example, between 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50.45 to 50, 35 to 45 or 40 to 45 bp) and no more than 100 bp apart from each other ( for example, no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). Homology arms length
[0446] [0446] The homology arm should extend, at least, to the region where the final resection can occur, for example, in order to allow the dry single chain overhang to find a complementary region within the donor mold. The total length can be limited by parameters such as plasmid size or viral packaging limits. In one embodiment, a homology arm does not extend into repeated elements, for example, ALU repetitions, LINE repetitions. A model can have two homology arms with the same or different lengths.
[0447] [0447] Exemplary homology arm lengths include at least 25, 50, 100, 250, 500, 750 or 1000 nucleotides.
[0448] [0448] The target position, as used herein, refers to a site on a target nucleic acid (eg, chromosome) that is modified by a process dependent on the Cas9 molecule. For example, the target position can be a cleavage modified by the Cas9 molecule of the target nucleic acid and directed modification of the template nucleic acid, e.g., correction, from the target position. In one embodiment, a target position can be a site between two nucleotides, for example, adjacent nucleotides, in the target nucleic acid to which one or more nucleotides are added. The target position may comprise one or more nucleotides that are altered, for example, corrected, by a template nucleic acid. In one embodiment, the target position is within a target sequence (for example, the sequence to which the gRNA binds). In one embodiment, a target position is upstream or downstream of a target sequence (for example, the sequence to which the gRNA binds).
[0449] [0449] Typically, the template sequence undergoes break-mediated or catalyzed recombination with the target sequence. In one embodiment, the template nucleic acid includes a sequence that corresponds to a site in the target sequence that is cleaved by a Cas9-mediated cleavage event. In one embodiment, the template nucleic acid includes a sequence that corresponds to both, a first site in the target sequence that is cleaved in a first Cas9-mediated event, and a second site in the target sequence that is cleaved in a second event mediated by Cas9.
[0450] [0450] In one embodiment, the template nucleic acid may include a sequence that results in a change in the coding sequence of a translated sequence, for example, one that results in the substitution of one amino acid for another in a protein product, for example, transformation of a mutant allele into a wild-type allele, transformation of a wild-type allele into a mutant allele and / or the introduction of a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a mutation meaningless.
[0451] [0451] In other embodiments, the template nucleic acid may include a sequence that results in a change in a non-coding sequence, for example, a change in an exon or in an untranslated or non-transcribed 3 'or 5 region '. Such changes include a change in a control element, for example, a promoter, an intensifier, and a change in a trans action or cis action control element.
[0452] [0452] The template nucleic acid may include a sequence that, when integrated, results in: decreased activity of a positive control element; increased activity of a positive control element; decreased activity of a negative control element; increased activity of a negative control element; decreased expression of a gene;
[0453] [0453] The template nucleic acid may include a sequence that results in: a change in the sequence of 1, 2,3,4,5,6,7,8,9,10,11,120 or more nucleotides in the target sequence.
[0454] [0454] In one embodiment, the template nucleic acid is 20 +/- 10, 30 +/- 10, 40 +/- 10, 50 +/- 10, 60 +/- 10, 70 +/- 10, 80+ / - 10, 90 +/- 10, 100 +/- 10, 1 10 +/- 10, 120 +/- 10, 130 +/- 10, 140 +/- 10, 150 +/- 10, 160 + / - 10, 170 +/- 10, 1 80 +/- 10, 190 +/- 10, 200 +/- 10, 210 +/- 10, 220 +/- 10, 200-300, 300-400, 400- 500, 500-600, 600-700, 700-800, 800-900, 900- 1000, 1000-2000, 2000-3000 or more than 3000 nucleotides in length.
[0455] [0455] A template nucleic acid comprises the following components: [5 'homology arm] - [insertion sequence] - [homology arm
[0456] [0456] The homology arms provide recombination on the chromosome, which can replace the unwanted element, for example, a mutation or signature, with the replacement sequence. In one embodiment, the homology arms flank the most distal cleavage sites.
[0457] [0457] In one embodiment, the 3 'end of the 5' homology arm is the position close to the 5 'end of the substitution sequence. In one embodiment, the 5 'homology arm can extend at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5 'from the 5' end of the substitution sequence.
[0458] [0458] In one embodiment, the 5 'end of the homology arm 3' is the position close to the 3 'end of the substitution sequence. In one embodiment, the 3 'homology arm can extend at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3 'from the 3' end of the substitution sequence.
[0459] [0459] It is contemplated that one or both homology arms can be shortened to avoid the inclusion of certain elements of sequence repetition, for example, LINE elements, Alu repetitions. For example, a 5 'homology arm can be shortened to avoid a sequence repetition element. In other embodiments, a 3 'homology arm can be shortened to avoid a sequence repetition element. In some embodiments, the 5 'and 3' homology arms can be shortened to avoid the inclusion of certain sequence repetition elements.
[0460] [0460] It is contemplated here that template nucleic acids to correct a mutation can be created for use as a single-stranded oligonucleotide (ssODN, from single-stranded oligonucleotide). When using an ssSODN, the 5 'and 3' homology arms can vary up to about 200 base pairs (bp) in length, for example, at least 25, 50, 75, 100, 125, 150, 175 or 200 bp in length. Longer homology arms are also contemplated for ssSODNs as improvements in oligonucleotide synthesis continue to be made. NHEJ Approaches to Gene Targeting
[0461] [0461] As described here, the nuclease-induced non-homologous end-joining (NHEJ) can be used to target specific gene inactivations. Nuclease-induced NHEJ can also be used to remove (for example, delete) a sequence in a gene of interest.
[0462] [0462] Although not wanting to be limited by theory, it is believed that, in one modality, the genomic changes associated with the methods described here depend on nuclease-induced NHEJ and the error-prone nature of the NHEJ repair pathway. NHEJ repairs a double-stranded DNA break by joining the two ends together; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by breaking the double strand, are perfectly connected. The DNA ends of the double strand break are often subjected to enzymatic processing, resulting in the addition or removal of nucleotides, in one or both strands, before rejoining the ends. This results in the presence of insertion and / or deletion (indel) mutations in the DNA sequence at the NHEJ repair site. Two-thirds of these mutations can alter the reading frame and therefore produce a non-functional protein. Additionally, mutations that maintain the reading frame, but that insert or delete a significant amount of sequence, can destroy the functionality of the protein. This is locus dependent, since mutations in critical functional domains are probably less tolerable than mutations in non-critical regions of the protein.
[0463] [0463] The indelible mutations generated by NHEJ are of an unpredictable nature; however, at a given break site, certain indelible sequences are favored and are over-represented in the population. Deletion lengths can vary widely; most commonly in the 1-50 bp range, but they can easily reach over 100-200 bp. The inserts tend to be shorter and generally include short duplications of the sequence immediately around the site of the break. However, it is possible to obtain large insertions and, in these cases, the inserted sequence was often attributed to other regions of the genome or to the plasmid DNA present in the cells.
[0464] [0464] As NHEJ is a mutagenic process, it can also be used to delete small sequence motifs, as long as the generation of a specific final sequence is not necessary. If a double strand break is targeted near a short target sequence, the deletion mutations caused by NHEJ repair often extend and therefore remove unwanted nucleotides. For the deletion of larger DNA segments, the introduction of two double strand breaks, one on each side of the sequence, can result in NHEJ between the ends with the removal of the entire intervening sequence. Both approaches can be used to delete specific DNA sequences; however, the error prone nature of NHEJ can still produce indelible mutations at the site of repair.
[0465] [0465] Both double-stranded Cas9 molecules and single-stranded or nickase Cas9 molecules can be used in the methods and compositions described herein to generate NHEJ-mediated indels. NHEJ-mediated indels targeting the gene, for example, a coding region, for example, an initial coding region of a gene of interest can be used to inactivate (i.e., eliminate the expression of) a gene of interest. For example, the initial coding region of a gene of interest includes sequence immediately after a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (for example, lower at 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).
[0466] [0466] Placement of double-chain or single-chain breaks in relation to the target position
[0467] [0467] In one embodiment, in which a Cas9 gRNA and nuclease generate a double strand break in order to induce NHEJ-mediated indels, a gRNA, for example, a unimolecular (or chimeric) or modular gRNA molecule, is configured to position a double-stranded break in close proximity to a nucleotide at the target position. In one embodiment, the cleavage site is between 0-500 bp away from the target position (for example, less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9,8,7,6, 5, 4, 3, 20U 1 bp from the target position).
[0468] [0468] In one embodiment, in which two gRNAs complexing with Cas9 nickases induce two single chain breaks for inducing NHEJ-mediated indels, two gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to position two single-strand breaks to provide NHEJ to repair a nucleotide from the target position. In one embodiment, gRNAs are configured to cut positions in the same position, or within a few nucleotides of each other, in different strands, essentially imitating a double strand break. In one embodiment, the closest nick is between 0-30 bp from the target position (for example, less than 30, 25, 20, 1, 10,9,8,7,6,5,4, 3, 2 or 1 bp from the target position), and the two nicks are between 25-55 bp from each other (for example, between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55 , 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50.40 to 50, 45 to 50, 35 to 45 or 40 to 45 bp) and not more than 100 bp from each other (for example, no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). In one embodiment, gRNAs are configured to place a single strand break on both sides of a nucleotide at the target position.
[0469] [0469] Both double-stranded Cas9 molecules and single-stranded or nickase Cas9 molecules can be used in the methods and compositions described here to generate breaks on both sides of a target position.
[0470] [0470] In other embodiments, the insertion of template nucleic acid can be mediated by microhomology end joining (MMEJ, from microhomology end joining). See, for example, Saksuma et al., "MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems." Nature Protocols 11, 118—133 (2016) doi: 10.1038 / nprot.2015.140 Published online on December 17, 2015, the contents of which are incorporated by reference in their entirety. VIII. Systems comprising more than one gRNA molecule
[0471] [0471] While not wanting to be limited by theory, it has been surprisingly shown in the present document that the targeting of two target sequences (for example, by two complexes of gRNA molecule / Cas9 molecule that induce each single chain break or double at or near their respective target sequences) located next to a continuous nucleic acid induces excision (for example, deletion) of the nucleic acid sequence (or at least 80%, 85%, 90%, 95%, 96 %, 97%, 98%, 99% of the nucleic acid sequence) located between the two target sequences. In some respects, the present invention provides for the use of two or more gRNA molecules that comprise target domains targeting target sequences in close proximity on a continuous nucleic acid, for example, a chromosome, for example, a gene or locus gene , including their introns, exons and regulatory elements. The use can be, for example, by introducing the two or more gRNA molecules, together with one or more Cas9 molecules (or nucleic acid that encodes the two or more gRNA molecules and / or one or more Cas9 molecules) in one cell.
[0472] [0472] In some respects, the target sequences of the two or more gRNA molecules are located at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80 , 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, or 15,000 nucleotides in a continuous nucleic acid, but not more than 25,000 nucleotides in a continuous nucleic acid. In embodiments, the target sequences are located about 4000 and about 6000 nucleotides apart. In one embodiment, the target sequences are located about 4000 nucleotides apart. In one embodiment, the target sequences are located about 5000 nucleotides apart. In one embodiment, the target sequences are located about 6000 nucleotides apart.
[0473] [0473] In some respects, the plurality of gRNA molecules each directs to sequences within the same gene or gene locus. In an exemplary aspect, the target sequences of the two or more gRNA molecules are located in the HBG1 promoter region. In an exemplary aspect, the target sequences of the two or more gRNA molecules are located in the HBG2 promoter region. In another aspect, the plurality of gRNA molecules each directs to sequences within 2 or more different genes or loci. In an exemplary aspect, the target sequence of one or more of the gRNA molecules is located in the HBG1 promoter region, and the target sequence of one or more of the other gRNA molecules is located in the HBG2 promoter region.
[0474] [0474] In some respects, the invention provides compositions and cells comprising a plurality, for example, 2 or more, for example, 2, gRNA molecules of the invention, wherein the plurality of gRNA molecules target sequences of less than 15,000, less than 14,000, less than 13,000, less than 12,000, less than
[0475] [0475] In one embodiment, the invention provides a method for excising (for example, deletion) nucleic acid disposed between two gRNA binding sites disposed less than 25,000, less than
[0476] [0476] In one aspect, the two or more gRNA molecules comprise target domains targeting target sequences flanking a gene regulatory element, for example, a promoter binding site, an enhancer region or a repressor region, such that excision of the intervening sequence (or a portion of the intervening sequence) causes upward or downward regulation of a gene of interest. In other embodiments, the two or more gRNA molecules comprise target domains that target sequences that flank a gene, such that excision of the intervening sequence (or its portion) causes the deletion of the gene of interest.
[0477] [0477] In one embodiment, the two or more gRNA molecules each include a target domain, for example, consisting of a target domain sequence from Table 1, for example, from Table 2 or, for example , from Table 3a or Table 3b. In embodiments, the two or more gRNA molecules each include a target domain comprising, for example, that consisting of, the target domain of a gRNA molecule that results in at least 15% up-regulation of the number of F cells from a population of differentiated red blood cells (for example, on the 7th day after editing) of HSPCs edited by said gRNA ex vivo using the methods described herein. In two respects, the two or more gRNA molecules comprise target domains that are complementary with sequences in the same gene or region, for example, the HBG1 promoter region or the HBG2 promoter region. In two respects, the two or more gRNA molecules comprise target domains that are complementary with sequences from different genes or regions, for example, one in the HBG1 promoter region and one in the HBG2 promoter region.
[0478] [0478] In one aspect, the two or more gRNA molecules comprise target domains targeting target sequences flanking a gene regulatory element, for example, a promoter binding site, an enhancer region or a repressor region, such that excision of the intervening sequence (or a portion of the intervening sequence) causes upward or downward regulation of a gene of interest. In another aspect, the two or more gRNA molecules comprise target domains targeting sequences that flank a gene, such that excision of the intervening sequence (or a portion of the intervening sequence) causes the deletion of the gene of interest. For example, the two or more gRNA molecules comprise target domains targeting target sequences flanking the HBG1 gene (for example, a gRNA molecule targeting a target sequence in the HBG1 promoter region, and a second molecule of gRNA targeting a target sequence in the HBG2 promoter region), such that the HBG1 gene is excised.
[0479] [0479] In one embodiment, the two or more gRNA molecules comprise target domains that comprise, for example, consist of target domains selected from Table 1.
[0480] [0480] In two respects, the two or more gRNA molecules comprise target domains that comprise, for example, consisting of sequences of the target domain listed in Table 2, above. In two respects, the two or more gRNA molecules comprise target domains which comprise, for example, consisting of sequences of the target domain of the gRNAs listed in Table 3a, above. In two respects, the two or more gRNA molecules comprise target domains which comprise, for example, consisting of sequences of the target domain listed in Table 3b, above.
[0481] [0481] GRNA molecules comprising target domains that target sequences within a non-deleting HPHP region, for example, an HBG1 and / or HBG2 promoter region, may additionally be used with a gRNA molecule comprising a domain complementary target to a target sequence within, for example, a BCL11a enhancer region (for example, a +55, +58 or +62 BCL11a enhancer region) and / or a gRNA molecule comprising a target domain complementary to a target of an HPFH deletion locus. Such deletion HPFH loci are known in the art, for example, those described in Sankaran VG et al. NEJM (2011) 365: 807-814 (hereby incorporated by reference in its entirety). IX. GRNA properties
[0482] [0482] It was also surprisingly shown here that single gRNA molecules can have target sequences in more than one loci (for example, loci with high sequence homology), and that, when such loci are present on the same chromosome, for example , within less than about 15,000 nucleotides, less than about 14,000 nucleotides, less than about 13,000 nucleotides, less than about 12,000 nucleotides, less than about 11,000 nucleotides, less than about 10,000 nucleotides, less than about 9,000 nucleotides, less than about 8,000 nucleotides, less than about 7,000 nucleotides, less than about 6,000 nucleotides, less than about 5,000 nucleotides, less than about 4,000 nucleotides or less than about 3,000 nucleotides (for example, about
[0483] [0483] It has also surprisingly been shown that gRNA molecules that comprise a target domain complementary to a sequence in only one gene or region, for example, that is complementary to a target sequence in the HBG1 promoter region (but not in the promoter region HBG2), or that is complementary to a target sequence in the HBG2 promoter region (but not the HBG1 promoter region), can result in significant upregulation of fetal hemoglobin in erythroid cells differentiated from modified HSPCs (as described here) . In aspects, the invention thus provides gRNA molecules that comprise a target domain that is complementary to a target sequence in a single non-deletion HPFH region, for example, within an HBG1 or HBG 2 promoter region (for example, as described in Table 1), but which does not have a complete complementary target sequence (for example, 100%) in any other gene or region.
[0484] [0484] It has also surprisingly been shown in this document that gRNA molecules and CRISPR systems comprising said gRNA molecules produce similar or identical indel patterns over multiple experiments using the same cell type, delivery method and gRNA components / tracr. Without being limited by theory, it is believed that some patterns of indel may be more advantageous than others. For example, indels that include predominantly insertions and / or deletions that result in a "reading frame shift mutation" (for example, 1- or 2- base pair insertions or deletions, or any insertion or deletion where n / 3 is not an integer (where n = the number of nucleotides in the insertion or deletion)) can be beneficial in reducing or eliminating the expression of a functional protein. Likewise, indels that predominantly include "large deletions" (deletions of more than 10, 11, 12, 13, 14, 15, 20, 25 or 30 nucleotides, for example, more than 1 kb, more than 2 kb, more than 3 kb, more than 5 kb or more than 10 kb, for example, comprising a sequence arranged between a first and second binding site for a gRNA, for example, as described herein) can also be beneficial, for example, in the removal of critical regulatory sequences such as promoter binding sites, or by altering the structure or function of a locus, which may similarly have an effect on the expression of the functional protein. Although the indel patterns induced by a given gRNA / CRISPR system have been surprisingly reproduced for a given cell type, gRNA and CRISPR system, as described here, no single indelible structure will inevitably be produced in a given cell after the introduction of a system gRNA / CRISPR.
[0485] [0485] The invention thus provides gRNA molecules that create a beneficial indelible pattern or structure, for example, which have indelible patterns or structures predominantly composed of large deletions. Such gRNA molecules can be selected by evaluating the indelible pattern or structure created by a candidate gRNA molecule in a test cell (for example, a HEK293 cell) or in the cell of interest, for example, an HSPC cell by NGS, as described herein. As shown in the Examples, gRNA molecules have been discovered which, when introduced into the desired cell population, result in a cell population comprising a significant fraction of the cells having a large deletion at or near the target sequence of the gRNA. In some cases, the rate of large deletion indel formation is as high as 75%, 80%, 85%, 90% or more. The invention thus provides cell populations that comprise at least about 40% of cells (for example, at least about 45%, at least about 50%, at least about 55%, at least about 60% at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) having a large deletion, for example as described herein, at or near the target site of a gRNA molecule described herein. The invention also provides cell populations that comprise at least about 50% of cells (for example, at least about 55%, at least about 60%, at least about 65%, at least about 70% at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) having a large deletion, for example as described herein, at or near the target site of a gRNA molecule described herein.
[0486] [0486] The invention thus provides methods for selecting gRNA molecules for use in the therapeutic methods of the invention, comprising: 1) providing a plurality of gRNA molecules to a target of interest, 2) evaluating the indelible pattern or structure created by using said gRNA molecules, 3) selecting a gRNA molecule that forms an indelible pattern or structure composed predominantly of reading frame displacement mutations, large deletions or a combination thereof, and 4) using said gRNA selected in methods of the invention.
[0487] [0487] The invention thus provides methods of selecting gRNA molecules for use in the therapeutic methods of the invention, comprising: 1) providing a plurality of gRNA molecules to a target of interest, for example, which have target sequences in more than one location, 2) evaluate the indelible pattern or structure created by the use of said gRNA molecules, 3) select a gRNA molecule that forms a sequence excision comprising the nucleic acid sequence located between the two target sequences, for example example, in at least about 25% or more of the cells in a population of cells that are exposed to said gRNA molecules, and 4) using said gRNA molecule selected in methods of the invention.
[0488] [0488] The invention also provides methods of altering cells and altered cells, in which a particular indel pattern is constantly produced with a given gRNA / CRISPR system in that type of cell. The patterns of indel, including the top 5 indels that occur most frequently observed with the gRNA / CRISPR systems described herein can be determined using the methods of the examples and are disclosed, for example, in the Examples. As shown in the Examples, cell populations are generated, where a significant fraction of cells comprise one of the top 5 indels (for example, cell populations where one of the main indels is present in more than 30%, more than 40% , more than 50%, more than 60% or more of the population cells. Thus, the invention provides cells, for example, HSPCs (as described here), which comprise an indel of any of the 5 main indels observed with a given system gRNA / CRISPR In addition, the invention provides populations of cells, for example, HSPCs (as described here), which when evaluated by, for example, NGS, comprise a high percentage of cells comprising one of the 5 main indels described here for a given gRNA / CRISPR system.When used in connection with indelible standard analysis, a "high percentage" refers to at least 50% (for example, at least 55%, at least 60%, at least 65%, at least 70%, at least about 75% at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%) of the population cells comprising one of the 5 main indels described here for a given gRNA / CRISPR system. In other embodiments, the cell population comprises at least about 25% (for example, from about 25% to about 60%, for example, from about 25% to about 50%, for example, about from 25% to about 40%, for example from about 25% to about 35%) of cells that have one of the 5 main indices described here for a given gRNA / CRISPR system.
[0489] [0489] It has also been found that certain gRNA molecules do not create indels in off-target sequences (for example, off-target sequences outside the HBG1 and / or HBG2 promoter region) within the genome of the target cell type, or produce indels at off-target sites (for example, off-target sequences outside the HBG1 and / or HBG2 promoter region) at very low frequencies (for example, <5% of cells within a population) in relation to the frequency of breeding indel at the target site.
[0490] [0490] The components, for example, a Cas9 molecule or gRNA molecule, or both, can be delivered, formulated or administered in a variety of ways. As a non-limiting example, the gRNA molecule and the Cas9 molecule can be formulated (in one or more compositions), directly delivered or administered to a cell in which a genome editing event is desired. Alternatively, the nucleic acid encoding one or more components, for example, a Cas9 molecule or gRNA molecule, or both, can be formulated (in one or more compositions), delivered or administered. In one aspect, the gRNA molecule is provided as DNA encoding the gRNA molecule and the Cas9 molecule is provided as DNA encoding the Cas9 molecule. In one embodiment, the gRNA molecule and the Cas9 molecule are encoded in separate nucleic acid molecules. In one embodiment, the gRNA molecule and the Cas9 molecule are encoded in the same nucleic acid molecule. In one aspect, the gRNA molecule is provided as RNA and the Cas9 molecule is provided as DNA that encodes the Cas9 molecule. In one embodiment, the gRNA molecule is provided with one or more modifications, for example, as described herein. In one aspect, the gRNA molecule is provided as RNA and the Cas9 molecule is provided as mRNA that encodes the Cas9 molecule. In one aspect, the gRNA molecule is provided as RNA and the Cas9 molecule is provided as a protein. In one embodiment, the gRNA molecule and the Cas9 molecule are supplied as a ribonuclear protein complex (RNP, from the English "ribonuclear protein complex"). In one aspect, the gRNA molecule is provided as DNA that encodes the gRNA molecule and the Cas9 molecule is provided as a protein.
[0491] [0491] Delivery, for example, delivery of RNP, (for example, HSPC cells as described herein) can be achieved by, for example, electroporation (for example, as known in the art) or another method that makes the cell membrane permeable to nucleic acid and / or polypeptide molecules. In modalities, the CRISPR system, for example, the RNP as described here, is delivered by electroporation using a Nucleofector-4D (Lonza), for example, using the program CM-137 in Nucleofector-4D (Lonza). In modalities, the CRISPR system, for example, the RNP as described here, is delivered by electroporation using a voltage of about 800 volts to about 2000 volts, for example, from about 1000 volts to about 1800 volts, for example , from about 1200 volts to about 1800 volts, for example, from about 1400 volts to about 1800 volts, for example, from about 1600 volts to about 1800 volts, for example, about 1700 volts, for example , at a voltage of 1700 volts. In modalities, the pulse width / length is from about 10 ms to about 50 ms, for example, from about 10 ms to about 40 ms, for example, from about 10 ms to about 30 ms, for example for example, from about 15 ms to about 25 ms, for example, about 20 ms, for example, ms. In modalities, 1, 2, 3, 4, 5, or more, for example, 2, for example, 1 pulse, are used. In one embodiment, the CRISPR system, for example, the RNP, as described here, is delivered by electroporation using a voltage of about 1700 volts (for example, 1700 volts), a pulse width of about 20 ms (for example, example, 20 ms), using a single pulse (1). In modalities, electroporation is performed using a Neon electroporator. Additional techniques for making the membrane permeable are known in the art and include, for example, cell compression (for example, as described in WO2015 / 023982 and WO2013 / 059343, the content of which is incorporated herein by reference in its entirety), nano-needles ( for example, as described in Chiappini et al., Nat. Mat., 14; 532-39, or US2014 / 0295558, the content of which is hereby incorporated by reference in its entirety) and nano-straws (for example, as described in Xie , ACS Nano, 7 (5); 4351-58, the content of which is incorporated herein by reference in its entirety).
[0492] [0492] When a component is delivered encoded in DNA, the
[0493] [0493] DNA encoding Cas9 molecules and / or gRNA molecules can be administered to subjects or delivered to cells by methods known in the art or as described herein. For example, DNA encoding Cas9 and / or encoding gRNA can be delivered, for example, by vectors (for example, viral or non-viral vectors), non-vector based methods (for example, using naked DNA or DNA complexes ), or a combination of these.
[0494] [0494] In some embodiments, the DNA encoding Cas9 and / or gRNA is delivered by a vector (for example, vector / virus, plasmid, minicircle or nanoplasmid).
[0495] [0495] A vector can comprise a sequence that encodes a Cas9 molecule and / or a gRNA molecule. A vector may also comprise a sequence encoding a signal peptide (for example, for nuclear, nucleolar, mitochondrial location), fused, for example, to a sequence of Cas9 molecules. For example, a vector can comprise one or more nuclear localization sequences (for example, from SV40) fused to the sequence encoding the Cas9 molecule.
[0496] [0496] One or more regulatory / control elements, for example, a promoter, an intensifier, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence and an acceptor or splice donor can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase | 1 (for example, a CMV promoter). In other embodiments, the promoter is recognized by RNA polymerase Ill (for example, a U6 promoter). In some embodiments, the promoter is a regulated promoter (for example, inducible promoter). In other embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is a viral promoter. In other embodiments, the promoter is a non-viral promoter.
[0497] [0497] In some modalities, the delivery vector or vehicle is a mini-circle. In some embodiments, the delivery vector or vehicle is a nanoplasmid.
[0498] [0498] In some embodiments, the delivery vector or vehicle is a viral vector (for example, for the generation of recombinant viruses). In some embodiments, the virus is a DNA virus (for example, dsDNA or ssDNA virus). In other embodiments, the virus is an RNA virus (for example, an sSRNA virus).
[0499] [0499] Exemplary viral / victim vectors include, for example, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology.
[0500] [0500] In some embodiments, the virus infects dividing cells. In other modalities, the virus infects cells that are not dividing. In some embodiments, the virus infects both dividing and non-dividing cells. In some embodiments, the virus can integrate into the host's genome. In some embodiments, the virus is manipulated to have reduced immunity, for example, in humans. In some embodiments, the virus is competent for replication. In other embodiments, the virus is deficient in replication, for example, having one or more coding regions for the genes necessary for additional cycles of replication and / or packaging of virions replaced by other genes or deleted. In some embodiments, viruses cause transient expression of the Cas9 molecule and / or the gRNA molecule. In other embodiments, viruses cause long-term expression, for example, at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, | year, 2 years, or permanent, of the Cas9 molecule and / or the gRNA molecule. The packaging capacity of viruses can vary, for example, from at least about 4 kb to at least about 30 kb, for example, at least 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb or 50 kb.
[0501] [0501] In some embodiments, the DNA encoding Cas9 and / or gRNA is delivered by a recombinant retrovirus. In some embodiments, the retrovirus (for example, the Moloney murine leukemia virus) comprises a reverse transcriptase, for example, which allows integration into the host genome. In some embodiments, the retrovirus is competent for replication. In other embodiments, the retrovirus is deficient in replication, for example, having one of the most coding regions for the genes needed for additional cycles of replication and packaging of virions replaced by other genes, or deleted.
[0502] [0502] In some embodiments, DNA encoding Cas9 and / or gRNA is supplied by a recombinant lentivirus. For example, lentivirus is defective in replication, for example, it does not comprise one or more genes necessary for viral replication.
[0503] [0503] In some embodiments, the DNA encoding Cas9 and / or gRNA is delivered by a recombinant adenovirus. In some embodiments, adenovirus is manipulated to have reduced immunity in humans.
[0504] [0504] In some embodiments, the DNA encoding Cas9 and / or gRNA is delivered by a recombinant AAV. In some embodiments, AAV may incorporate its genome into that of a host cell, for example, a target cell, as described here. In some embodiments, AAV is a self-complementing adeno-associated virus (SCAAV) -complementary adeno-associated virus "), for example, a scAAV that packages both strands that pair together to form double-stranded DNA. AAV serotypes that can be used in the disclosed methods include, for example, AAV1, AAV2, modified AAV2 (for example, modifications in Y444F, Y500F, Y730F and / or S662V), AAV3, modified AAV3 (for example, modifications in Y705F , Y73 1 F and / or T492V), AAVA4, AAV5, AAVG6, modified AAV6 (for example, modifications in S663V and / or T492V), AAV8. AAV 8.2, AAV9, AAV rh 10 and pseudotyped AAV, such as AAV2 / 8, AAV2 / 5 and AAV2 / 6 can also be used in the disclosed methods.
[0505] [0505] In some embodiments, the DNA encoding Cas9 and / or gRNA is distributed by a hybrid virus, for example, a hybrid of one or more of the viruses described here.
[0506] [0506] A packaging cell is used to form a virus particle that is capable of infecting a host or target cell. Such a cell includes a 293 cell, which can package adenovirus, and a y2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimum viral sequences necessary for packaging and subsequent integration into a host or target cell (if applicable), other viral sequences being replaced by an expression cassette encoding the protein to be expressed. For example, an AAV vector used in gene therapy typically only has inverted terminal repeat (ITR) sequences from the AAV genome that are necessary for packaging and gene expression in the host or target cell. The absent viral functions are provided in trans by the packaging cell line. Hereafter, the viral DNA is packaged in a cell line, which contains an auxiliary plasmid encoding the other AAV genes, namely, rep and cap, but without the ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of the helper plasmid AAV genes. The helper plasmid is not packaged in significant quantities due to a lack of ITR sequences. Contamination with adenovirus can be reduced, for example, by heat treatment to which adenovirus is more sensitive than AAV.
[0507] [0507] In one embodiment, the viral vector has the ability to recognize the cell type and / or tissue type. For example, the viral vector can be pseudotyped with a different / alternative viral envelope glycoprotein; manipulated with a cell type-specific receptor (e.g., genetic modification of glycoproteins in the viral envelope to incorporate targeting ligands, such as a peptide ligand, a single chain antibody, a growth factor); and / or modified to have a molecular bridge with dual specificities, with one end recognizing a viral glycoprotein and the other end recognizing a portion of the target cell surface (for example, ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation ).
[0508] [0508] In one embodiment, the viral vector achieves cell type specific expression. For example, a tissue-specific promoter can be constructed to restrict the expression of the transgene (Cas 9 and gRNA) in the target cell only. The specificity of the vector can also be mediated by microRNA-dependent control of transgene expression. In one embodiment, the viral vector has increased efficiency of the fusion of the viral vector and a membrane of the target cell. For example, a fusion protein, such as fusion-competent hemagglutin (HA), can be incorporated to increase virus absorption in cells. In one embodiment, the viral vector has the capability of nuclear localization. For example, a virus that requires the cell wall to break (during cell division) and therefore will not infect a non-dividing cell can be changed to incorporate a nuclear-located peptide into the virus's matrix protein, thus allowing the transduction of non-proliferating cells.
[0509] [0509] In some embodiments, the DNA encoding Cas9 and / or gRNA is delivered by a non-vector-based method (for example, using DNA complexes or naked DNA). For example, DNA can be delivered, for example, by modified silica or silicate (Ormosil) - organically, electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates or a combination of these .
[0510] [0510] In some embodiments, the DNA encoding Cas9 and / or gRNA is delivered by a combination of a vector-based method and a non-vector-based method. For example, a virosome comprises a liposome combined with an inactivated virus (for example, HIV or influenza virus), which can result in more efficient gene transfer, for example, in a respiratory epithelial cell, than a viral or liposomal method alone. .
[0511] [0511] In one embodiment, the delivery vehicle is a non-viral vector. In one embodiment, the non-viral vector is an inorganic nanoparticle (for example, linked to the payload on the surface of the nanoparticle). Exemplary inorganic nanoparticles include, for example, magnetic nanoparticles (for example, Fe IvlO2), or silica. The outer surface of the nanoparticle can be conjugated to a positively charged polymer (for example, polyethyleneimine, polylysine, polyserine) that allows the attachment (for example, conjugation or trapping) of payload. In one embodiment, the non-viral vector is an organic nanoparticle (for example, trapping the payload inside the nanoparticle). Exemplary organic nanoparticles include, for example, SNALP liposomes that contain cationic lipids in conjunction with neutral auxiliary lipids that are coated with polyethylene glycol (PEG) and protamine and lipid-coated nucleic acid complex.
[0512] [0512] Exemplary lipids and / or polymers for transferring CRISPR systems or nucleic acid, for example, vectors encoding CRISPR systems or their components include, for example, those described in WOZ2011 / 076807, WO2014 / 136086, WO2005 / 060697, WO2014 / 140211, WO2012 / 031046, WO2013 / 103467, WO2013 / 006825, WO2012 / 006378, WO 2015/095340 and WO2015 / 095346, the contents of each of the above are hereby incorporated by reference in their entirety. In one embodiment, the vehicle has targeting modifications to increase the update of the target cell of nanoparticles and liposomes, for example, cell-specific antigens, monoclonal antibodies,
[0513] [0513] In one embodiment, the delivery vehicle is a non-viral biological delivery vehicle. In one embodiment, the vehicle is an attenuated bacterium (for example, naturally or artificially manipulated to be invasive, but attenuated to prevent pathogenesis and expressing the transgene (for example, Listeria monocytogenes, certain strains of Salmonella, Bifidobacterium longum and modified Escherichia coli) , bacteria having nutritional tropism and tissue-specific to target specific tissues, bacteria with surface proteins modified to alter the specificity of the target tissue). In one embodiment, the vehicle is a genetically modified bacteriophage (for example, modified phages that have great packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequences and having targeting ligands incorporated). In one embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (for example, by purifying the "empty" particles followed by ex vivo assembly of the virus with the desired charge). The vehicle can also be manipulated to incorporate targeting ligands to alter the specificity of the target tissue. In one embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (eg, erythrocyte ghosts, which are red blood cells divided into spherical structures derived from the subject (eg, tissue targeting can be achieved by linking several specific tissues or cell ligands), or secretory exosomes - subject (i.e., patient) derived from membrane-bound nanovesicles (30 - 100 nm) of endocytic origin (for example, it can be produced from various types of cells and can therefore be absorbed by cells without the need for targeting ligands).
[0514] [0514] In one embodiment, one or more nucleic acid molecules (for example, DNA molecules), with the exception of the components of a Cas system, for example, the component of the Cas9 molecule and / or the component of the gRNA molecule described here, are delivered. In one embodiment, the nucleic acid molecule is delivered at the same time as one or more components of the Cas system are delivered. In one embodiment, the nucleic acid molecule is delivered before or after (for example, less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks or 4 weeks) of one or more components of the Cas9 system to be delivered. In one embodiment, the nucleic acid molecule is delivered by a different medium than one or more of the components of the Cas9 system, for example, the Cas9 molecule component and / or the gRNA molecule component, is delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, for example, an integration-deficient lentivirus, and the component of the Cas9 molecule and / or the component of the gRNA molecule can be delivered by electroporation, for example, from such that the toxicity caused by nucleic acids (for example, DNAs) can be reduced.
[0515] [0515] RNA encoding Cas9 molecules (for example, active Cas9 molecules, inactive Cas9 molecules or inactive Cas9 fusion proteins) and / or gRNA molecules, can be delivered to cells, for example, the target cells described here, for example methods known in the art or as described herein. For example, RNA encoding Cas9 and / or encoding gRNA can be delivered, for example, by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery of the Cas9 molecule as protein
[0516] [0516] Cas9 molecules (for example, active Cas9 molecules, inactive Cas9 molecules or inactive Cas9 fusion proteins) can be delivered to cells by methods known in the art or as described herein. For example, Cas9 protein molecules can be delivered, for example, by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, cell compression or abrasion (for example, by nano-needles) or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA, for example, by pre-complexing the gRNA and the Cas9 protein into a ribonuclear protein complex (RNP).
[0517] [0517] In one aspect, the Cas9 molecule, for example, as described here, is delivered as a protein and the gRNA molecule is delivered as one or more RNAs (for example, as a dgRNA or SgRNA, as described here) . In modalities, the Cas9 protein is complexed with the gRNA molecule before delivery to a cell,
[0518] [0518] The above procedure can be modified for use with saRNA molecules by omitting step 2, above, and step 1, providing the Cas9 molecule and the saRNA molecule in solution at high concentration, and allowing the components to balance.
[0519] [0519] In aspects, the components of the gene editing system (eg, CRISPR system) and / or nucleic acid encoding one or more components of the gene editing system (eg, CRISPR system) are introduced into cells by mechanical disturbance of cells, for example, passing said cells through a pore or channel that constricts the cells. Such disturbance can be carried out in a solution comprising the components of the gene editing system (eg, CRISPR system) and / or nucleic acid encoding one or more components of the gene editing system (eg, CRISPR system), for example example, as described here. In embodiments, the disturbance is carried out using a TRIAMF system, for example, as described herein, for example, in the Examples and in the PCT patent application PCT / US17 / 54110 (incorporated herein by reference in its entirety). Bi-Modal or Differential Component Delivery
[0520] [0520] The separate delivery of the components of a Cas system, for example, the component of the Cas9 molecule and the component of the gRNA molecule and, more particularly, the delivery of the components in different ways, can improve performance, for example, by improving the specificity and safety of the fabric.
[0521] [0521] In one embodiment, the Cas9 molecule and the gRNA molecule are delivered in different ways, or as sometimes referred to here as differential modes. Different or differential modes, as used herein, refer to delivery modes that confer different pharmacodynamic or pharmacokinetic properties on the object component molecule, for example, a Cas9 molecule, gRNA molecule or template nucleic acid. For example, modes of delivery may result in different tissue distribution, different half-life or different temporal distribution, for example, in a selected compartment, tissue or organ.
[0522] [0522] Some modes of delivery, for example, delivery by a nucleic acid vector that persists in a cell, or in offspring of a cell, for example, by autonomous replication or insertion into cellular nucleic acid, result in expression and presence persistent effects of a component. XI. Treatment Methods
[0523] [0523] Cas9 systems, for example, one or more gRNA molecules and one or more Cas9 molecules, described herein, are useful for the treatment of disease in a mammal, for example, in a human.
[0524] [0524] Systems —Cas9 comprising gRNA molecules comprising the target domains described herein, for example, in Table 1 and the methods and cells (for example, as described herein) are useful for the treatment of hemoglobinopathies. Hemoglobinopathies
[0525] [0525] Hemoglobinopathies encompass a number of anemias of genetic origin in which there is decreased production and / or increased destruction (hemolysis) of red blood cells (erythrocytes). These also include genetic defects that result in the production of hemoglobins - abnormal with an impaired ability to maintain the concentration of oxygen. Some of these disorders involve an inability to produce normal B-globin in sufficient quantities, while others involve an inability to produce normal B-globin completely. These disorders associated with the B-globin protein are generally referred to as B-hemoglobinopathies. For example, B-thalassemias result from a partial or complete defect in the expression of the B-globin gene, leading to deficient or absent HbA. Sickle cell anemia results from a point mutation in the structural gene of B-globin, leading to the production of an abnormal hemoglobin (sickle cell) (HbS). HbS is prone to polymerization, particularly under deoxygenated conditions. HbS red blood cells are more fragile than normal red blood cells and undergo hemolysis more readily, eventually leading to anemia.
[0526] [0526] In one embodiment, a genetic defect in alpha globin or beta globin is corrected, for example, by homologous recombination, using Cas9 molecules and gRNA molecules, for example, CRISPR systems, described here.
[0527] [0527] In one embodiment, a gene encoding a copy of wild-type alpha or beta globin (eg, without mutation) is inserted into the cell's genome, for example, in a safe haven site, for example, in a AAVS1 safe harbor site, by homologous recombination, using a CRISPR system and methods described here.
[0528] [0528] In one embodiment, a gene associated with hemoglobinopathies is targeted, using the Cas9 molecule and the gRNA molecule described here. Exemplary targets include, for example, genes associated with the control of gamma globin genes. In one embodiment, the target is a non-deletion HPFH region.
[0529] [0529] Fetal hemoglobin (also hemoglobin F or HbF or a2y2) is a tetramer of two adult alpha-globin polypeptides and two beta-like gamma-globin polypeptides. HbF is the main oxygen-carrying protein in the human fetus during the last seven months of development in the uterus and in the newborn up to approximately 6 months of age. Functionally, fetal hemoglobin differs more from adult hemoglobin, as it is able to bind to oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream.
[0530] [0530] In newborns, fetal hemoglobin is almost completely - * replaced by adult hemoglobin in approximately 6 months after birth. In adults, the production of fetal hemoglobin can be reactivated pharmacologically, which is useful in the treatment of diseases such as hemoglobinopathies. For example, in certain patients with hemoglobinopathies, higher levels of gamma globin expression may partially offset the production of defective or impaired beta-globin genes, which can improve the clinical severity of these diseases. Increased levels of HbF or numbers of F cells (erythrocytes containing HbF) can improve the severity of hemoglobinopathies disease, for example, beta-thalassemia major and sickle cell anemia.
[0531] [0531] As has been surprisingly discovered, increased levels of HbF or F cell counts may be associated with indel formation in one or more non-deletion HPFH regions in cells, for example, HSPCs and / or cells differentiated from HSPCs (for example, HSPCs modified by one or more gRNA molecules described here). In one embodiment, the cell is a hemopoietic stem cell or progenitor cell. Sickle cell disease
[0532] [0532] Sickle cell disease is a group of disorders that affects hemoglobin. People with this disorder have atypical hemoglobin molecules (hemoglobin S), which can distort red blood cells into a sickle or crescent shape. Characteristics of this disorder include a low number of red blood cells (anemia), repeated infections and periodic episodes of pain.
[0533] [0533] Mutations in the HBB gene cause sickle cell disease. The T and HBB gene provides instructions for the production of beta-globin. Several versions of beta-globin result from different mutations in the HBB gene. A particular mutation in the HBB gene produces an abnormal version of beta-globin known as hemoglobin S (HbS). Other mutations in the HBB gene lead to additional abnormal versions of beta-globin, such as hemoglobin C (HbC) and hemoglobin E (HbE). Mutations in the HBB gene can also result in an unusually low level of beta-globin, that is, beta thalassemia.
[0534] [0534] In people with sickle cell disease, at least one of the subunits of beta-globin in hemoglobin is replaced by hemoglobin S. In sickle cell anemia, which is a common form of sickle cell disease, hemoglobin S replaces the two subunits of beta- globin in hemoglobin. In other types of sickle cell disease,
[0535] [0535] Beta thalassemia is a blood disorder that reduces hemoglobin production. In people with beta thalassemia, low hemoglobin levels lead to a lack of oxygen in many parts of the body. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue and more serious complications. People with beta thalassemia are at an increased risk of developing abnormal blood clots.
[0536] [0536] Beta thalassemia is classified into two types, depending on the severity of the symptoms: major thalassemia (also known as Cooley's anemia) and intermediate thalassemia. Of the two types, thalassemia major is more severe.
[0537] [0537] Mutations in the HBB gene cause beta thalassemia. The HBB gene provides instructions for the production of beta-globin. Some mutations in the HBB gene prevent the production of any beta-globin. The absence of beta-globin is called beta-zero thalassemia (B Ӽ). Other mutations in the HBB gene allow the production of some beta-globin, but in small quantities, that is, beta-plus thalassemia (B *). People with both types were diagnosed with major thalassemia and intermediate thalassemia.
[0538] [0538] In one embodiment, a complex of Cas9 molecule / gRNA molecule targeting a first gene or locus is used to treat a disorder characterized by a second gene, for example, a mutation in a second gene. As an example, targeting the first gene, for example, by editing or delivery of charge, can compensate or inhibit further damage from the effect of a second gene, for example, a second mutant gene. In one embodiment, the allele (s) of the first gene carried by the subject is not the cause of the disorder. For example, as shown here, gRNA molecules that induce indel formation in a non-deletion region of HPFH, for example, an HBG1 and / or HBG2 promoter region, can result in upregulation of fetal hemoglobin in differentiated erythroid cells of modified HSPCs (as described here), and without being limited by theory, this positive regulation of fetal hemoglobin compensates for and corrects the HBB gene carrying a sickle cell mutation.
[0539] [0539] In one aspect, the invention relates to the treatment of a mammal, for example, a human, in need of an increase in fetal hemoglobin (HbF).
[0540] [0540] In one aspect, the invention relates to the treatment of a mammal, for example, a human being, who has been diagnosed with, or is at risk of developing, a hemoglobinopathy.
[0541] [0541] In one aspect, hemoglobinopathy is a hemoglobinopathy B. In one aspect, hemoglobinopathy is sickle cell disease. In one aspect, hemoglobinopathy is beta thalassemia. Hemoglobinopathy Treatment Methods
[0542] [0542] In another aspect, the invention provides treatment methods. In some respects, gRNA molecules, CRISPR systems and / or cells of the invention are used to treat a patient in need of them. In aspects, the patient is a mammal, for example, a human being. In aspects, the patient has hemoglobinopathy. In modalities, the patient has sickle cell disease.
[0543] [0543] In one aspect, the method of treatment comprises administering to a mammal, for example, a human, one or more gRNA molecules, for example, one or more gRNA molecules comprising a target domain described in the Table 1 and one or more Cas9 molecules described herein.
[0544] [0544] In one aspect, the treatment method comprises administering to a mammal a cell population, wherein the cell population is a cell population of a mammal, for example, a human, which has been administered with one or more molecules of gRNA, for example, one or more gRNA molecules comprising a target domain described in Table 1 and one or more Cas9 molecules described herein, for example, a CRISPR system as described herein. In one embodiment, the administration of one or more gRNA molecules or CRISPR systems to the cell is performed in vivo. In one embodiment, the administration of one or more gRNA molecules or CRISPR systems to the cell is performed ex vivo.
[0545] [0545] In one aspect, the method of treatment comprises administering to the mammal, for example, human, an effective amount of a population of cells comprising cells that comprise or once comprised one or more gRNA molecules, for example, one or more gRNA molecules comprising a target domain described in Table 1, and one or more Cas9 molecules described herein or the progeny of said cells. In one embodiment, the cells are allogeneic to the mammal. In one embodiment, the cells are autologous to the mammal. In one embodiment, cells are harvested from the mammal, manipulated ex vivo and returned to the mammal.
[0546] [0546] In aspects, cells comprising or once comprised one or more gRNA molecules, for example, one or more gRNA molecules comprising a target domain described in Table 1 and one or more Cas9 molecules described herein, or the progeny of said cells, comprise stem cells or progenitor cells. In one aspect, stem cells are hematopoietic stem cells. In one aspect, the progenitor cells are hematopoietic progenitor cells. In one aspect, the cells comprise hematopoietic stem cells and hematopoietic progenitor cells, for example, are HSPCs. In one aspect, the cells comprise, for example, consist of CD34 + cells. In one aspect, the cells are substantially free of CD34- cells. In one aspect, cells comprise, for example, consist of stem cells - CD34 + / CD90 +. In one aspect, the cells comprise, for example, consist of CD34 + / CD90- cells. In one aspect, cells are a population comprising one or more of the cell types described above or described herein.
[0547] [0547] In one embodiment, the disclosure provides a method for the treatment of a hemoglobinopathy, for example, sickle cell disease or beta-thalassemia, or a method for increasing the expression of fetal hemoglobin in a mammal, for example, a human, in need thereof, the method comprising: a) providing, for example, by harvesting or isolation, a population of HSPCs (for example, CD34 + cells) from a mammal; b) providing said cells ex vivo, for example, in a cell culture medium, optionally in the presence of an effective amount of a composition comprising at least one stem cell expander, wherein said population of HSPCs ( for example, CD34 + cells) expands to a greater degree in an untreated population; c) contacting the population of HSPCs (for example, CD34 + cells) with an effective amount of: a composition comprising at least one gRNA molecule comprising a target domain described here, for example, a target domain described in Table 1, or a nucleic acid encoding said gRNA molecule and at least one Cas9 molecule, for example, described herein, or a nucleic acid encoding said Cas9 molecule, for example, one or more RNPs as described herein for example, with a CRISPR system described here; d) cause at least one modification in at least a portion of the population's cells (for example, at least a portion of HSPCs, for example, CD34 + cells, in the population), so, for example, when the said HSPCs are differentiated into cells of an erythroid lineage, for example, red blood cells, the expression of fetal hemoglobin is increased, for example, with respect to cells not contacted according to step c); and f) returning a population of cells comprising said modified HSPCs (e.g., CD34 + cells) to the mammal.
[0548] [0548] In one respect, HSPCs are allogeneic to the mammal to which they are returned. In one respect, HSPCs are autologous to the mammal to which they are returned. In some respects, HSPCs are isolated from bone marrow. In some respects, HSPCs are isolated from peripheral blood, for example, mobilized peripheral blood. In aspects, the mobilized peripheral blood is isolated from a subject who has been given a G-CSF. In aspects, the peripheral blood mobilized is isolated from a subject who has been given a mobilization agent other than G-CSF, for example, Plerixafor & (AMD3100). In other respects, the mobilized peripheral blood is isolated from a subject who has been given a combination of G-CSF and Plerixafor & (AMD3100). In some respects, HSPCs are isolated from umbilical cord blood. In modalities, the cells are derived from a patient with hemoglobinopathy, for example, a patient with sickle cell disease or a patient with thalassemia, for example,
[0549] [0549] In other embodiments of the method, the method further comprises, after providing a population of HSPCs (for example, CD34 + cells), for example, from a source described above, the step of enriching the cell population for HSPCs (for example, CD34 + cells). In method embodiments, after said enrichment, the cell population, for example, HSPCs, is substantially free of CD34- cells.
[0550] [0550] In modalities, the cell population that is returned to the mammal includes at least 70% viable cells. In embodiments, the population of cells that is returned to the mammal includes at least 75% viable cells. In embodiments, the cell population that is returned to the mammal includes at least 80% viable cells. In embodiments, the cell population that is returned to the mammal includes at least 85% viable cells. In embodiments, the cell population that is returned to the mammal includes at least 90% viable cells. In embodiments, the cell population that is returned to the mammal includes at least 95% viable cells. In embodiments, the cell population that is returned to the mammal includes at least 99% viable cells. Viability can be determined by staining a representative portion of the cell population for a cell viability marker, for example, as is known in the art.
[0551] [0551] In another embodiment, the disclosure provides a method for the treatment of a hemoglobinopathy, for example, sickle cell disease or beta-thalassemia, or a method for increasing the expression of fetal hemoglobin in a mammal, for example, a human , in need thereof, comprising the method, the steps of: a) providing, for example, by harvest or isolation, a population of HSPCs (for example, CD34 + cells) from a mammal, for example, from a mammal's bone marrow ; b) isolating CD34 + cells from the cell population of step a); c) providing said CD34 + cells ex vivo, and culturing said cells, for example, in a cell culture medium, in the presence of an effective amount of a composition comprising at least one stem cell expander, for example , (S) -2- (6- (2- (1H-indol-3-yI) ethylamino) -2- (5-fluoropyridin-3-i1) -9H- purin-9-yl) propan-1-ol , for example, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) - 2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan- 1-0l, in a concentration of about 0.5 to about 0.75 micromolar, so that said population of CD34 + cells expands to a greater degree than an untreated population; d) introducing into the cells of the CD34 + cell population an effective amount of: a composition comprising a Cas9 molecule, for example, as described herein, and a gRNA molecule, for example, as described herein, for example, optionally where the Cas9 molecule and the gRNA molecule is in the form of an RNP, for example, as described herein, and optionally where said introduction is by electroporation, for example, as described herein, of said RNP in said cells; e) cause at least one genetic modification in at least a portion of the population's cells (for example, at least a portion of HSPCs, for example, CD34 + cells, of the population), so an indel, for example , as described here, is created at or near the genomic site complementary to the target domain of the gRNA introduced in step d); f) optionally, further cultivating said cells after said introduction, for example, in a cell culture medium, in the presence of an effective amount of a composition comprising at least one stem cell expander, for example, ( S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H- purin-9-yl) propan-1-ol, for example example, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) - 2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1- 10, in a concentration of about 0.5 to about 0.75 micromolar, such that the cells expand at least 2 times, for example, at least 4 times, for example, at least 5 times; g) cryopreserve said cells; and h) returning the cells to the mammal, wherein, the cells returned to the mammal comprise cells that 1) maintain the ability to differentiate into cells of the erythroid lineage, for example, red blood cells; 2) when differentiated into red blood cells, they produce an increased level of fetal hemoglobin, for example, relative to cells not modified by the gRNA in step e), for example, they produce at least 6 picograms of fetal hemoglobin per cell.
[0552] [0552] In one respect, HSPCs are allogeneic to the mammal to which they are returned. In one respect, HSPCs are autologous to the mammal to which they are returned. In some respects, HSPCs are isolated from bone marrow. In some respects, HSPCs are isolated from peripheral blood, for example, mobilized peripheral blood. In aspects, the mobilized peripheral blood is isolated from a subject who has been given a G-CSF. In aspects, the peripheral blood mobilized is isolated from a subject who has been given a mobilization agent other than G-CSF, for example, Plerixafor & (AMD3100). In other respects, the mobilized peripheral blood is isolated from a subject who has been given a combination of G-CSF and Plerixafor & (AMD3100). In some respects, HSPCs are isolated from umbilical cord blood. In modalities, the cells are derived from a patient with hemoglobinopathy, for example, a patient with sickle cell disease or a patient with thalassemia, for example, beta thalassemia.
[0553] [0553] In modalities of the above method, the recited step b) results in a cell population that is substantially free of CD34- cells.
[0554] [0554] In other embodiments of the method, the method further comprises, after providing a population of HSPCs (for example, CD34 + cells), for example, from a source described above, the cell population enriched for HSPCs (for example, CD34 + cells).
[0555] [0555] In additional modalities of these methods, the population of modified HSPCs (eg, CD34 + stem cells) that have the ability to differentiate the increased expression of fetal hemoglobin is cryopreserved and stored before being reintroduced into the mammal. In modalities, the cryopreserved population of HSPCs having the ability to differentiate into cells of the erythroid lineage, for example, red blood cells and / or when differentiated into cells of the erythroid lineage, for example, red blood cells, produces an increased level of fetal hemoglobin, thawed and then reintroduced into the mammal. In an additional embodiment of these methods, the method comprises chemotherapy and / or radiation therapy to remove or reduce the endogenous hematopoietic parent or stem cells in the mammal. In an additional embodiment of these methods, the method does not comprise a chemotherapy and / or radiation therapy step to remove or reduce the endogenous hematopoietic parent or stem cells in the mammal. In an additional embodiment of these methods, the method comprises chemotherapy and / or radiation therapy to partially reduce (e.g., partial lymphodepletion) the endogenous hematopoietic parent or stem cells in the mammal. In modalities, the patient is treated with a full dose of busulfan lymphodepletion before the reintroduction of the modified HSPCs in the mammal. In modalities, the patient is treated with a partial dose of busulfan lymphodepletion before the reintroduction of the modified HSPC in the mammal.
[0556] [0556] In modalities, cells are contacted with RNP comprising a Cas9 molecule, for example, as described herein, complexed with a gRNA for a non-deletion HPFH region, for example, as described herein (for example, comprising a target domain listed in Table 1).
[0557] [0557] In modalities, the stem cell expander is Compound 1. In modalities, the stem cell expander is Compound 2. In modalities, the stem cell expander is Compound 3. In modalities, the expander stem cells is (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-i1) -9H-purin-9-yl) propan - 1-o0l. In embodiments, the stem cell expander is (S) -2- (6- (2- (1H- indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin- 9-yl) propan-1-ol and is present at a concentration of 2-0.1 micromolar, for example, 1-0.25 micromolar, for example, 0.75-0.5 micromolar. In embodiments, the stem cell expander is a molecule described in WO2010 / 059401 (for example, the molecule described in Example 1 of WO2010 / 059401).
[0558] [0558] In embodiments, cells, for example, HSPCs, for example, as described herein, are grown ex vivo for a period of about 1 hour to about 15 days, for example, a period of about 12 hours at about 12 days, for example, a period of about 12 hours to 4 days, for example, a period of about 1 day to about 4 days, for example, a period of about 1 day to about 2 days , for example, a period of about 1 day or a period of about 2 days before the step of contacting the cells with a CRISPR system, for example, described herein. In embodiments, said culture, before said contact step is in a composition (for example, a cell culture medium), which comprises a stem cell expander, for example, described here, for example, (S) - 2- (6- (2- (11H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-i) propan-1-
[0559] [0559] In embodiments, the cell population comprising the modified HSPCs returned to the mammal comprises at least about 1 million cells (for example, at least about 1 million CD34 + cells) per kg. In embodiments, the cell population comprising the modified HSPCs returned to the mammal comprises at least about 2 million cells (for example, at least about 2 million CD34 + cells) per kg. In embodiments, the cell population comprising the modified HSPCs returned to the mammal comprises at least about 3 million cells (for example, at least about 3 million CD34 + cells) per kg. In embodiments, the cell population comprising the modified HSPCs returned to the mammal comprises at least about 4 million cells (for example, at least about 4 million CD34 + cells) per kg. In embodiments, the cell population comprising the modified HSPCs returned to the mammal comprises at least about 5 million cells (for example, at least about 5 million CD34 + cells) per kg. In embodiments, the cell population comprising the modified HSPCs returned to the mammal comprises at least about 6 million cells (for example, at least about 6 million CD34 + cells) per kg.
[0560] [0560] In modalities, it is the result of any of the methods described above the patient has at least 80% of his circulating CD34 + cells comprising an indelible at or near the genomic site complementary to the target domain of the gRNA molecule used in the method , for example, as measured, at least 15 days, for example, at least 20, at least 30, at least 40, at least 50 or at least 60 days after the reintroduction of cells in the mammal. Without being limited by theory, it was surprisingly found here that the indelible and indelible patterns (including large deletions) observed when gene editing systems, for example, CRISPR systems, for example, CRISPR systems comprising a gRNA molecule targeting the HBG1 region and / or HBG2, for example, as described here, are introduced into HSPCs, and these cells are transplanted into organisms, certain gRNAs produce cells comprising indels and indelible patterns (including large indels) that remain detectable in the edited cell population and their progeny, in the body, and persist for more than 8 weeks, 12 weeks, 16 weeks or 20 weeks. Without being bound by theory, a cell population comprising a particular indelible or indelible pattern (including large deletion) that persists within a detectable cell population, for example, more than 16 weeks or more than 20 weeks after introduction into a organism (for example, patient), could be beneficial for producing a long-term improvement of a disease or condition, for example, described here (for example, a hemoglobinopathy, for example, sickle cell disease or thalassemia) than cells ( or their offspring) who, after being introduced into an organism or patient, lose one or more indels (including large deletions). In embodiments, the persistent indel or indel pattern is associated with positively regulated fetal hemoglobin (for example, in erythroid offspring of said cells). Thus, in embodiments, the present disclosure provides populations of cells, for example, HSPCs, for example, as described herein, which comprise one or more indels (including large deletions) that persist (for example, remain detectable, for example, in a population of cells or their offspring) in the blood and / or bone marrow for more than 8 weeks, more than 12 weeks, more than 16 weeks or more than 20 weeks after introduction into an organism, for example, a patient.
[0561] [0561] In modalities, it is the result of any of the methods described above the patient has at least 20% of his bone marrow CD34 + cells comprising an indel in or near the genomic site complementary to the target domain of the used gRNA molecule in the method, for example, as measured, at least 15 days, for example, at least 20, at least 30, at least 40, at least 50 or at least 60 days after the reintroduction of cells in the mammal.
[0562] [0562] In modalities, HSPCs that are reintroduced into the mammal are able to differentiate in vivo into cells of the erythroid lineage, for example, red blood cells, and said differentiated cells exhibit increased levels of fetal hemoglobin, for example, produce at least 6 picograms of fetal hemoglobin per cell, for example, at least 7 picograms of fetal hemoglobin per cell, at least 8 picograms of fetal hemoglobin per cell, at least 9 picograms of fetal hemoglobin per cell, at least 10 picograms of fetal hemoglobin per cell, for example, between about 9 and about 10 picograms of fetal hemoglobin per cell, for example, such that hemoglobinopathy is treated in the mammal.
[0563] [0563] It will be understood that when a cell is characterized as having increased fetal hemoglobin, this includes modalities in which a progeny, for example, a differentiated progeny, from that cell exhibits increased fetal hemoglobin. For example, in the methods described here, the altered or modified CD34 + cell (or cell population) may not express increased fetal hemoglobin, but when differentiated into cells of the erythroid lineage, for example, red blood cells, the cells express increased fetal hemoglobin, for example , fetal hemoglobin increased over an unmodified or unchanged cell under similar conditions. XII. Culture Methods and Cell Production Methods
[0564] [0564] The description provides cell culture methods, for example, HSPCs, for example, hematopoietic stem cells, for example, CD34 + cells modified, or to be modified, with the gRNA molecules described herein. DNA repair pathway inhibitors
[0565] [0565] Without being limited by theory, it is believed that the pattern of indels produced by a given gRNA molecule in a particular target sequence is a product of each of the active DNA repair mechanisms within the cell (for example, non-homologous end joint, microhomology-mediated end joint, etc.). Without being limited by theory, it is believed that a particularly favorable indel can be selected for or enriched by placing the cells to be edited in contact with an inhibitor of a DNA repair pathway that does not produce the desired indel. Thus, gRNA molecules, CRISPR systems, methods and other aspects of the invention can be performed in combination with such inhibitors. Examples of such inhibitors include those described in, for example, WOZ2014 / 130955, the content of which is incorporated herein by reference in its entirety. In embodiment, the inhibitor is a DNAPKc inhibitor, for example, NU7441. Stem Cell Expander
[0566] [0566] In one aspect, the invention relates to cultivating cells, for example, HSPCs, for example, CD34 + cells modified, or to be modified, with the gRNA molecules described herein, with one or more agents that result in an increased rate of expansion, increased level of expansion, or increased graft compared to cells not treated with the agent. Such agents are referred to herein as stem cell expanders.
[0567] [0567] In one aspect, the one or more agents that result in an increased rate of expansion or an increased level of expansion, relative to cells not treated with the agent, for example, the stem cell expander, comprises an agent which is an inhibitor of the aryl hydrocarbon receptor (AHR) pathway. In aspects, the stem cell expander is a compound described in WOZ2013 / 110198 or a compound described in WO2010 / 059401, the contents of which are incorporated by reference in their entirety.
[0568] [0568] In one aspect, the one or more agents that results in an increased rate of expansion or increased level of expansion, relative to cells not treated with the agent, is a derivative of indole pyrimido [4,5-b] , for example, as disclosed in WO 2013/110198, the content of which is incorporated herein by reference in its entirety. In one embodiment, the agent is compound 1 ((1r, 4r) - N- (2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b] indol-4-yl) cyclohexane-1,4-diamine): Compound 1 / = - ty Cs - == Nn
[0569] [0569] In another aspect, the agent is Compound 2 (methyl 4- (3-piperidin-1-ylpropylamine) -9H-pyrimido [4,5-b] indole-7-carboxylate): Compound 2: THE
[0570] [0570] In another aspect, the one or more agents that result in an increase in the rate of expansion or increase in the level of expansion, relative to cells not treated with the agent, is an agent disclosed in WOZ2010 / 059401, whose content is incorporated herein by reference in its entirety.
[0571] [0571] In one embodiment, the stem cell expander is compound 3: 4- (2- (2- (benzo [b] thiophene-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, that is, the compound is from example 1 of WO 2010/059401, with the following structure: Compound 3:
[0572] [0572] In another aspect, the stem cell expander is (S) -2- (6- (2- (11H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) - 9H-purin-9-yl) propan- 1-ol ((8) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-i1) -9H- purin-9-yl) propan-1-ol, i.e. it is compound 157S according to WO 2010/059401), having the following structure: (S8) -2- (6- (2- ( 1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol:
[0573] [0573] In modalities, the population of HSPCs is placed in contact with the stem cell expander, for example, compound 1, compound 2, compound 3, (S) -2- (6- (2- (11H-indole -3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-0l, or combinations thereof (for example, a combination of compound 1 and (S) -2- (6- (2-11H-indol-3- i) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-01) before the introduction of CRISPR system (e.g., gRNA molecule and / or Cas9 molecule of the invention) in said HSPCs. In modalities, the population of HSPCs is placed in contact with the stem cell expander, for example, compound 1, compound 2, compound 3, (S) - 2- (6- (2- (1H-indole-3- il) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, or combinations thereof (for example, a combination of compound 1 and (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -
[0574] [0574] In modalities, the stem cell expander is present in an amount effective to increase the level of expansion of HSPCs, compared to HSPCs in the same medium, but for the absence of the stem cell expander. In embodiments, the stem cell expander is present in a concentration ranging from about 0.01 to about 10 µM, for example, from about 0.1 µM to about 1 µM. In embodiments, the stem cell expander is present in the cell culture medium at a concentration of about 1 µM, about 950 nM, about 900 nM, about 850 nM, about 800 nM, about 750 nM , about 700 nM, about 650 nM, about 600 nM, about 550 nM, about 500 nM, about 450 nM, about 400 nM, about 400 nM, about 350 nM, about 300 nM, about 250 nM , about 200 nM, about 150 nM, about 100 nM, about 50 nM, about 25 nM, or about nM. In embodiments, the stem cell expander is present in a concentration ranging from about 500 nM to about 750 nM.
[0575] [0575] In embodiments, the stem cell expander is (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H -purin-9-yl) propan-1-ol, which is present in the cell culture medium in a concentration ranging from about 0.01 to about 10 micromolar (uM). In embodiments, the stem cell expander is (S) -2- (6- (2- (1H-
[0576] [0576] In embodiments, the stem cell expander is a mixture of compound 1 and (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridine- 3-i1) -9H-purin-9-yl) propan-1-ol.
[0577] [0577] In embodiments, the cells of the invention are placed in contact with one or more stem cell expander molecules for a sufficient time and in an amount sufficient to cause a 2 to 10,000 fold expansion of CD34 + cells, for example, a 2-1000 fold expansion of CD34 + cells, for example, a 2-100 fold expansion of CD34 + cells, for example, a 20-200 fold expansion of CD34 + cells. As described here, contact with one or more stem cell expanders can be before the cells are contacted with a CRISPR system, for example, as described here, after the cells are contacted with a CRISPR system, for example, as described here described, or combination of these. In one embodiment, cells are contacted with one or more stem cell expander molecules, for example, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5 -fluoropyridin-3-i1) -9H- purin-9-yl) propan-1-ol, for a sufficient time and in an amount sufficient to cause at least a 2-fold expansion of CD34 + cells, for example, cells CD34 + comprising an indel at or near the target site having complementarity with the CRISPR / Cas9 system gRNA target domain introduced into said cell.
[0578] [0578] In embodiments, cells, for example, HSPCs, for example, as described herein, are cultured ex vivo for a period of about 1 hour to about 10 days, for example, a period of about 12 hours at about 5 days, for example, a period of about 12 hours to 4 days, for example, a period of about 1 day to about 4 days, for example, a period of about 1 day to about 2 days , for example, a period of about 1 day or a period of about 2 days before the step of putting the cells in contact with a CRISPR system, for example, described here. In embodiments, said culture, before said contact step is in a composition (for example, a cell culture medium), which comprises a stem cell expander, for example, described here, for example, (S) - 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H- purin-9-yl) propan-1-ol, for example, ( S) -2- (6- (2- (1H-indol-3-yl) ethylamino) - 2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-0l at a concentration from about 0.25 µM to about 1 µM, for example, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3- il) - 9H-purin-9-yl) propan-1-0l at a concentration of about 0.75-0.5 micromolar.
[0579] [0579] In embodiments, the cell culture medium is a chemically defined medium. In embodiments, the cell culture medium may additionally contain, for example, StemSpan SFEM (StemCell Technologies; Cat no. 09650). In embodiments, the cell culture medium may, alternatively or additionally, contain, for example, fermented HSC, GMP (Miltenyi). In embodiments, the cell culture medium is serum-free. In modalities, the medium can be supplemented with thrombopoietin (TPO), human FIt3 ligand (FlIt-3L), human stem cell factor (SCF), human interleukin-6, L-glutamine and / or penicillin / streptomycin. In modalities, the medium is supplemented with thrombopoietin (TPO), human FIt3 ligand (FIlt-3L), human stem cell factor (SCF), human interleukin-6 and L-glutamine. In other modalities, the medium is supplemented with thrombopoietin (TPO), human FIt3 ligand (FIt-3L), human stem cell factor (SCF) and human interleukin-6. In other modalities, the medium is supplemented with thrombopoietin (TPO), human FIt3 ligand (FIt-3L) and human stem cell factor (SCF), but not human interleukin-6. In other embodiments, the medium is supplemented with human FIt3 ligand (FIt-3L), human stem cell factor (SCF), but not human thrombopoietin (TPO) or human interleukin-6. When present in the medium, thrombopoietin (TPO), human FIt3 ligand (FIt-3L), human stem cell factor (SCF), human interleukin-6 and / or L-glutamine are each present in a concentration ranging from about 1 ng / mL to about 1000 ng / mL, for example, a concentration ranging from about 10 ng / mL to about
[0580] [0580] The present disclosure contemplates the use of the gRNA molecules described herein, or cells (for example, hematopoietic stem cells, for example, CD34 + cells) modified with the gRNA molecules described herein, in combination with one or more other modalities therapies and / or agent agents. Thus, in addition to the use of gRNA molecules or cells modified with the gRNA molecules described here, one or more "standard" therapies for the treatment of hemoglobinopathies can also be administered to the subject.
[0581] [0581] The one or more additional therapies for the treatment of hemoglobinopathies may include, for example, additional stem cell transplantation, for example, hematopoietic stem cell transplantation. Stem cell transplantation can be allogeneic or autologous.
[0582] [0582] The one or more additional therapies for the treatment of hemoglobinopathies may include, for example, blood transfusion and / or iron chelation therapy (for example, removal). Known iron chelating agents include, for example, deferoxamine and deferasirox.
[0583] [0583] The one or more additional therapies for the treatment of hemoglobinopathies may include, for example, folic acid or hydroxyurea supplements (eg, 5-hydroxyurea). The one or more additional therapies for treating hemoglobinopathies can be hydroxyurea. In embodiments, hydroxyurea can be administered at a dose of, for example, 10-35 mg / kg per day, for example, 10-20 mg / kg per day. In modalities, hydroxyurea is administered at a dose of 10 mg / kg per day. In modalities, hydroxyurea is administered at a dose of 10 mg / kg per day. In modalities, hydroxyurea is administered at a dose of 20 mg / kg per day. In embodiments, hydroxyurea is administered before and / or after the cell (or population of cells), for example, CD34 + cells (or population of cells) of the invention, for example, as described herein.
[0584] [0584] The one or more additional therapeutic agents may include,
[0585] [0585] The one or more additional agents may include, for example, a small molecule that positively regulates fetal hemoglobin. Examples of such molecules include TN1 (for example, as described in Nam, T. et al., ChemMedChem 2011, 6, 777-7180, DOI: 10.1002 / cmdc.201000505, incorporated herein by reference).
[0586] [0586] One or more additional therapies may also include irradiation or other bone marrow ablation therapy known in the art. An example of such a therapy is busulfan. Such additional therapy can be performed prior to introducing the cells of the invention into the subject. In one embodiment, the treatment methods described herein (for example, treatment methods that include the administration of cells (for example, HSPCs) modified by the methods described herein (for example, modified with a CRISPR system described herein, for example, to increase HbF production)), the method does not include the bone marrow ablation step. In modalities, the methods include a stage of partial bone marrow ablation.
[0587] [0587] The therapies described herein (for example, comprising administering a population of HSPCs, for example, HSPCs modified using a CRISPR system described herein) can also be combined with an additional therapeutic agent. In one embodiment, the additional therapeutic agent is an HDAC inhibitor, for example, panobinostat. In one embodiment, the additional therapeutic agent is a compound described in PCT Publication No. WO '2014/150256, for example, a compound described in Table 1 of WO2014 / 150256, for example, GBT440. Other examples of HDAC inhibitors include, for example, hydroxamic suberoylanilide acid (SAHA). The one or more additional agents can include, for example, a DNA methylation inhibitor. Such agents have been shown to increase HbF induction in cells with reduced BCL11a activity (eg, Jian Xu et al., Science 334, 993 (2011); DO !:
[0588] [0588] The gRNA molecules described herein, or cells (e.g., hematopoietic stem cells, e.g., CD34 + cells) modified with the gRNA molecules described herein, and the co-therapeutic or co-therapy may be administered in the same formulation or separately. In the case of separate administration, the gRNA molecules described herein, or cells modified with the gRNA molecules described herein, can be administered before, after or concurrently with co-therapy or co-therapy. An agent can precede or follow the administration of the other agent at intervals ranging from minutes to weeks. In modalities in which two or more different types of therapeutic agents are applied separately to a subject, it can generally be ensured that a significant period of time has not expired between the time of each delivery, in such a way that these different types of agents would still be capable of exerting a beneficially combined effect on target tissues or cells. XIII. Nucleosides, Nucleotides and Modified Nucleic Acids
[0589] [0589] The modified nucleosides and modified nucleotides can be present in nucleic acids, for example, particularly gRNA, but also other forms of RNA, for example, mRNA, RNAi or siRNA. As described herein, "nucleoside" is defined as a compound containing a sugar molecule with five carbon atoms (a pentose or ribose) or its derivative, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, "nucleotide" is defined as a nucleoside further comprising a phosphate group.
[0590] [0590] Modified nucleosides and nucleotides may include one or more of: (i) alteration, for example, replacement of one or both of the non-binding phosphate oxygen and / or one or more of the phosphate-binding oxygen in the skeleton bond phosphodiester; (ii) altering, for example, substituting, a ribose sugar constituent, for example, 2 'hydroxyl in ribose sugar; (ili) total replacement of the phosphate fraction with "defosfo" ligands; (iv) altering or replacing a naturally occurring nucleobase, including with a non-canonical nucleobase; (v) replacement or modification of the ribose-phosphate backbone; (vi) modification of the 3 or 5 end of the oligonucleotide, for example, removal, replacement or modification of a terminal phosphate group or conjugation of a fraction, cap or linker; and (vii) sugar modification or replacement.
[0591] [0591] The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In one embodiment, each base of a gRNA is modified, for example, all bases have a modified phosphate group, for example, all are phosphorothioate groups. In one embodiment, all or substantially all of the phosphate groups on a unimolecular or modular gRNA molecule are replaced with phosphorothioate groups. In embodiments, one or more of the five 3'-terminal bases and / or one or more of the five 5'-terminal bases of the gRNA are modified with a phosphorothioate group.
[0592] [0592] In one embodiment, the modified nucleotides, for example, nucleotides with modifications as described herein, can be incorporated into a nucleic acid, for example, a "modified nucleic acid". In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30% at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65 %, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotide.
[0593] [0593] Unmodified nucleic acids may be prone to degradation by, for example, cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Therefore, in one aspect, the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, for example, to introduce stability with respect to the nucleases.
[0594] [0594] In some embodiments, the modified nucleosides, modified nucleotides and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a cell population, both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single-stranded nucleic acids, usually of viral or bacterial origin, which involves inducing the expression and release of cytokines, particularly interferons and cell death. In some embodiments, the modified nucleosides, modified nucleotides and modified nucleic acids described herein can disrupt the binding of a partner that interacts with the main groove with the nucleic acid. In some embodiments, the modified nucleosides, modified nucleotides and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a cell population, both in vivo and ex vivo, and also disrupt the binding of a partner that interacts with the groove main with nucleic acid. Definitions of Chemical Groups
[0595] [0595] As used herein, "alkyl" is intended to refer to a saturated hydrocarbon group that is straight or branched. Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (eg, n-propyl and isopropyl), butyl (eg, n-butyl, isobutyl, t-butyl), pentyl (eg, n- pentila, isopentila, neopentila) and the like. An alkyl group may contain from 1 to about 20, from 2a to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about of 3 carbon atoms.
[0596] [0596] As used herein, "aryl" refers to monocyclic or polycyclic aromatic hydrocarbons (for example, having 2, 3 or 4 fused rings) such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl and similar. In some embodiments, aryl groups have 6 to about 20 carbon atoms.
[0597] [0597] As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized by having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl and 3-hexynyl.
[0598] [0598] As used herein, "arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl and trityl groups.
[0599] [0599] As used herein, "cycloalkyl" refers to non-aromatic cyclic, bicyclic, tricyclic or polycyclic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl fractions include, but are not limited to, cyclopropyl, cyclopentyl and cyclohexyl.
[0600] [0600] As used herein, "heterocyclyl" refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclics include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl and morpholinyl.
[0601] [0601] As used herein, "heteroaryl" refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl fractions include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenylpyrazolyl, pyridinyl,
[0602] [0602] In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygen with a different substituent. In addition, the modified nucleotide, for example, modified nucleotide present in a modified nucleic acid, may include the total replacement of an unmodified phosphate fraction with a modified phosphate as described herein. In some embodiments, modification of the phosphate backbone may include changes that result in an unloaded binder or a charged binder with non-symmetrical charge distribution.
[0603] [0603] Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borane phosphates, borane phosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging oxygen atoms in the phosphate backbone fraction can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (where R can be, for example, hydrogen , alkyl or aryl) ,, C (for example, an alkyl group, an aryl group and the like), H, NR2 (where R can be, for example, hydrogen, alkyl or aryl), or OR (where R can be, for example, alkyl or aryl). The phosphoric atom in an unmodified phosphate group is achiral. However, replacing one of the non-binding oxygen with one of the atoms or groups of atoms above can make the phosphorus atom chiral; that is, a phosphoric atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphoric atom can have the "R" configuration (here Rp) or the "S" configuration (here Sp).
[0604] [0604] Phosphorodithioates have both non-binding oxygen substituted by sulfur. The phosphorus center in the phosphorodithioates is aquira, which prevents the formation of oligoribonucleotide diastereoisomers. In some embodiments, modifications to one or both of the non-bridge oxygen may also include the replacement of the non-bridge oxygen with a group independently selected from S, Se, B, C, H, N, and OR (R can be , for example, alkyl or aryl).
[0605] [0605] The phosphate ligand can also be modified by substituting a bridged oxygen, (ie, the oxygen that binds the phosphate to the nucleoside), with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates) and carbon (methylene phosphonates) bridged). The substitution can occur either in the binding oxygen or in both the binding oxygen. Replacement of the Phosphate Group
[0606] [0606] The phosphate group can be replaced by connectors containing no phosphorus. In some embodiments, the charge phosphate group can be replaced by a neutral fraction.
[0607] [0607] Examples of fractions that can replace the phosphate group may include, without limitation, for example, methyl phosphonate, hydroxylamine, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide binder, sulfonate, sulfonamide, thiophormacetal , formacetal, oxime, methyleneimine, methylenomethylimine, methylenehydrozo, methylenedimethylhydrozo and methyleneoxymethylimine. Replacement of the Ribophosphate Skeleton
[0608] [0608] Molds that can mimic nucleic acids can also be constructed in which the phosphate linker and ribose sugar are replaced with nucleoside-resistant nucleotide or nucleotide substitutes. In some embodiments, the nucleobases can be attached by a substitute skeleton. Examples can include, without limitation, nucleoside substitutes morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA).
[0609] [0609] The modified nucleosides and modified nucleotides can include one or more modifications in the sugar group. For example, the 2 'hydroxyl group (OH) can be modified or substituted by several different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2 'hydroxyl group can increase the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the binding phosphorus atom.
[0610] [0610] Examples of modifications of the "oxy" -2 'hydroxyl group may include alkoxy or aryloxy (OR, where "R" can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethylene glycols (PEG), 0 (CH2CH20) / CH2CH2O0R where R can be, for example, H or optionally substituted alkyl, and n can be an integer from 0 to 20 (for example, from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4d to 2 to 8, from 2 to 10 from 2 to 16, from 2 to 20, 4 to 8, ded4 to 10, from 4 to 1, 6, 4, 20 ). In some embodiments, modification of the "oxy" -2 'hydroxyl group may include "blocked" nucleic acids (LNA) in which the 2' hydroxyl can be linked, for example, by a C 1 -6 alkylene or C 1 -6 heteroalkylene bridge , to the 4 'carbon of the same ribose sugar, wherein exemplary bridges may include methylene, propylene, ether or amine bridges; O-amine (where amine can be, for example, NH;> z; alkylamine, - dialguylamine, - heterocyclic, arylamine, diarylamine, heteroarylamine or diheteroarylamine, ethylenediamine or polyamine) and aminoalkoxy, O (CH2)) - amine , (where amine can be, for example, NH> z, alkylamine, dialkylamine, heterocyclyl, arylamine, diarylamine,
[0611] [0611] "Deoxy" modifications may include hydrogen (ie, deoxyribose sugars, for example, in the overhang portions of partially ds RNA); halo (for example, bromine, chlorine, fluorine or iodine); amine (where amine can be, for example, NH>; alkylamine, dialkylamine, heterocyclyl, arylamine, diarylamine, heteroarylamine, di-heteroarylamine or amino acid); amine NH (CH2CHaNH) .CH2CH2- (where amine can be, for example, as described herein) NHC (O) R (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyan; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which can be optionally substituted with, for example, an amine as described herein.
[0612] [0612] The sugar group can also contain one or more carbons that have the stereochemical configuration opposite to the corresponding carbon in the ribose. Thus, a modified nucleic acid can include nucleotides containing, for example, arabinose, such as sugar. The nucleotide "monomer" may have an alpha bond at the | in sugar, for example, alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which do not have a C- nucleobase. These abasic sugars can also be further modified to one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, for example, L-nucleosides.
[0613] [0613] RNA usually includes the ribose sugar group, which is a 5-membered ring with an oxygen. The modified nucleosides and exemplary modified nucleotides may include, without limitation, the substitution of oxygen in the ribose (for example, with sulfur (S), selenium (Se) or alkylene, such as, for example, methylene or ethylene); adding a double bond (for example, to replace ribose with cyclopentenyl or cyclohexenyl); contraction of the ribose ring (for example, to form a 4-membered ring of cyclobutane or oxetane); expansion of the ribose ring (for example, to form a 6 or 7-membered ring having an additional carbon or heteroatom, such as, for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl and morpholino which also has phosphoramidate skeleton). In some embodiments, the modified nucleotides may include multicyclic forms (for example, tricycles; and "unlocked" forms, such as nucleic acid glycol (GNA) (for example, R-GNA or S-GNA, where ribose is replaced by units glycol linked to phosphodiester bonds), nucleic acid threose (TNA, where ribose is replaced by aL-treofuranosyl- (3 '-> 2')). Modifications in Nucleobase
[0614] [0614] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C) and uracil (U). These nucleobases can be modified or completely replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, pyrimidine, purine or pyrimidine analogue. In some embodiments, the nucleobase may include, for example, naturally occurring synthetic derivatives of a base. Uracil
[0615] [0615] In some embodiments, the modified nucleobase is a modified uracil.
[0616] [0616] In some embodiments, the modified nucleobase is a modified cytosine. Examples of nucleobases and nucleosides having a modified cytosine include, without limitation, 5-aza-cytidine, 6-aza-cytidine, pseudo-isocytidine, 3-methyl-cytidine (mMºC), N4-acetyl-cytidine (act), 5- formyl-cytidine (fºC), N4-methyl-cytidine (mºC), 5-methyl-cytidine (mºC), - 5-halo-cytidine (for example, - 5-iodo-cytidine), 5-hydroxymethyl-cytidine ( hm5C), 1-methyl-pseudo-isocitidine, pyrrolo-cytidine, pyrrole-pseudo-isocitidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudo-isocitidine, - 4 -tio-1-methyl-pseudo-isocitidine, - 4-thio-1-methyl-1- deaza- - pseudo-isocytidine, 1-methyl- 1-deaza-pseudo-isocitidine, zebularine, —5-aza-zebularine, —5-methyl-zebularine, 5-aza-2-thiozebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudo-isocytidine, 4-methoxy -1-methyl-pseudo-isocytidine, lysidine (KkPC), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (mºCm), N4-acetyl- 2-0- methyl-cytidine (acºCm), N4,2'-0-dimethyl-cytidine (mºCm), 5-for mil-2'-O0-methyl-cytidine (f SCm), N4, N4,2'-O0-trimethyl-cytidine (mé2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F -citidine and 2-0H- ara-cytidine. Adenine
[0617] [0617] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides with a modified adenine include without limitation 2-amine-purine, 2,6-diaminapurine, 2-amine-6-halo-purine (eg, 2-amine-6-chloro-purine), 6-halo -purine (for example, 6-chloro-purine), 2-amine-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza- 8-aza-adenine, 7-deaza-2 -amine-purine, 7-deaza-8-aza-2-amine-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (mM 'A), 2-methyl-adenine (m A), N6-methyl-adenosine (mêA), 2-methylthio-N6-methyl-adenosine (ms2mºA), N6-isopentenyl-adenosine (iºA), 2-methylthio- N6 -isopentenyl-adenosine (ms iºA), N6- (cis-hydroxy-isopentenyl) adenosine (ioºA), 2-methylthio-N6- (cis-hydroxy-isopentenyl) adenosine (ms2i0ºA), N6-glycinylcarbamoyl-adenosine (gºA) , N6-threonylcarbamoyl-adenosine (t6A),) N6-methyl-N6-threonylcarbamoyl-adenosine (mºtºA), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms GºA), N6, N6-dimethyl-adenosine (MÔ2A), N6-hydroxinorvalylcarbamoyl-adenosine (hnºA), 2- methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hnºA), N6-acetyl-adenosine (acºA), 7-methyl-adenine, 2-methylthioadenine, 2-methoxy-adenine, a-thio-adenosine, 2'-0-methyl -adenosine (Am), No., 2'-0-dimethyl-adenosine (MºAm), No.-Methyl-2 "-deoxyadenosine, N6, N6,2'-0-trimethyl-adenosine (mMº2Am), 1, 2-0 -dimethyl-adenosine (m 'Am), 2'-0-ribosyladenosine (phosphate) (Ar (p)), 2-amine-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2' -F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine and N6- (19-amine-pentaoxanonadecyl) -adenosine. Guanine
[0618] [0618] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with a modified guanine include, without limitation, inosine (1), 1-methyl-inosine (m '!), Wiosin (IMG), methylwiosin (mimG), 4-demethyl-wiosin (iMG-14), isowiosin (iMG2), wibutosine (yW), peroxiwibutosine (o02yW), hydroxywibutosine (OHyW), sub-modified hydroxywibutosine (OHyW "*), 7-deaza-guanosine, queuosine (Q), epoxy-quosin (0Q), galactosyl
[0619] [0619] In some embodiments, the modified nucleic acids can be modified gRNAs. In some embodiments, the gRNAs can be modified at the 3 'end. In this embodiment, the gRNAs can be modified at the 3 'terminal U ribose. For example, the two terminal hydroxyl groups of ribose U can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to provide a modified nucleoside, where U can be an unmodified or modified uridine.
[0620] [0620] In another embodiment, the 3 'U-terminal can be modified with a 2' 3 cyclic phosphate, where U can be an unmodified or modified uridine. In some embodiments, the gRNA molecules may contain 3 'nucleotides that can be stabilized against degradation, for example, by incorporating one or more of the modified nucleotides described herein. In this embodiment, for example, uridines can be replaced with modified uridines, for example, 5- (2-amino) propyl-uridine and 5-bromo-uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced by modified adenosines and guanosines, for example, with changes in position 8, for example, 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, the deaza nucleotides, for example, 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, the O- and N-alkylated nucleotides, for example, N6-methyl adenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, for example, in which the OH-2º group is replaced by a group selected from H, -OR, -R (where R can be, for example, methyl, alkyl , cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amine (where amine can be, for example NH> z; alkylamine, dialkylamine, heterocyclyl, arylamine, —diarylamine, heteroarylamine, di-heteroarylamine or amino acid); or cyan (-CN). In some embodiments, the phosphate backbone can be modified as described herein, for example, with a phosphotioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each be independently a modified or unmodified nucleotide, including, but not limited to, modified 2'-sugar, such as 2-F 2'-0-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo) and any combinations thereof.
[0621] [0621] In one embodiment, one or more or all of the single chain overhang nucleotides of an RNA molecule, for example,
[0622] [0622] The pharmaceutical compositions of the present invention may comprise a gRNA molecule described herein, for example, a plurality of gRNA molecules, as described herein, or a cell (e.g., a population of cells, e.g., a population hematopoietic stem cells, e.g. CD34 + cells) comprising one or more cells modified with one or more gRNA molecules described herein, in combination with one or more pharmaceutically or physiologically acceptable vehicles, diluents or excipients. Such compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates, such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives. The compositions of the present invention are in one aspect formulated for intravenous administration.
[0623] [0623] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's illness, although appropriate dosages can be determined by clinical trials.
[0624] [0624] In one embodiment, the pharmaceutical composition is substantially free of, for example, there are no detectable levels of a contaminant, for example, selected from the group consisting of endotoxin, mycoplasma, mouse antibodies, pooled human serum, bovine serum albumin , bovine serum, components of the culture medium, unwanted components of the system
[0625] [0625] The administration of the present compositions can be carried out in any convenient way, including by aerosol inhalation, injection, ingestion, transfusion, implant or transplant. The compositions described herein can be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullarily, intramuscularly, by intravenous (i.v.) or intraperitoneal injection. In one aspect, the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the cellular compositions of the present invention are administered by injection | V.
[0626] [0626] The dosage of the above treatments to be administered to a patient will vary with the exact nature of the condition being treated and with the recipient of the treatment. Scaling of dosages for human administration can be carried out according to practices accepted in the art. Cells
[0627] [0627] The invention also relates to cells comprising a gRNA molecule of the invention, or nucleic acid that encodes said gRNA molecules.
[0628] [0628] In one aspect, cells are cells made by a process described here.
[0629] [0629] In modalities, the cells are hematopoietic stem cells (for example, hematopoietic and progenitor stem cells; HSPCs), for example, CD34 + stem cells. In modalities, the cells are CD34 + / CD90 + stem cells. In modalities, the cells are CD34 + / CD90- stem cells. In modalities, the cells are human hematopoietic stem cells. In modalities, the cells are autologous. In modalities, the cells are allogeneic.
[0630] [0630] In modalities, cells are derived from bone marrow, for example, autologous bone marrow. In modalities, the cells are derived from peripheral blood, for example, mobilized peripheral blood, for example, autologous mobilized peripheral blood. In modalities that use mobilized peripheral blood, cells are isolated from patients who have been administered with a mobilizing agent. In modalities, the mobilization agent is the G-CSF. In modalities, the mobilization agent is Plerixafor & (AMD3100). In modalities, the mobilizing agent comprises a combination of G-CSF and Plerixafor & (AMD3100). In modalities, cells are derived from umbilical cord blood, for example, allogeneic umbilical cord blood. In modalities, the cells are derived from a patient with hemoglobinopathy, for example, a patient with sickle cell disease or a patient with thalassemia, for example, beta thalassemia.
[0631] [0631] In modalities, the cells are from mammals. In modalities, the cells are human. In modalities, the cells are derived from a patient with hemoglobinopathy, for example, a patient with sickle cell disease or a patient with thalassemia, for example, beta thalassemia.
[0632] [0632] In one aspect, the invention provides a cell comprising a modification or alteration, for example, an indelible at or near (for example, within 20, 19, 18, 17, 16, 15, 14, 13, 12 , 11,10,9,8,7,6,5,4,3,2 or 1 nucleotides of) a nucleic acid sequence having complementarity with a gRNA molecule or gRrRNA molecules, for example, as described herein, introduced in said cells, for example, as part of a CRISPR system as described herein.
[0633] [0633] In one aspect, the invention provides a population of cells comprising cells having a modification or alteration, for example, an indel at or close to (for example, within 20, 19, 18, 17, 16, 15, 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2U1 nucleotides of) a nucleic acid sequence having complementarity with a gRNA molecule or gRNA molecules, for example, as herein described, introduced into said cells, for example, as part of a CRISPR system as described here. In modalities, at least 50%, for example, at least 60%, at least 70%, at least 80% or at least 90% of the population cells have the modification or alteration (for example, have at least one modification or alteration ), for example, as measured by NGS, for example, as described herein, for example, on day two after the introduction of the gRNA and / or CRISPR system of the invention. In modalities, at least 90%, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the population cells have the modification or alteration (for example, they have at least one modification or alteration), for example, as measured by NGS, for example, as described here, for example, on day two after introduction of gRNA and / or CRISPR system of the invention.
[0634] [0634] In embodiments, the cell population, for example, as described herein, comprises at least about 1 and 3 cells. In embodiments, the cell population, for example, as described herein, comprises at least about 1 and 4 cells.
[0635] [0635] In embodiments, the cell population, for example, as described herein, comprises at least about 1 and 6 HSPCs, for example, CD34 + cells, per kilogram of patient body weight to which they are to be administered.
[0636] [0636] In modalities, the cell population, for example, as described herein, comprises about 1 and 6 HSPCs, for example, CD34 + cells, per kilogram of patient body weight to which they are to be administered. In embodiments, the cell population, for example, as described herein, comprises about
[0637] [0637] In modalities, the cell population, for example, as described here, comprises from about 2e6 to about 10e6 HSPCs, for example, CD34 + cells, per kilogram of the patient's body weight to which they must be administered. In embodiments, the cell population, for example, as described herein, comprises from 2e6 to 10e6 HSPCs, for example, CD34 + cells, per kilogram of patient body weight to which they are to be administered.
[0638] [0638] The cells of the invention can comprise a gRNA molecule of the present invention, or nucleic acid encoding said gRNA molecule and a Cas9 molecule of the present invention, or nucleic acid encoding said Cas9 molecule. In one embodiment, the cells of the invention may comprise a ribonuclear protein (RNP) complex comprising a gRNA molecule of the invention and a Cas9 molecule of the invention.
[0639] [0639] The cells of the invention are preferably modified to comprise a gRNA molecule of the invention ex vivo, for example, by a method described herein, for example, by electroporation or by TRIAMF (as described in PCT / patent application US2017 / 54110, incorporated herein by reference in its entirety).
[0640] [0640] The cells of the invention include cells in which the expression of one or more genes has been altered, for example, reduced or inhibited, by introducing a CRISPR system comprising a gRNA of the invention. For example, the cells of the present invention may have a reduced level of beta globin expression (e.g., hemoglobin beta comprising a sickle cell mutation) relative to unmodified cells. As another example, the cells of the present invention may have an increased level of fetal hemoglobin expression compared to unmodified cells. Alternatively, or in addition, a cell of the invention can give rise, for example, to differentiate into another cell type, for example, an erythrocyte, which has an increased level of fetal hemoglobin expression relative to differentiated cells from unmodified cells. In embodiments, the increase in the level of fetal hemoglobin is at least about 20%, at least about 30%, at least about 40% or at least about 50%. Alternatively, or in addition, a cell of the invention can give rise, for example, to differentiate into another cell type, for example, an erythrocyte, which has a reduced level of beta globin expression (for example, beta hemoglobin comprising a mutation sickle cell, also referred to here as beta sickle globin) in relation to differentiated cells from unmodified cells. In embodiments, the decrease in the level of beta sickle globin is at least about 20%, at least about 30%, at least about 40% or at least about 50%.
[0641] [0641] The cells of the invention include cells in which the expression of one or more genes has been altered, for example, reduced or inhibited, by introducing a CRISPR system comprising a gRNA of the invention. For example, cells of the present invention may have a reduced level of beta hemoglobin expression, for example a mutated or wild-type hemoglobin beta, compared to unmodified cells. In another aspect, the invention provides cells that are derived from, for example, differentiated from cells into which a CRISPR system comprising a gRNA of the invention has been introduced. In such aspects, cells in which the CRISPR system comprising the gRNA of the invention has been introduced may not exhibit the reduced level of beta hemoglobin, for example, a mutated or wild-type beta hemoglobin, but cells derived from, for example, differentiated of said cells exhibit a reduced level of beta hemoglobin, for example, a wild-type or mutated beta hemoglobin. In modalities, the derivation, for example, differentiation, is performed in vivo (for example, in a patient, for example, in a patient with hemoglobinopathy, for example, in a patient with sickle cell disease or thalassemia, for example, thalassemia beta) . In embodiments, the cells in which the CRISPR system comprising the gRNA of the invention was introduced are CD34 + cells and the derived cells, for example, differentiated, from there are of the erythroid lineage, for example, red blood cells.
[0642] [0642] The cells of the invention include cells in which the expression of one or more genes has been altered, for example, increased or promoted, by the introduction of a CRISPR system comprising a gRNA of the invention. For example, the cells of the present invention may have an increased level of fetal hemoglobin expression compared to unmodified cells. In another aspect, the invention provides cells that are derived from, for example, differentiated from cells into which a CRISPR system comprising a gRNA of the invention has been introduced. In these respects, cells into which the CRISPR system comprising the gRNA of the invention has been introduced may not exhibit the increased level of fetal hemoglobin, but cells derived from, for example, differentiated from said cells exhibit the increased level of fetal hemoglobin. In modalities, the derivation, for example, differentiation, is performed in vivo (for example, in a patient, for example, in a patient with hemoglobinopathy, for example, in a patient with sickle cell disease or thalassemia, for example, thalassemia beta) . In embodiments, the cells in which the CRISPR system comprising the gRNA of the invention was introduced are CD34 + cells and the derived cells, for example, differentiated, from there are of the erythroid lineage, for example, red blood cells.
[0643] [0643] In another aspect, the invention relates to cells that include an indel in (for example, in) or close to (for example, within 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6, 5, 4,3, 2 or 1 nucleotides of) a nucleic acid sequence having complementarity with the gRNA molecule (for example, the target sequence of the gRNA molecule ) or gRNA molecules introduced into said cells. In modalities, the indel is an indel of displacement of the reading frame. In embodiments, the cell includes a large deletion, for example, a deletion of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb or more. In embodiments, the large deletion comprises nucleic acids arranged between two binding sites for the gRNA molecule or gRNA molecules introduced into said cells. In embodiments, the deletion comprises, for example, consists of about 4900 nt arranged between the target sequence of a gRNA described herein arranged in the HBG1 promoter region and the target sequence of a gRNA described herein arranged in the HBG2 promoter region . In embodiments, the indel, for example, the deletion, does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, - chain (hg38).
[0644] [0644] In one aspect, the invention relates to a population of cells (for example, as described herein), for example, a population of HSPCs, which comprises cells that include an indel in or near (for example, within of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of) a nucleic acid sequence having complementarity with a gRNA molecule or gRNA molecules, for example, as described herein, introduced into said cells, for example, as described herein. In modalities, the indel is an indel of displacement of the reading frame. In embodiments, the cell population includes cells that comprise a large deletion, for example, a deletion of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb or more. In embodiments, the large deletion comprises nucleic acids arranged between two binding sites for the gRNA molecule or gRNA molecules introduced into said cells. In embodiments, the deletion comprises, for example, consists of about 4900 nt arranged between the target sequence of a gRNA described herein arranged in the HBG1 promoter region and the target sequence of a gRNA described herein arranged in the HBG2 promoter region . In modalities, less than 1%, 0.5%, 0.1% or 0.001% of the population cells (for example, no cell in the population) comprises a deletion of a nucleotide arranged between 5,250,092 and 5,249,833, - chain (hg38) . In modalities, 20% -100% of the population's cells include that large, indelible or indelible deletion. In modalities, 30% -100% of the population's cells include the said large, indelible or indelible deletion. In modalities, 40% -100% of the population's cells include that large, indelible or indelible deletion. In modalities, 50% -100% of the population's cells include said large, indelible or indelible deletion. In modalities, 60% -100% of the population's cells include that large, indelible or indelible deletion. In modalities,
[0645] [0645] In one embodiment, the invention provides a population of cells, for example, CD34 + cells, of which at least 90%, for example, at least 95%, for example, at least 98%, of the cells of the population comprise a large deletion or one or more indels, for example, as described herein. Without being bound by theory, it is believed that the introduction of a gRNA molecule or CRISPR system as described here in a population of cells produces a pattern of indels and / or large deletions in that population, and thus, each cell of the population that comprises an indel and / or large deletion may not exhibit the same indel and / or large deletion. In embodiments, the indel and / or large deletion comprises one or more nucleic acids at or near a site complementary to the target domain of a gRNA molecule described herein; cells maintain the ability to differentiate into cells of an erythroid lineage, for example, red blood cells; and / or in which said cells differentiated from the cell population have an increased level of fetal hemoglobin (e.g., the population has a higher% F cells) compared to cells differentiated from a similar population of unmodified cells. In embodiments, the cell population has undergone at least a 2-fold expansion ex vivo, for example, in media comprising one or more stem cell expanders, for example, comprising (S) -2- (6- (2- ( 1H-indol-3-iN) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol. In embodiments, the cell population has undergone at least a 5-fold expansion ex vivo, for example, in media comprising one or more stem cell expanders, for example, comprising (S) -2- (6- (2- ( 1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol.
[0646] [0646] In embodiments, the indel is less than about 50 nucleotides, for example, less than about 45, less than about 40, less than about 35, less than about 30 or less than about 25 nucleotides. In embodiments, the indel is less than about 25 nucleotides. In embodiments, the indel is less than about 20 nucleotides. In embodiments, the indel is less than about 15 nucleotides. In embodiments, the indel is less than about 10 nucleotides. In embodiments, the indel is less than about 9 nucleotides. In embodiments, the indel is less than about 9 nucleotides. In embodiments, the indel is less than about 7 nucleotides. In embodiments, the indel is less than about 6 nucleotides. In embodiments, the indel is less than about 5 nucleotides. In embodiments, the indel is less than about 4 nucleotides. In embodiments, the indel is less than about 3 nucleotides. In embodiments, the indel is less than about 2 nucleotides. In any of the aforementioned embodiments, the indel is at least 1 nucleotide. In embodiments, the indel is 1 nucleotide. In modalities, the large deletion comprises about 1 kb of DNA. In modalities, the large deletion comprises about 2 kb of DNA. In modalities, the large deletion comprises about
[0647] [0647] In embodiments, a cell population (for example, as described herein) comprises a pattern of indels and / or large deletions comprising any 1, 2, 3, 4, 5 or 6 of the most frequently detected indels associated with a CRISPR system comprising a gRNA molecule described herein, for example, comprises 1, 2, 3, 4, 5 or 6 of the indels and large deletions described in Table 7-2 (for example, comprises 1, 2, 3, 4, 5 or 6 of the indels and large deletions detected in or near the HBG1 target sequence and / or comprise 1, 2, 3, 4, 5 or 6 of the indels and large deletions detected in or near the HBG2 target sequence). In modalities, indels and / or large deletions are detected by a method described here, for example, by NGS or qPCR.
[0648] [0648] In one aspect, the cell or cell population (for example, as described herein) does not comprise an indel or large deletion at an off-target site, for example, as detected by a method described herein.
[0649] [0649] In modalities, offspring for example, differentiated offspring, for example, erythroid offspring (for example, red blood cells) of the cell or cell population described here (for example, derived from a patient with sickle cell disease) produces a lower level beta-sickle globin and / or a higher level of gamma globin than unmodified cells. In embodiments, the offspring, for example, differentiated offspring, for example, erythroid offspring (for example, red blood cells) of the cell or cell population described here (for example, derived from a patient with sickle cell disease) produces a lower level of globin sickle beta and a higher level of gamma globin than unmodified cells. In embodiments, beta sickle globin is produced at a level of at least about 20%, at least about 30%, at least about 40% or at least about 50% less than unmodified cells. In embodiments, gamma globin is produced at a level of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% or at least about 70% higher than unmodified cells.
[0650] [0650] In one aspect, the invention provides a population of modified HSPCs or erythroid cells differentiated from said HSPCs (for example, differentiated ex vivo or in a patient), for example, as described herein, in which at least 15%, at least least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the cells are F cells. In embodiments, the cell population contains (or is capable of differentiating, for example, in vivo, in a population of erythrocytes containing) a higher percentage of F cells than a similar population of cells that did not have a gRNA molecule or gRNA molecules, for example, as described herein, introduced into said cells . In modalities, the cell population has (or is able to differentiate, for example, in vivo, into a population of erythrocytes that it has) at least a 20% increase, for example, at least a 21% increase , at least an increase of 22%, at least an increase of 23%, at least an increase of
[0651] [0651] In embodiments, including any of the embodiments and aspects described herein, the invention relates to a cell, for example, a population of cells, for example, as modified by any of the CRISPR gRNA, methods and / or systems described herein, comprising F cells that produce at least 6 picograms of fetal hemoglobin per cell. In modalities, F cells produce at least 7 picograms of fetal hemoglobin per cell. In modalities, F cells produce at least 8 picograms of fetal hemoglobin per cell. In modalities, F cells produce at least 9 picograms of fetal hemoglobin per cell. In modalities, F cells produce at least 10 picograms of fetal hemoglobin per cell. In modalities, F cells produce an average of between 6.0 and 7.0 picograms, between 7.0 and 8.0, between 8.0 and 9.0, between 9.0 and 10.0, between 10.0 and 11.0, or between 11.0 and 12.0 picograms of fetal hemoglobin per cell.
[0652] [0652] In embodiments, a cell or population of cells, for example, as described herein (for example, comprising an indel, for example, a large deletion or indel described in Table 7-2) (or its progeny), it is detectable in the cells of a subject to which it is introduced, for example, it remains detectable by detecting the indel, for example, using a method described herein. In embodiments, the cell or cell population (or its progeny) is detectable in a subject to which it is introduced for at least 10 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks or more after said cell or cell population is introduced into said subject.
[0653] [0653] In modalities, one or more indels (for example, a large deletion or indel described in Table 7-2), is detectable in cells (for example, cells, for example, CD34 +, bone marrow and / or cells peripheral blood) of a subject to which the cells or cell population described herein have been introduced, for example, remains detectable by a method described herein, for example, NGS. In embodiments, the one or more indels is detectable in the cells (for example, cells, for example, CD34 + cells, bone marrow and / or peripheral blood) of a subject to which the cells or cell population described here were introduced during at least 10 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, at least 50 weeks or more after the cell or cell population described here be introduced in that subject. In modalities, the level of detection of said one or more indels does not decrease over time, or a reduction of less than 5%, less than 10%, less than 15%, less than 20%, less than than 30%, less than 40% or less than 50% (for example, in relation to the level of detection of pre-transplant indel or relative to the level of detection in week 2 post-transplant or week 8 post-transplant) , for example, when measured at week 20 post-transplant in relation to the level of detection (for example, percentage of cells comprising the one or more indels) measured before transplant or measured at week 2 after transplant or at week 8 after transplant transplant.
[0654] [0654] In embodiments, including any of the above mentioned embodiments, the cell and / or cell population of the invention includes, for example, consists of cells that do not comprise the nucleic acid encoding a Cas9 molecule. Treatment Methods Delivery time
[0655] [0655] In one embodiment, one or more nucleic acid molecules (for example, DNA molecules), with the exception of the components of a Cas system, for example, the component of the Cas9 molecule and / or the component of the gRNA molecule described here, are delivered. In one embodiment, the nucleic acid molecule is delivered at the same time as one or more components of the Cas system are delivered. In one embodiment, the nucleic acid molecule is delivered before or after (for example, less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks or 4 weeks) of one or more components of the Cas system to be delivered. In one embodiment, the nucleic acid molecule is delivered by a different medium than one or more of the components of the Cas system, for example, the Cas9 molecule component and / or the gRNA molecule component, is delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be administered by a viral vector, for example, an integration deficient lentivirus, and the component of the Cas9 molecule and / or the component of the gRNA molecule can be administered by electroporation, for example, to Toxicity caused by nucleic acids (eg, DNAs) can be reduced. In one embodiment, the nucleic acid molecule encodes a therapeutic protein, for example, a protein described herein. In one embodiment, the nucleic acid molecule encodes an RNA molecule, for example, an RNA molecule described herein. Bi-modal or Differential Component Delivery
[0656] [0656] The separate delivery of the components of a Cas system,
[0657] [0657] Some modes of delivery, for example, delivery by a nucleic acid vector that persists in a cell, or in offspring of a cell, for example, by autonomous replication or insertion into cellular nucleic acid, result in a more persistent expression the presence of a component. Examples include viral delivery, for example, adeno-associated viruses or lentiviruses.
[0658] [0658] By way of example, components, for example, a Cas9 molecule and a gRNA molecule, can be delivered in ways that differ in terms of the resulting or persistent half-life of the component delivered in the body, or in a particular compartment, tissue or organ. In one embodiment, a gRNA molecule can be delivered by these modes. The component of the Cas9 molecule can be delivered in a way that results in less persistence or less exposure to the body or to a specific compartment or tissue or organ.
[0659] [0659] More generally, in one embodiment, a first delivery mode is used to deliver a first component and a second delivery mode is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, for example, distribution, persistence or exposure, of the component, or of a nucleic acid encoding the component, in the body, in a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, for example, distribution, persistence or exposure, of the component, or of a nucleic acid encoding the component, in the body, in a compartment, tissue or organ.
[0660] [0660] In one embodiment, the first pharmacodynamic or pharmacokinetic property, for example, distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.
[0661] [0661] In one embodiment, the first mode of delivery is selected to optimize, for example, minimize, a pharmacodynamic and pharmacokinetic property, for example, distribution, persistence or exposure.
[0662] [0662] In one embodiment, the second mode of delivery is selected to optimize, for example, maximize, a pharmacodynamic and pharmacokinetic property, for example, distribution, persistence or exposure.
[0663] [0663] In one embodiment, the first mode of delivery comprises the use of a relatively persistent element, for example, a nucleic acid, for example, a plasmid or viral vector, for example, an AAV or a lentivifus. As such vectors are relatively persistent, the product transcribed from it would be relatively persistent.
[0664] [0664] In one embodiment, the second mode of delivery comprises a relatively transient element, for example, an RNA or protein.
[0665] [0665] In one embodiment, the first component comprises gRNA, and the mode of delivery is relatively persistent, for example, the gRNA is transcribed from a plasmid or viral vector, for example, an AAV or lentivirus. The transcription of these genes would have little physiological consequence, because the genes do not code for a protein product, and gRAs are unable to act in isolation. The second component, a Cas9 molecule, is distributed in a transient manner, for example as MRNA or as a protein, ensuring that the complete Cas9 molecule / gRNA molecule complex is present and active only for a short period of time.
[0666] [0666] In addition, components can be delivered in different molecular forms or with different delivery vectors that complement each other to increase tissue safety and specificity.
[0667] [0667] The use of differential delivery modes can improve performance, safety and effectiveness. For example, the probability of a possible off-target modification can be reduced. Delivery of immunogenic components, for example, Cas9 molecules, by less persistent ways can reduce immunogenicity, as peptides from the bacteria-derived Cas enzyme are presented on the cell surface by MHC molecules. A two-part delivery system can alleviate these disadvantages.
[0668] [0668] Differential delivery modes can be used to deliver components to different but overlapping target regions. The active formation complex is minimized outside the overlap of the target regions. Thus, in one embodiment, a first component, for example, a gRNA molecule, is delivered by a first delivery mode that results in a first spatial distribution, for example, of tissue. A second component, for example, a Cas9 molecule, is delivered by a second mode of delivery that results in a second spatial distribution, for example, of tissue. In one embodiment, the first mode comprises a first element selected from a liposome, a nanoparticle, for example, a polymeric nanoparticle and a nucleic acid, for example, a viral vector. The second mode comprises a second element selected from the group. In one embodiment, the first mode of delivery comprises a first targeting element, for example, a specific cell receptor or an antibody, and the second mode of delivery does not include that element. In one embodiment, the second mode of delivery comprises a second targeting element, for example, a second specific cell receptor or second antibody.
[0669] [0669] When the Cas9 molecule is delivered in a virus, liposome or polymeric nanoparticle delivery vector, there is the potential for delivery and therapeutic activity in multiple tissues, when it may be desirable that only a single tissue be reached. A two-part delivery system can solve this challenge and improve tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separate delivery vehicles with separate but overlapping tissue tropism, the fully functional complex will only be formed in the tissue that is the target of both vectors.
[0670] [0670] Candidate Cas molecules, for example, Cas9 molecules, candidate gRNA molecules, candidate complexes of Cas9 / gRNA molecules and candidate CRISPR systems, can be evaluated in ways known in the art or as described herein. For example, exemplary methods for evaluating the endonuclease activity of the Cas9 molecule are described, for example, in Jinek el al, SCIENCE
[0671] [0671] Additional aspects are described in the modalities listed below.
[0672] [0672] Modalities:
[0673] [0673] 1. A gRNA molecule comprising tracr and cr »RNA, wherein the cr» RNA comprises a target domain that: a) is complementary to a target sequence from a non-deletion HFPH region (for example, an HPFH region human deletion); b) is complementary to a target sequence in the genomic nucleic acid sequence at Chr11: 5,249,833 to Chr11: 5,250,237, - strand, hg38; c) is complementary to a target sequence in the genomic nucleic acid sequence at Chr11: 5,254,738 to Chr11: 5,255,164, - strand, hg38; d) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,250,094-5,250,237, - strand, ho38; e) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,255,022-5,255,164, - strand, hg38; f) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,249,833-5,249,927, - strand, hg38; g) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,254,738-5,254,851, - strand, hg38; h) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,250,139-5,250,237, - strand, hg38; or i) their combinations.
[0674] [0674] 2. The mode 1 gRNA molecule, where the target domain comprises, for example, consists of either SEQ ID NO: 1 through SEQ ID NO: 72, or a fragment thereof.
[0675] [0675] 3. The mode 2 gRNA molecule, where the target domain comprises, for example, consists of any of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 67, or a fragment thereof.
[0676] [0676] 4, The mode 2 gRNA molecule, wherein the target domain comprises, for example, consists of any one of a) SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 67, or a fragment thereof; or b) SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 54, or a fragment thereof.
[0677] [0677] 5. The gRNA molecule of any of the 2-4 modalities, where the target domain comprises, for example, consists of, 17, 18, 19 or 20 consecutive nucleic acids from any of the domain-sequences target recited.
[0678] [0678] 6. The mode 5 gRNA molecule, where the 17, 18, 19 or 20 consecutive nucleic acids from any of the target domain sequences recited are the 17, 18, 19 or 20 consecutive nucleic acids arranged in the 3 ”end of the target domain string recited.
[0679] [0679] 7. The mode 5 gRNA molecule, in which the 17,18, 19 or 20 consecutive nucleic acids of any of the target domain sequences recited are the 17, 18, 19 or 20 consecutive nucleic acids arranged in the 5 'end of the recited target domain sequence.
[0680] [0680] 8. The mode 5 gRNA molecule, where the 17, 18, 19 or 20 consecutive nucleic acids of any of the recited target domain sequences do not comprise nucleic acid 5 or 3 of the target domain sequence recited.
[0681] [0681] 9. The gRNA molecule of any of the 2-8 modalities, wherein the target domain consists of the recited target domain sequence.
[0682] [0682] 10. The gRNA molecule of any of the above embodiments, wherein a portion of the crRNA and a portion of the tracr hybridize to form a mast comprising SEQ ID NO: 182 or
[0683] [0683] 11. The mode 10 gRNA molecule, wherein the mast additionally comprises a first extension of the mast, located 3 'with respect to the crRNA portion of the mast, wherein said first extension of the mast comprises SEQ ID NO: 184.
[0684] [0684] 12. The gRNA molecule of modality 10 or 11, in which the mast additionally comprises a second extension of the mast located 3 "in relation to the crRNA portion of the mast and, if present, the first extension of the mast, in which said second mast extension comprises SEQ ID NO: 185.
[0685] [0685] 13. The gRNA molecule of any of modalities 1-12, wherein the tracr comprises SEQ ID NO: 224, or a SEQ ID NO:
[0686] [0686] 14. The gRNA molecule of any of modalities 1-13, wherein the tracr comprises SEQ ID NO: 232, optionally further comprising, at the 3 ”, 1, 2, 3,4,5,6 or 7 additional uracil (U) nucleotides.
[0687] [0687] 15. The gRNA molecule of any of modalities 1-14, in which the crRNA comprises, from 5 to 3 ”, [target domain] -:
[0688] [0688] 16. The gRNA molecule of any of the modalities 1-9 or 15, in which the tracr comprises, from 5 to 3 ": a) SEQ ID NO: 187; b) SEQ ID NO: 188; c) SEQ ID NO: 203; d) SEQ ID NO: 204; e) SEQ ID NO: 224; f) SEQ ID NO: 225; g) SEQ ID NO: 232; h) SEQ ID NO: 227; i) (SEQ ID NO: 228; j) SEQ ID NO: 229; k) any one of a) to j), above, further comprising, at the 3 'end, at least 1, 2, 3, 4, 5, 6 or 7 nucleotides of uracil (U), e.g., 1, 2 , 3, 4, 5, 6 or 7 uracil (U) nucleotides; |) any of a) to k), above, further comprising, at the 3 'end, at least 1, 2, 3, 4, 5, 6 or 7 adenine nucleotides (A), e.g., 1, 2 , 3, 4, 5, 6 or 7 adenine nucleotides (A); or m) any one of a) to |), above, further comprising, at the 5 'end (e.g., at the 5' end), at least 1, 2, 3, 4, 5, 6 or 7 nucleotides of adenine (A), e.g., 1, 2, 3, 4, 5, 6 or 7 adenine nucleotides (A).
[0689] [0689] 17. The gRNA molecule of any of the modalities
[0690] [0690] 18. The gRNA molecule of any of the 13-14 modalities, wherein the mast crRNA portion comprises SEQ ID NO: 201, or a SEQ ID NO: 202.
[0691] [0691] 19. The gRNA molecule of any of modalities 1-12, wherein the tracr comprises SEQ ID NO: 187 or 188 and, optionally, if a first mast extension is present, a first tracr extension, arranged in 5 'to SEQ ID NO: 187 or 188, said first tracer extension comprising SEQ ID NO: 189.
[0692] [0692] 20. The gRNA molecule of any of modalities 1-19, wherein the target domain and tracr are arranged in separate nucleic acid molecules.
[0693] [0693] 21. The gRNA molecule of any of the modalities 1-19, in which the target domain and the tracr are arranged in a single nucleic acid molecule and in which the tracr is arranged 3 'in relation to the target.
[0694] [0694] 22. The gRNA molecule of modality 21, additionally comprising a loop, arranged 3 "in relation to the target domain and 5º in relation to tracr.
[0695] [0695] 23. The mode 22 gRNA molecule, wherein the loop comprises SEQ ID NO: 186.
[0696] [0696] 24. To the gRNA molecule of any of modalities 1-9, comprising, from 5 to 3 ”, [target domain] -: (a) SEQ ID NO: 195;
[0697] [0697] 25. The gRNA molecule of any of the modalities 1-9 or 21-24, in which the target domain and tracr are arranged in a single nucleic acid molecule and in which said nucleic acid molecule comprises o, e.g., consists of said target domain and SEQ ID NO: 231, optionally immediately arranged 3 'with respect to said target domain.
[0698] [0698] 26. The gRNA molecule of any one of embodiments 1-25, wherein one, or optionally more than one, of the nucleic acid molecules comprising the gRNA molecule comprises: a) one or more, e.g. ., three, phosphorothioate modifications at the 3 'end of said molecule or nucleic acid molecules; b) one or more, e.g., three, phosphorothioate modifications at the 5 'end of said molecule or nucleic acid molecules; c) one or more, e.g., three, 2'-O-methyl modifications at the 3 'end of said molecule or nucleic acid molecules; d) one or more, e.g., three, 2'-O-methyl modifications at the 5 'end of said molecule or nucleic acid molecules; e) a 2 'O-methyl modification in each of the 4th, 3rd and 2nd residues in relation to the 3' terminal of said molecule or nucleic acid molecules; f) a 2 'O-methyl modification in each of the 4th, 3rd and 2nd residues in relation to the 5' terminal of said molecule or nucleic acid molecules; or f) any combination thereof.
[0699] [0699] 27. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 74; (b) SEQ ID NO: 75; or (c) SEQ ID NO: 76.
[0700] [0700] 28. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 77 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 77 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 78 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 78 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0701] [0701] 29. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 79; (b) SEQ ID NO: 80; or (c) SEQ ID NO: 81.
[0702] [0702] 30. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 82 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID
[0703] [0703] 31. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 84; (b) SEQ ID NO: 85; or (c) SEQ ID NO: 86.
[0704] [0704] 32. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 87 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 87 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 88 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 88 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0705] [0705] 33. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 89;
[0706] [0706] 34. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 92 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 92 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 93 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 93 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0707] [0707] 35. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 94; (b) SEQ ID NO: 95; or (c) SEQ ID NO: 96.
[0708] [0708] 36. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 97 and a tracr comprising, for example, consisting of, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 97 and a tracer comprising, for example, consisting of SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID
[0709] [0709] 37. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 99; (b) SEQ ID NO: 100; or (c) SEQ ID NO: 101.
[0710] [0710] 38. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 102 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 102 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 103 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 103 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0711] [0711] 39. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 104; (b) SEQ ID NO: 105; or (c) SEQ ID NO: 106.
[0712] [0712] 40. A mode 1 gRNA molecule,
[0713] [0713] 41. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 109; (b) SEQ ID NO: 110; or (c) SEQ ID NO: 111.
[0714] [0714] 42. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 112 and a tracr comprising, for example, consisting of, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 112 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 113 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a cr »RNA comprising, for example, consisting of, SEQ ID
[0715] [0715] 43. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 114; (b) SEQ ID NO: 115; or (c) SEQ ID NO: 116.
[0716] [0716] 44. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 117 and a tracr comprising, for example, consisting of, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 117 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 118 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 118 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0717] [0717] 45. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 119; (b) SEQ ID NO: 120; or (c) SEQ ID NO: 121.
[0718] [0718] 46. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 122 and a tracer comprising, for example, consisting of, SEQ
[0719] [0719] 47. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 124; (b) SEQ ID NO: 125; or (c) SEQ ID NO: 126.
[0720] [0720] 48. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 127 and a tracr comprising, for example, consisting of, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 127 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 128 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 128 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0721] [0721] 49. A modality 1 gRNA molecule,
[0722] [0722] 50. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 132 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 132 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 133 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 133 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0723] [0723] 51. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 134; (b) SEQ ID NO: 135; or (c) SEQ ID NO: 136.
[0724] [0724] 52. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 137 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 137 and a tracer comprising, for example, consisting of, SEQ
[0725] [0725] 53. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 139; (b) SEQ ID NO: 140; or (c) SEQ ID NO: 141.
[0726] [0726] 54. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 142 and a tracr comprising, for example, consisting of, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 142 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 143 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 143 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0727] [0727] 55. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 144; (b) SEQ ID NO: 145; or
[0728] [0728] 56. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 147 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 147 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 148 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 148 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0729] [0729] 57. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 149; (b) SEQ ID NO: 150; or (c) SEQ ID NO: 151.
[0730] [0730] 58. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 152 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 152 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 153 and a tracer comprising, for example, consisting of, SEQ
[0731] [0731] 59. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 154; (b) SEQ ID NO: 155; or (c) SEQ ID NO: 156.
[0732] [0732] 60. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 157 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 157 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 158 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 158 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0733] [0733] 61. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 159; (b) SEQ ID NO: 160; or (c) SEQ ID NO: 161.
[0734] [0734] 62. A mode 1 gRNA molecule, comprising, for example, consisting of:
[0735] [0735] 63. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 164; (b) SEQ ID NO: 165; or (c) SEQ ID NO: 166.
[0736] [0736] 64. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 167 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 167 and a tracer comprising, for example, consisting of SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 168 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 168 and a tracer comprising, for example, consisting of, SEQ
[0737] [0737] 65. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 169; (b) SEQ ID NO: 170; or (c) SEQ ID NO: 171.
[0738] [0738] 66. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 172 and a tracr comprising, for example, consisting in, SEQ ID NO: 224; (b) a crRNA comprising, for example, consisting of, SEQ ID NO: 172 and a tracer comprising, for example, consisting of, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, consisting of, SEQ ID NO: 173 and a tracer comprising, for example, consisting of, SEQ ID NO: 224; or (d) a crRNA comprising, for example, consisting of, SEQ ID NO: 173 and a tracer comprising, for example, consisting of SEQ ID NO: 73.
[0739] [0739] 67. A mode 1 gRNA molecule, comprising, for example, consisting of the sequence: (a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ ID NO: 176.
[0740] [0740] 68. A mode 1 gRNA molecule, comprising, for example, consisting of: (a) a crRNA comprising, for example, consisting of, SEQ ID NO: 177 and a tracr comprising, for example, consisting of, SEQ ID NO: 224;
[0741] [0741] 69. A gRNA molecule of any of modalities 1-68, wherein a) when a CRISPR system (for example, an RNP as described here) comprising a gRNA molecule is introduced into a cell, an indel is formed at or near the target sequence complementary to the target domain of the gRNA molecule; and / or b) when a CRISPR system (for example, an RNP as described herein) comprising the gRNA molecule is introduced into a cell, a deletion is created comprising the sequence, for example, comprising substantially the entire sequence, between a sequence complementary to the gRNA target domain (for example, at least 90% complementary to the gRNA target domain, for example, fully complementary to the gRNA target domain) in the HBG1 promoter region and a complementary sequence to the gRNA target domain ( for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region.
[0742] [0742] 70. The 69 mode gRNA molecule, where the indel does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, - strand (hg38), optionally where the indel does not comprise a nucleotide from a site binding of the transcription factor or non-deletion HPFH.
[0743] [0743] 71. A gRNA molecule of any of modalities 1-70, wherein, when a CRISPR system (eg, an RNP as described here) comprising the gRNA molecule is introduced into a population of cells, an indel is formed at or near the target sequence complementary to the target domain of the gRNA molecule by at least about 15%, for example, at least about 17%, for example, at least about 20%, for example at least about 30%, for example, at least about 40%, for example, at least about 50%, for example, at least about 55%, for example, at least about 60%, for example at least about 70%, for example, at least about 75%, for example, at least about 80%, for example, at least about 85%, for example, at least about 90%, for example at least about 95% of the population cells.
[0744] [0744] 72. A gRNA molecule of any of the 69-71 modalities, wherein the indel comprises at least one nucleotide from an HBG1 promoter region or at least one nucleotide from an HBG2 promoter region.
[0745] [0745] 73. A gRNA molecule of any of the 71-72 modalities, wherein at least about 15% of the population's cells comprise an indel that comprises at least one nucleotide from an HBG1 promoter region and an indel that comprises at least one nucleotide from an HBG 2 promoter region.
[0746] [0746] 74. A gRNA molecule of any of the 71-73 modalities, in which the percentage of cells in the population, which comprise an indel that comprises at least one nucleotide from an HBG1 promoter region, differs from the percentage of cells in the population comprising an indel comprising at least one nucleotide from an HBG2 promoter region by at least about 5%, for example, at least about 10%, for example, at least about 20%, for example, at least about 30%.
[0747] [0747] 75. The gRNA molecule of any of the 69-74 modalities, where indel is as measured by next generation sequencing (NGS).
[0748] [0748] 76. A gRNA molecule of any one of modalities 1-75, wherein, when a CRISPR system (eg, an RNP as described here) comprising the gRNA molecule is introduced into a cell, the expression fetal hemoglobin is increased in said cell or its progeny, eg, its erythroid progeny, eg, its red blood cell progeny.
[0749] [0749] 77. A modality 76 gRNA molecule, where when a CRISPR system (for example, an RNP, as described herein), comprising the gRNA molecule, is introduced into a population of cells, the percentage of cells F in said population, or population of its offspring, for example, its erythroid offspring, for example, its red blood cell offspring is increased by at least about 15%, for example, at least about 17%, for example, at least about 20%, for example, at least about 25%, for example, at least about 30%, for example, at least about 35%, for example, at least about 40%, with respect to the percentage of F cells in a cell population to which the gRNA molecule has not been introduced or a population of its progeny, for example, its erythroid progeny, for example, its red blood cell progeny.
[0750] [0750] 78. A gRNA molecule of any of the modalities 76, in which said cell or its progeny, eg, its erythroid progeny, eg, its red blood cell progeny,
[0751] [0751] 79. The gRNA molecule of any of modalities 1-78, wherein, when a CRISPR system (eg, an RNP as described here) comprising the gRNA molecule is introduced into a cell, none are indelible outside of the target are formed in said cell, for example, indels outside the target do not form outside the HBG1 and / or HBG2 promoter regions, for example, as detectable by next generation sequencing and / or a nucleotide insertion assay.
[0752] [0752] 80. The gRNA molecule of any of modalities 1-78, wherein, when a CRISPR system (eg, an RNP as described here) comprising the gRNA molecule is introduced into a cell population, none indel off-target, for example, no indel off-target outside the HBG1 and / or HBG2 promoter regions, is detected by more than about 5%, e.g., more than about 1%, e.g. more than about 0.1%, e.g., more than about 0.01%, of the cells in the cell population, e.g., as detectable by next generation sequencing and / or a nucleotide insertion assay.
[0753] [0753] 81. The gRNA molecule of any of the 69-80 embodiments, wherein the cell is (or population of cells comprises) a mammalian, primate or human cell, e.g., is a human cell.
[0754] [0754] 82. The mode 81 gRNA molecule, wherein the cell is (or population of cells comprises) an HSPC.
[0755] [0755] 83. The modality 82 gRNA molecule, where HSPC is CD34 +.
[0756] [0756] 84. The modality 83 gRNA molecule, where HSPC is CD34 + CD90 +.
[0757] [0757] 85. The gRNA molecule of any of the 69-84 modalities, wherein the cell is autologous with respect to a patient being administered that cell.
[0758] [0758] 86. The gRNA molecule of any of the 69-84 modalities, wherein the cell is allogeneic with respect to a patient being administered that cell.
[0759] [0759] 87. Composition comprising: 1) one or more gRNA molecules (including a first gRNA molecule), of any of the modalities 1-86, and a Cas9 molecule; 2) one or more gRNA molecules (including a first gRNA molecule), of any of modalities 1-86, and nucleic acid encoding a Cas9 molecule; 3) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule), of any of modalities 1-86, and a Cas9 molecule; 4) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule), of any of modalities 1-86, and nucleic acid encoding a Cas9 molecule; or 5) any one of 1) to 4), above, and a template nucleic acid; or 6) any one of 1) to 4) above, and nucleic acid comprising sequence encoding a model nucleic acid.
[0760] [0760] 88. A composition comprising a first gRNA molecule, of any of modalities 1-86, optionally further comprising a Cas9 molecule.
[0761] [0761] 89. The composition of modality 87 or 88, in which the Cas9 molecule is an active or inactive S. pyogenes Cas9.
[0762] [0762] 90. The composition of modality 87-89, in which the molecule
[0763] [0763] 91. The composition of modality 87-89, wherein the Cas9 molecule comprises, for example, consists of: (a) SEQ ID NO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e) SEQ ID NO: 237; (f) SEQ ID NO: 238; (g) SEQ ID NO: 239; (h) SEQ ID NO: 240; (1) SEQ ID NO: 241; (1) SEQ ID NO: 242; (k) SEQ ID NO: 243; or (1) SEQ ID NO: 244.
[0764] [0764] 92. The composition of any of the 88-91 modalities, in which the first gRNA molecule and the Cas9 molecule are present in a ribonuclear protein complex (RNP).
[0765] [0765] 93. The composition of any of the 87-92 modalities, further comprising a second gRNA molecule; a second gRNA molecule and a third gRNA molecule; or a second gRNA molecule, optionally a third gRNA molecule and, optionally, a fourth gRNA molecule, wherein the second gRNA molecule, the optional third gRNA molecule, and the optional fourth gRNA molecule are a gRNA molecule , in any of embodiments 1-68, and wherein each gRNA molecule in the composition is complementary to a different target sequence.
[0766] [0766] 94. The composition of modality 93, in which two or more of the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule and the optional fourth gRNA molecule are complementary with target sequences within the same gene or region.
[0767] [0767] 95. The 93 or 94 modality composition, wherein the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule and the optional fourth gRNA molecule are complementary with target sequences separated by no more than 6000, no more than 5000 nucleotides, no more than 500, no more than 400 nucleotides, no more than 300 nucleotides, no more than 200 nucleotides, no more than 100 nucleotides, no more than 90 nucleotides, not more than 80 nucleotides, not more than 70 nucleotides, not more than 60 nucleotides, not more than 50 nucleotides, not more than 40 nucleotides, not more than 30 nucleotides, not more than 20 nucleotides or not more than 10 nucleotides.
[0768] [0768] 96. The composition of modality 93, wherein two or more of the first gRNA molecule, the second gRNA molecule, the optional third gRNA molecule and the optional fourth grRNA molecule comprise at least one gRNA molecule that comprises a target domain complementary to a target sequence of an HBG1 promoter region and at least one gRNA molecule comprising a target domain complementary to a target sequence of an HBG2 promoter region.
[0769] [0769] 97. The composition of any of the 94-95 modalities, comprising a first gRNA molecule and a second gRNA molecule, wherein the first gRNA molecule and the second gRNA molecule are: (a) independently selected from gRNA molecules, as defined in modality 1, and are complementary to different target sequences; (b) independently selected from the gRNA molecules,
[0770] [0770] 98. The composition of any of the 94-96 modalities, comprising a first gRNA molecule and a second gRNA molecule, in which: a) the first gRNA molecule is complementary to a target sequence comprising - at least 1 nucleotide (for example, comprising 20 consecutive nucleotides) in: i) Chr11: 5,249,833 to Chr11: 5,250,237 (hg38); ii) Chr11: 5,250,094-5,250,237 (hg38); iii) Chr11: 5,249,833-5,249,927 (hg38); or iv) Chr11: 5,250,139-5,250,237 (hg38); b) the second gRNA molecule is complementary to a target sequence comprising - at least 1 nucleotide (for example, comprising 20 consecutive nucleotides) in: i) Chr11: 5,254,738 to Chr11: 5,255,164 (hg38); ii) Chr11: 5,255,022-5,255,164 (h9g38); or iii) Chr11: 5,254,738-5,254,851 (hg38).
[0771] [0771] 99. The composition of any of the 87-108 modalities, wherein, with respect to the components of the composition's gRNA molecule, the composition consists of a first gRNA molecule and a second gRNA molecule.
[0772] [0772] 100. The composition of any of the modalities 87-109, wherein each of said gRNA molecules is in a ribonuclear protein (RNP) complex with a Cas9 molecule described here, e.g., a Cas9 molecule, any of the 90 or 91 modalities.
[0773] [0773] 101. The composition of any of the 87-100 embodiments, comprising a template nucleic acid, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide at or near the target sequence of the first gRNA molecule.
[0774] [0774] 102. The composition of any of the modalities 101, wherein the template nucleic acid comprises nucleic acid encoding: (a) human beta globin, e.g., human beta globin comprising one or more of the G16D, E22A and T87Q or its fragment; or (b) human globin gamma or fragment thereof.
[0775] [0775] 103. The composition of any of the modalities 87-102, formulated in a suitable medium for electroporation.
[0776] [0776] 104. The composition of any of the modalities 87-103, wherein each of said gRNA molecules is in an RNP with a Cas9 molecule described here and in which each of said RNP is at a concentration of less than that about 10 UM, for example, less than about 3 µM, for example, less than about 1 µM, for example, less than about 0.5 µM, for example, less than about 0 , 3 µM, for example, less than about 0.1 µM, optionally wherein the concentration of said RNP is about 2 µM or is about 1 µM, optionally where the composition further comprises a population of cells, for example, HSPCs.
[0777] [0777] 105. A nucleic acid sequence that encodes one or more gRNA molecules, of any of modalities 1-68.
[0778] [0778] 106. The nucleic acid sequence of mode 105, wherein the nucleic acid comprises a promoter operably linked to the sequence encoding one or more gRNA molecules.
[0779] [0779] 107. The nucleic acid sequence of mode 106, wherein the promoter is a promoter recognized by an RNA polymerase | 1 or RNA polymerase III.
[0780] [0780] 108. The 107 nucleic acid sequence, wherein the promoter is a U6 promoter or an HI promoter.
[0781] [0781] 109. The nucleic acid sequence of any of the 105-108 embodiments, wherein the nucleic acid further encodes a Cas9 molecule.
[0782] [0782] 110. The 109 nucleic acid sequence, wherein the Cas9 molecule comprises any of SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO:
[0783] [0783] 111. The nucleic acid sequence of any of the 109-110 embodiments, wherein said nucleic acid comprises a promoter operably linked to the sequence encoding a Cas9 molecule.
[0784] [0784] 112. The 111 nucleic acid sequence, wherein the promoter is an EF-1 promoter, a CMV IE gene promoter, an EF-1a promoter, a ubiquitin C promoter or a phosphoglycerate kinase promoter (PGK).
[0785] [0785] 113. A vector comprising nucleic acid, in any of the modalities 105-112.
[0786] [0786] 114. The 113 mode vector, in which the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), a herpes simplex virus (HSV) vector, a plasmid , a minicircle, a nanoplasmid and an RNA vector.
[0787] [0787] 115. Method of altering a cell (eg a population of cells), (eg altering the structure (eg, sequence) of nucleic acid) at or near a target sequence within said cell , comprising contacting (e.g., introducing into) said cell (e.g., cell population) with: 1) one or more gRNA molecules, of any of modalities 1-68, and a Cas9 molecule; 2) one or more gRNA molecules, of any of the modalities 1-68, and nucleic acid encoding a Cas9 molecule; 3) nucleic acid encoding one or more gRNA molecules, of any of the modalities 1-68, and a Cas9 molecule; 4) nucleic acid encoding one or more gRNA molecules, of any of modalities 1-68, and nucleic acid encoding a Cas9 molecule; 5) any one of 1) to 4), above, and a template nucleic acid; 6) any one of 1) to 4) above, and nucleic acid comprising a sequence encoding a model nucleic acid; 7) the composition, of any of the 87-104 modalities; or 8) the vector, of any of the 113-114 modalities.
[0788] [0788] 116. The 115 method, wherein the gRNA molecule or nucleic acid encoded the gRNA molecule, and the Cas9 molecule or nucleic acid encoding the Cas9 molecule, are formulated into a single composition.
[0789] [0789] 117. The method of modality 115, wherein the gRNA or nucleic acid molecule encoding the gRNA molecule, and the Cas9 molecule or nucleic acid encoding the Cas9 molecule, are formulated in more than one composition.
[0790] [0790] 118. The 117 method, in which more than one composition is administered simultaneously or sequentially.
[0791] [0791] 119. The method of any of the 115-118 modalities, in which the cell is an animal cell.
[0792] [0792] 120. The method of any of the modalities 115-118, wherein the cell is a mammalian, primate or human cell.
[0793] [0793] 121. The method of modality 120, in which the cell is a hematopoietic stem cell or progenitor stem cell (HSPC) (eg, a population of HSPCs).
[0794] [0794] 122. The method of any of the 115-121 modalities, wherein the cell is a CD34 + cell.
[0795] [0795] 123. The method of any of the 115-122 modalities, wherein the cell is a CD34 + CD90 + cell.
[0796] [0796] 124. The method of any of the 115-123 modalities, wherein the cell is arranged in a composition comprising a population of cells that has been enriched for CD34 + cells.
[0797] [0797] 125. The method of any of the 115-124 modalities, in which the cell (eg, cell population) was isolated from the bone marrow, mobilized peripheral blood or cord blood.
[0798] [0798] 126. The method of any of the modalities 115-125, in which the cell is autologous or allogeneic in relation to a patient to be administered with said cell, optionally, in which the patient is a hemoglobinopathy patient, optionally , in which the patient has sickle cell disease or thalassemia, optionally, beta thalassemia.
[0799] [0799] 127. The method of any of the 115-126 modalities, in which: (a) the change results in an indelible at or near a complementary genomic DNA sequence with the target domain of one or more gRNA molecules ; and / or b) the change results in a deletion comprising a sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of one or more gRNA molecules (for example, at least 90% complementary to the target domain of gRNA, for example, completely complementary to the gRNA target domain) in the HBG1 promoter region and a sequence complementary to the target domain of one or more gRNA molecules (for example, at least 90% complementary to the gRNA target domain , for example, completely complementary to the target domain of gRNA) in the HBG2 promoter region, optionally, where the deletion does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, - strand (hg38).
[0800] [0800] 128. The 127 method, wherein the indel is an insertion or deletion of less than about 40 nucleotides, e.g., less than 30 nucleotides, e.g., less than 20 nucleotides , e.g., less than 10 nucleotides.
[0801] [0801] 129. The 128 method, wherein the indel is a single nucleotide deletion.
[0802] [0802] 130. The method of any of the 127-129 modalities, wherein the method results in a population of cells in which at least about 15%, for example, at least about 17%, for example, at least about 20%, for example, at least about 30%, for example, at least about 40%, for example, at least about 50%, for example, at least about 55%, for example, at least about 60%, for example, at least about 70%, for example, at least about 75%, for example at least about 80%, for example, at least about 85%, for example, at least about 90%, for example, at least about 95% of the population has changed, for example, comprising an indel, optionally where the indel is selected from an indel listed in Table 2-7,
[0803] [0803] 131. The method of any of the 115-130 modalities, in which the change results in a cell (eg, cell population) that is able to differentiate into a cell differentiated from an erythroid lineage (p a red blood cell) and wherein said differentiated cell exhibits an increased level of fetal hemoglobin, e.g., relative to an unchanged cell (e.g., cell population).
[0804] [0804] 132. The method of any of the modalities 115-131, in which the change results in a population of cells that is able to differentiate into a population of differentiated cells, eg, a population of cells from one erythroid lineage (eg, a population of red blood cells) and where said population of differentiated cells has an increased percentage of F cells (eg, at least about 15%, at least about 20%, at least at least about 25%, at least about 30% or at least about 40% higher percentage of F cells), e.g., relative to a population of unchanged cells.
[0805] [0805] 133. The method of any of the 115-131 modalities, in which the change results in a cell that is able to differentiate into a differentiated cell, eg, a cell of an erythroid lineage (eg ., a red blood cell) and wherein said differentiated cell produces at least about 6 picograms (e.g., at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or about 8 to about 9 picograms or about 9 to about 10 picograms) of fetal hemoglobin per cell.
[0806] [0806] 134. A cell, altered by the method of any of the 115-133 modalities, or a cell obtainable by the method, of any of the 115-133 modalities.
[0807] [0807] 135. Cell, comprising an indel described in Table 7-2, optionally, in which the cell does not comprise a deletion of a nucleotide arranged between 5,250,092 and 5,249,833, - strand (hg38).
[0808] [0808] 136. A cell, comprising a first gRNA cell, of any of the modalities 1-68, or a composition, of any of the modalities 87-104, a nucleic acid, of any of the modalities 105-112, or a vector, of any of the 113-114 modalities.
[0809] [0809] 137. The 136 mode cell, comprising a Cas9 molecule.
[0810] [0810] 138. The 137 mode cell, wherein the Cas9 molecule comprises any of SEQ ID NO: 205, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243 or SEQ ID NO: 244.
[0811] [0811] 139. The cell of any of modalities 134-138, wherein the cell comprises, comprises or will comprise a second gRNA molecule, of any of modalities 1-68, or a nucleic acid encoding a second gRNA molecule , of any of embodiments 1-68, wherein the first gRNA molecule and the second gRNA molecule comprise non-identical target domains.
[0812] [0812] 140. The cell of any of the modalities 134-139, in which the expression of fetal hemoglobin is increased in said cell or its progeny (eg, its erythroid progeny, eg, its blood cell progeny red) in relation to a cell or its progeny of the same cell type that has not been modified to comprise a gRNA molecule.
[0813] [0813] 141. The cell of any of the 134-139 modalities, in which the cell is able to differentiate into a differentiated cell, eg, a cell of an erythroid lineage (eg, a red blood cell ) and wherein said differentiated cell exhibits an increased level of fetal hemoglobin, e.g., relative to a cell of the same type that has not been modified to comprise a gRNA molecule.
[0814] [0814] 142. The cell of any of the modalities 140-141, in which the differentiated cell (eg, cell of an erythroid lineage, eg, red blood cell) produces at least about 6 picograms (eg eg at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or from about 8 to about 9 picograms or from about 9 to about picograms ) of fetal hemoglobin, e.g., in relation to a differentiated cell of the same type that has not been modified to comprise a gRNA molecule.
[0815] [0815] 143. The cell of any of the modalities 134-142, which was placed in contact with a stem cell expander.
[0816] [0816] 144. The 143 mode cell, in which the stem cell expander is: a) (1r, Ar) -N '- (2-benzyl-7- (2-methyl-2H-tetrazole-5- il) -9H-pyrimido [4,5-blindol-4-yl) cyclohexane-1,4-diamine; b) methyl 4- (3S-piperidin-1-ylpropylamine) -9H-pyrimido [4,5-b] indole-7-carboxylate; c) 4- (2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol; d) (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9- yl) propan-1- ol; or e) their combinations (for example, a combination of (1r, 4r) -N '- (2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5- b] indol-4-yl) cyclohexane-1,4-diamine and (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3 -yl) -9H-purin-9-yl) propan-1-01).
[0817] [0817] 145. The 144 mode cell, wherein the stem cell expander is (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridine -3-yl) -9H-purin-9-yl) propan-1-ol.
[0818] [0818] 146. A cell, for example, a cell of any of the 134-145 modalities, comprising: a) an indel in or near a genomic DNA sequence complementary to the target domain of a gRNA molecule, from any of modalities 1-68; and / or b) a deletion comprising a sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of a gRNA molecule, of any of modalities 1-68, (for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG1 promoter region and a sequence complementary to the target domain of a gRNA molecule, of any one of modalities 1-68, (for example , at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region, optionally where the deletion does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, - strand (hg38 ).
[0819] [0819] 147. The 146 mode cell, where the indel is an insertion or deletion of less than about 40 nucleotides, e.g., less than 30 nucleotides, e.g., less than 20 nucleotides , e.g., less than 10 nucleotides.
[0820] [0820] 148. The cell of any of the 146-147 modalities, where the indel is a single nucleotide deletion.
[0821] [0821] 149. The cell of any of the modalities 134-148, in which the cell is an animal cell.
[0822] [0822] 150. The 149 mode cell, wherein the cell is a mammalian, primate or human cell.
[0823] [0823] 151. The cell of any of the 134-150 modalities, where the cell is a hematopoietic or progenitor stem cell (HSPC) (eg, a population of HSPCs).
[0824] [0824] 152. The cell of any of the modalities 134-151, in which the cell is a CD34 + cell.
[0825] [0825] 153. The cell of mode 152, in which the cell is a CD34 + CD90 + cell.
[0826] [0826] 154. The cell of any of the 134-153 modalities, in which the cell (eg, cell population) was isolated from bone marrow, mobilized peripheral blood, or umbilical cord blood.
[0827] [0827] 155. The cell of any of the modalities 134-154, in which the cell is autologous in relation to a patient to be administered with said cell, optionally in which the patient is a hemoglobinopathy patient, optionally, in which patients have sickle cell disease or thalassemia, optionally, beta thalassemia.
[0828] [0828] 156. The cell of any of the modalities 134-154, wherein the cell is allogeneic with respect to a patient to be administered said cell.
[0829] [0829] 157. A population of cells comprising the cell, in any of the modalities 134-156.
[0830] [0830] 158. The cell population of modality 157, in which at least about 50%, e.g., at least about 60%, e.g., at least about 70%, e.g. at least about 80%, eg at least about 90% (eg, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%) of the population's cells are a cell, of any of the 134-156 modalities.
[0831] [0831] 159. The cell population of any of the 157-158 modalities, in which the cell population is able to differentiate into a population of differentiated cells, eg, a cell population of an erythroid lineage ( eg a population of red blood cells) and where said population of differentiated cells has an increased percentage of F cells (eg at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30% or at least about 40% higher percentage of F cells), e.g., in relation to a population of unmodified cells of the same type.
[0832] [0832] 160. The cell population of mode 159, where the F cells of the population of differentiated cells produce an average of at least about 6 picograms (eg, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or from about 8 to about 9 picograms or from about 9 to about 10 picograms) of fetal hemoglobin per cell.
[0833] [0833] 161. The cell population of any of the 157-160 modalities, comprising: 1) at least 1 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 2) at least 2 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 3) at least 3 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 4) at least 4e € 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 5) from 2e6 to 10e6 CD34 + cells / kg of body weight of the patient to which the cells are to be administered.
[0834] [0834] 162. The cell population of any of the 157-161 modalities, in which at least about 40%, eg, at least about 50% (eg, at least about 60% , at least about 70%, at least about 80% or at least about 90%)
[0835] [0835] 163. The cell population of mode 162, in which at least about 10%, e.g., at least about 15%, e.g., at least about 20%, e.g. , at least about 30% of the population's cells are CD34 + CD90 + cells.
[0836] [0836] 164. The cell population of any of the 157-163 modalities, in which the cell population is derived from umbilical cord blood, peripheral blood (for example, mobilized peripheral blood) or bone marrow, for example, is derived from bone marrow.
[0837] [0837] 165. The cell population of any of the 157-164 modalities, wherein the cell population comprises, for example, consists of mammalian cells, for example, human cells, optionally in which the cell population is obtained from a patient suffering from hemoglobinopathy, for example, sickle cell disease or thalassemia, for example, beta-thalassemia.
[0838] [0838] 166. The cell population of any of the 157-165 modalities, in which the cell population is (i) autologous in relation to a patient to be administered, or (ii) allogeneic in relation to a patient to be administered.
[0839] [0839] 167. The population of cells (for example, CD34 + cells), for example, of any of the 157-165 modalities, comprising an indelible pattern as described in Table 7-2, optionally where the indels of the indelible pattern described in Table 7-2 are detectable in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the population cells.
[0840] [0840] 168. A composition comprising a cell, of any of the 134-156 modalities, or the cell population of any of the 1557-167 modalities.
[0841] [0841] 169. The composition of embodiment 168, comprising a pharmaceutically acceptable medium, e.g., a pharmaceutically acceptable medium suitable for cryopreservation.
[0842] [0842] 170. A method of treating a hemoglobinopathy, comprising administering to a patient a cell, of any of the 134-156 modalities, a cell population, of any of the 157-167 modalities, or a composition, of any of the 168-169 modalities.
[0843] [0843] 171. A method of increasing fetal hemoglobin expression in a mammal, comprising administering to a patient a cell, of any of the 134-156 modalities, a cell population, of any of the 157-167 modalities, or a composition, of any of the 168-169 modalities.
[0844] [0844] 172. The 170 method, in which hemoglobinopathy is beta-thalassemia or sickle cell disease.
[0845] [0845] 173. A cell preparation method (e.g., a population of cells) comprising: (a) providing a cell (e.g., a population of cells) (e.g., an HSPC (e.g. a population of HSPCs)); (b) culturing said cell (e.g., said cell population) ex vivo in a cell culture medium comprising a stem cell expander; and (c) introducing into said cell a first gRNA molecule, of any of modalities 1-86, a nucleic acid molecule encoding a first gRNA molecule, of any of modalities 1-86, a composition, of any one of modalities 87-104 or 168-169, a nucleic acid, of any of the modalities 105-112, or a vector, of any of the modalities 113-114.
[0846] [0846] 174. The 173 method, in which, after the introduction of step (c), said cell (for example, cell population) is able to differentiate into a differentiated cell (for example, population of differentiated cells), for example, a cell of an erythroid lineage (for example, a cell population of an erythroid lineage), for example, a red blood cell (for example, a population of red blood cells) and in which said differentiated cell ( eg differentiated cell population) produces increased fetal hemoglobin, for example, in relation to the same cell that was not subjected to step (c).
[0847] [0847] 175. The method, in any of the 173-174 modalities, in which the stem cell expander is: a) (1r, 4r) -N1- (2-benzyl-7- (2-methyl-2H -tetrazol-5-i1) -9H-pyrimido [4,5-blindol-4-yl) cyclohexane-1,4-diamine; b) methyl 4- (3-piperidin-1-ylpropylamine) -9H-pyrimido [4,5-b] indole-7-carboxylate; c) 4- (2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol; d) (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9- yl) propan-1- ol; or e) their combinations (for example, a combination of (1r, 4r) -N1- (2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) -9H-pyrimido [4,5-b ] indol-4-i) cyclohexane-1,4-diamine and (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3- yl) -9H-purin-9-yl) propan-1-01).
[0848] [0848] 176. The 175 method, wherein the stem cell expander is (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridine -3- iI) -9H-purin-9-yl) propan-1-o |
[0849] [0849] 177. The method of any of the modalities 173-176, in which the cell culture medium comprises thrombopoietin (Tpo), FIt3 ligand (FIt-3L) and human stem cell factor (SCF).
[0850] [0850] 178. The 177 method, wherein the cell culture medium further comprises human interleukin-6 (IL-6).
[0851] [0851] 179. The method of any of the modalities 177-178, in which the cell culture medium comprises thrombopoietin (Tpo), FIt3 ligand (FIt-3L) and human stem cell factor (SCF) each to a concentration ranging from about 10 ng / ml to about 1000 ng / ml.
[0852] [0852] 180. The 179 method, in which the cell culture medium comprises thrombopoietin (Tpo), FIt3 ligand (FIt-3L) and human stem cell factor (SCF) each at a concentration of about 50 ng / ml, e.g., at a concentration of 50 ng / ml.
[0853] [0853] 181. The method of any of the 178-180 modalities, wherein the cell culture medium comprises human interleukin-6 (IL-6) at a concentration ranging from about 10 ng / mL to about 1000 ng / ml.
[0854] [0854] 182. The 181 method, wherein the cell culture medium comprises human interleukin-6 (IL-6) at a concentration of about 50 ng / ml, e.g., at a concentration of 50 ng / ml.
[0855] [0855] 183. The method of any of the 173-182 modalities, wherein the cell culture medium comprises a stem cell expander at a concentration ranging from about 1 nM to about 1 mM.
[0856] [0856] 184. The 183 method, wherein the cell culture medium comprises a stem cell expander at a concentration ranging from about 1 µM to about 100 µM.
[0857] [0857] 185. The 184 method, wherein the cell culture medium comprises a stem cell expander at a concentration ranging from about 500 nM to about 750 nM.
[0858] [0858] 186. The 185 method, wherein the cell culture medium comprises a stem cell expander at a concentration of about 500 nM, e.g., at a concentration of 500 nM.
[0859] [0859] 187. The method of mode 186, wherein the cell culture medium comprises a stem cell expander at a concentration of about 750 nM, e.g., at a concentration of 750 nM.
[0860] [0860] 188. The method of any of the modalities 173-187, in which the culture of step (b) comprises a culture period before the introduction of step (c).
[0861] [0861] 189. The 188 method, in which the culture period before the introduction of step (c) is at least 12 hours, for example, is for a period of about 1 day to about 12 days, for example example, it is for a period of about 1 day to about 6 days, for example, for a period of about 1 day to about 3 days, for example, it is for a period of about 1 day to about 2 days, for example, is for a period of about 2 days or for a period of about 1 day.
[0862] [0862] 190. The method of any of the modalities 173-189, in which the culture of step (b) comprises a period of culture after the introduction of step (c).
[0863] [0863] 191. The 190 method, in which the culture period after the introduction of step (c) is at least 12 hours, for example, is for a period of about 1 day to about 12 days , e.g., it is over a period of about 1 day to about 6 days, e.g., it is over a period of about 2 days to about 4 days, e.g., it is over a period about 2 days or over a period of about 3 days or over a period of about 4 days.
[0864] [0864] 192. The method of any of the 173-191 modalities, in which the cell population is expanded at least 4 times, eg, at least 5 times, eg, at least 10 times, eg eg, for cells that are not cultured according to step (b).
[0865] [0865] 193. The method of any of the 173-192 modalities, in which the introduction of step (c) comprises an electroporation.
[0866] [0866] 194. The 193 mode method, in which electroporation comprises 1 to 5 pulses, eg 1 pulse, and in which each pulse is a pulse voltage ranging from 700 volts to 2000 volts and has a duration pulse rates ranging from 10 ms to 100 ms.
[0867] [0867] 195. The 194 method, in which the electroporation comprises 1 pulse.
[0868] [0868] 196. The method of any of the 194-195 modalities, in which the pulse voltage varies from 1500 to 1900 volts, for example, is 1700 volts.
[0869] [0869] 197. The method of any of the 194-196 modalities, in which the pulse duration varies from 10 ms to 40 ms, for example, it is 20 ms.
[0870] [0870] 198. The method of any of the 173-197 modalities, wherein the cell (e.g., cell population) provided in step (a) is a human cell (e.g., a human cell population).
[0871] [0871] 199. The 198 method, in which the cell (eg cell population) provided in step (a) is isolated from the bone marrow, peripheral blood (eg mobilized peripheral blood) or umbilical cord blood .
[0872] [0872] 200. The method of modality 199, in which (i) the cell (eg cell population) provided in step (a) is isolated from the bone marrow, for example, it is isolated from the bone marrow of a patient who suffers from hemoglobinopathy, optionally where hemoglobinopathy is sickle cell disease or thalassemia, optionally, where thalassemia is beta-thalassemia; or (ii) the cell (e.g., cell population) provided in step (a) is isolated from the peripheral blood, for example, it is isolated from the peripheral blood of a patient suffering from hemoglobinopathy,
[0873] [0873] 201. The method of any of the 173-200 modalities, in which the cell population provided in step (a) is enriched for CD34 + cells.
[0874] [0874] 202. The method of any of the 173-201 modalities, in which, following the introduction of step (c), the cell (for example, cell population) is cryopreserved.
[0875] [0875] 203. The method of any of the modalities 173-202, wherein, subsequent to the introduction of step (c), the cell (for example, cell population) comprises: (a) an indel in or near a genomic DNA sequence complementary to the target domain of the first gRNA molecule; and / or b) deletion comprising sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of the first gRNA molecule (e.g., at least 90% complementary to the target domain of gRNA, for example, completely complementary to the target domain of gRNA) in the HBG1 promoter region and a sequence complementary to the target domain of the first gRNA molecule (for example, at least 90% complementary to the target domain of gRNA, for example, completely complementary to the targeting gRNA) in the HBG2 promoter region, optionally, where the indel, for example, the deletion, does not comprise a nucleotide arranged between
[0876] [0876] 204. The method of any of the modalities 173-203, in which, after the introduction of step (c), at least about 40%, at least about 50%, at least about 60%, at least least about
[0877] [0877] 205. A cell (for example, cell population), obtainable by the method, in any of the 173-204 modalities.
[0878] [0878] 206. A method of treating a hemoglobinopathy, comprising administering to a human patient a composition comprising a cell, of any of the 134-156 modalities, a cell population, of any of the 157-167 modalities, or a cell (for example, a cell population) of 205 mode.
[0879] [0879] 207. A method of increasing fetal hemoglobin expression in a human patient, comprising administering to said human patient a composition comprising a cell, of any of the 134-156 modalities, a population of cells, of any one of modalities 157-167, or cell (e.g., a population of cells) of modality 205.
[0880] [0880] 208. The 206 method, in which hemoglobinopathy is beta thalassemia or sickle cell disease.
[0881] [0881] 209. The method of any of the 206-208 modalities, wherein the human patient is administered a composition comprising at least about 1 and 6 cells, of the 205 modality, per kg of the human patient's body weight, e.g. , at least about 1 and 6 CD34 + cells, of modality 205, per kg of human patient's body weight.
[0882] [0882] 210. The method of modality 209, wherein the human patient is administered a composition comprising at least about 2 and 6 cells, of modality 205, per kg of body weight of the human patient, e.g., at least about 2e6 CD34 + cells, of modality 205, per kg of human patient's body weight.
[0883] [0883] 211. The method of modality 209, wherein the human patient is administered a composition comprising about 2e6 to about 10e6 cells, of modality 205, per kg of body weight of the human patient, e.g. at least about 2e6 to about 10e6 CD34 + cells, of modality 205, per kg of human patient's body weight.
[0884] [0884] 212. A gRNA molecule, of any of modalities 1-86, a composition, of any of modalities 87-114 or 168-169, a nucleic acid, of any of modalities 105-112, a vector , of any of the modalities 113-114, a cell, of any of the modalities 134-156 or 205, or a population of cells, of any of the modalities 157-167, for use as a medicament.
[0885] [0885] 213. A gRNA molecule, of any of modalities 1-86, a composition, of any of modalities 87-114 or 168-169, a nucleic acid, of any of modalities 105-112, a vector , of any of the modalities 113-114, a cell, of any of the modalities 134-156 or 205, or a population of cells, of any of the modalities 157-167, for use in the manufacture of a medicament.
[0886] [0886] 214. A gRNA molecule, of any of modalities 1-86, a composition, of any of modalities 87-114 or 168-169, a nucleic acid, of any of modalities 105-112, a vector , of any of the modalities 113-114, a cell, of any of the modalities 134-156 or 205, or a population of cells, of any of the modalities 157-167, for use in the treatment of a disease.
[0887] [0887] 215. A gRNA molecule, of any of modalities 1-86, a composition, of any of modalities 87-114 or 168-169, a nucleic acid, of any of modalities 105-112, a vector , of any of the modalities 113-114, a cell, of any of the modalities 134-156 or 205, or a population of cells, of any of the modalities 1157-167, for use in the treatment of a disease, where disease is a hemoglobinopathy.
[0888] [0888] 216. A gRNA molecule, of any of modalities 1-86, a composition of any of modalities 87-114 or 168-169, a nucleic acid of any of modalities 105-112, a vector of any of modalities 113-114, a cell of any of modalities 134-156 or 205, or a population of cells of any of modalities 157-167, for use in the treatment of a disease, in which hemoglobinopathy is beta thalassemia or sickle cell disease.
[0889] [0889] The selection of the initial guide was carried out in silico using a human reference genome and user-defined genomic regions of interest (eg a gene, a gene exon, non-coding regulatory region, etc.), to identify PAMs in the regions of interest. For each identified MAP, the analyzes were performed and the statistics reported. The gRNA molecules were further selected and classified based on a number of methods to determine efficiency and effectiveness, for example, as described herein. This example provides the experimental details for procedures that can be used to test the CRISPR systems, gRNAs and other aspects of the invention described here. Any changes to these general procedures that were employed in a specific experiment are noted in this example.
[0890] [0890] Throughout the Examples, in the experiments below, saRNA molecules or dgRNA molecules were used. Unless otherwise indicated, where the dgRNA molecules were used, gRNA includes the following: crRNA: [target domain] - [SEQ ID NO: 201] tracr (trRNA): SEQ ID NO: 224;
[0891] [0891] Unless otherwise indicated, in experiments employing a sgRNA molecule, the following sequence was used: [target domain] - [SEQ ID NO: 195]) - UUUU Next Generation Sequencing (NGS) and Analysis for Target Cleavage Efficiency and Indel Formation
[0892] [0892] To determine the efficiency of editing (for example, cleavage) of the target location in the genome, deep sequencing was used to identify the presence of insertions and deletions introduced by non-homologous end joining.
[0893] [0893] In summary, the PCR primers were designed around the target site and the genomic area of interest was amplified by PCR in edited and unedited samples. Resulting amplicons were converted to Illumina sequencing libraries and sequenced. The sequencing readings were aligned with the human genome reference and subjected to the so-called variant analysis, allowing us to determine the sequence variants and their frequency in the target region of interest. The data were submitted to several quality filters and the known variants or variants identified only in the unedited samples were excluded. The percentage of editing was defined as the percentage of all insertion or deletion events occurring at the target site of interest (that is, insertion and exclusion readings at the target site over the total number of readings (wild type and mutant readings ) at the target site A detailed description of the NGS analysis process is described in Example 2.1.
[0894] [0894] The addition of crRNA and trRNA to the Cas9 protein results in the formation of the active Cas9 ribonucleoprotein complex (RNP), which mediates binding to the target region specified by the crRNA and specific cleavage of the target genomic DNA. This complex was formed by the loading of trRNA and crRNA into Cas9, which is believed to cause conformational changes to Cas9 allowing it to bind and cleave dsDNA.
[0895] [0895] The crRNA and trRNA were denatured separately at 95 ° C for 2 minutes and allowed to reach room temperature. Cas9 protein (10 mg / mL) was added to 5X CCE buffer (20 mM HEPES, 100 mM KCI, 5 mM MgCl2, 1 MM DTT, 5% glycerol), to which trRNA and the various cr »RNAs ( in separate reactions) and incubated at 37 ºC for 10 minutes, thus forming the active RNP complex. The complex was delivered by electroporation and other methods in a wide variety of cells, including HEK-293 and CD34 + hematopoietic cells. Delivery of RNPs to CD34 + HSCs Cas9 RNPs were delivered in CD34 + HSCs.
[0896] [0896] CD34 + HSCs were thawed and cultured (at -500,000 cells / mL) overnight in StemSpan SFEM medium (StemCell Technologies) with the addition of I1L-12, SCF, TPO, FIt3L and Pen / Strep. Approximately 90,000 cells were aliquoted and pelleted for each RNP delivery reaction. The cells were then resuspended in P3 60 µL nucleofection buffer (Lonza), to which the active RNP was subsequently added. The HSCs were then electroporated (for example, nucleofected using the CA-137 program on a Lonza Nucleofector) in triplicate (20 uL / electroporation). Immediately after electroporation, StemSpan SFEM medium (with IL-12, SCF, TPO, FItàaL and Pen / Strep) was added to HSCs, which were cultured for at least 24 hours. The HSCs were then harvested and subjected to analysis of expression of T7E1, NGS and / or surface markers. HSC Functional Assay
[0897] [0897] CD34 + HSCs can be tested for the stem cell phenotype using known techniques, such as flow cytometry or the in vitro colony formation assay. As an example, the cells were assayed by the in vitro colony formation assay (CFC) using the Methocult H4034 Optimum kit (StemCell Technologies) using the manufacturer's protocol. Briefly, 500-2000 CD34 + cells in volume <= 100 µL are added to 1- 1,25 ml Methocult. The mixture was stirred vigorously for 4-5 seconds to mix thoroughly, then left to stand at room temperature for at least 5 minutes. Using a syringe, 1-1.25 ml of MethoCult + cells were transferred to a 35 mm disc or well on a 6-well plate. Colony numbers and morphology were evaluated after 12-14 days, according to the manufacturer's protocol. Xeno-transplantation in vivo
[0898] [0898] HSCs are functionally defined by their ability to self-renew and by differentiation of multiple lineages. This functionality can only be evaluated in vivo. The gold standard for determining human HSC function is through xeno-transplantation in the mouse gamma NOD-SCID (NSG) which through a series of mutations is severely immunocompromised and, therefore, can act as a receptor for human cells. The HSCs that follow the edition were transplanted into NSG mice to validate that the induced edition does not affect the HSC function. Periodic analysis of peripheral blood was used to assess the development of human lineage and chimerism and secondary transplantation, after 20 weeks it was used to establish the presence of functional HSCs, as described in more detail in these examples. Example 2.1 Editing of the non-deletion HPFH region in hematopoietic and progenitor stem cells (HSPCs) using CRISPR-Cas9 for the repression of fetal globin expression in adult erythroid cells Methods:
[0899] [0899] Human CD34 * cell culture. Human CD34 + cells were isolated from peripheral blood mobilized G-CSF from adult donors (AllCells) using immunoselection (Miltenyi), according to the manufacturer's instructions, and expanded for 4 to 6 days using StemSpan SFEM (StemCell Technologies; Cat No. 09650 ) supplemented with 50 ng / mL each of thrombopoietin (Tpo, Life Technologies, Cat. & PHC9514), FIt3 ligand (FIt3L, Life Technologies, Cat. tHPHC9413), human stem cell factor (SCF, Life Technologies, Cat. PHC2113) and human interleukin-6 (IL-6, Life Technologies, Cat. PHCO063), as well as 1x antibiotic / antimycotic (Gibco, Cat. 10378-016) and 500 nM of compound 4. Over of this example (including its sub-examples), where the protocol indicates that the cells were "expanded", this medium was used. This medium is also known as "stem cell expansion medium" or "expansion medium" throughout this example (including its sub-examples).
[0900] [0900] Assembly of Cas9 and ribonucleoprotein (RNP) complexes of guide RNA, preparation of HSPC and electroporation of RNP in
[0901] [0901] Erythropoiesis in vitro and FACS analysis for erythroid cells containing HbF. After electroporation, cells were immediately transferred to 250 µl of preheated erythroid differentiation medium (EDM), consisting of IMDM (GE Life Sciences, Cat. No. SH30228.01), 330 puo / mL holo-transferrin (Invitria Cat No. 777TRFO29), 10 pug / mL recombinant human insulin (Gibco Cat No. A1138211), 2 IU / mL heparin (Sigma, part No. H3393), 5% serum
[0902] [0902] From day 7 remaining, cells cultured 80,000 per condition were transferred to an EDM-Il culture medium for further differentiation until day 11. EDM-II consists of EDM-I supplemented with only 100 ng / ml of SCF. On day 11 the cells were counted and 200,000 cells per condition were transferred to EDM without additional supplements. On day 14, cells were stained for analysis of HbF expression similar to day 7, but surface markers were excluded from staining to prevent cell aggregation.
[0903] [0903] Genomic DNA preparation and next generation sequencing (NGS). Genomic DNA was prepared from edited and unedited HSPC, 7 days after electroporation, using Quick Extract DNA extraction solution (Epicenter Cat No. QE09050). To determine the editing efficiency and insertion and deletion patterns (indels), PCR products were generated using primers flanking the target sites, which were then subjected to next generation sequencing (NGS), as described in the literature. The percentage editing of corresponding strings in unedited samples (electroporated with RNP's consisting only of Cas9 and Tracr) was typically less than 1% and never exceeded 3%.
[0904] [0904] Preparation of the NGS library and sequencing of amplicons. The PCR amplicons were purified using 1.8x Agencourt AmpureXP beads (Beckman Coulter) following the manufacturer's recommendations. Amplicons were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) following the manufacturer's recommendations. The Illumina sequencing libraries were generated using the Nextera DNA Library Prep kit (Illumina) following the manufacturer's recommendations with the following changes. Tagmentation was carried out in a final volume of 5 µl using 5 ng of purified PCR product, 0.15 µl of Nextera tagment enzyme and tagmentation buffer previously described by Wang et al (PMID: 24071908; incorporated here by reference) . Tagmented amplicons were then amplified by PCR in a final volume of 50 µL using a final concentration of 0.2 mM dNTP (Life Technologies), Illumina index PCR primers 0.2 µM (Integrated DNA Technologies), 1x polymerase buffer of Phusion DNA (New England Biolabs) and 1U of Phusion DNA polymerase (New England Biolabs). The PCR cycling conditions used were as follows: 72 ºC for 3 min, 98 ºC for 2 min and 15 cycles of 98 ºC for 10 s, 63 ºC for 30 s and 72 ºC for 3 min. The sequencing libraries were then purified using 1.0x Agecourt AmpureXP beads (Beckman Coulter) following the manufacturers' recommendations. The sequencing libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) following the manufacturer's recommendations and grouped equimolar for sequencing. The sequencing libraries were sequenced with 150 base pair end readings on a MiSeq sequencer following the manufacturer's recommendations (Illumina). A minimum of 1000 times sequencing coverage was generated by amplicon.
[0905] [0905] NGS QC sequencing data and analysis of variants. Using standard parameters, the Illumina MiSeq analysis software (MiSegq reporter, version 2.6.2, Illumina) was used to generate amplicon-specific FASTQ sequencing data files (Cock et al, Nucleic Acids Res. 2010, 38 (6): 1767-71, PMID: 20015970). The FASTQ files were then processed through a variant pipeline analysis developed in-house, consisting of a series of public domain software packages joined using a standard Perl script wrapper. The workflow used was divided into five stages.
[0906] [0906] Step 1, PCR primer and QC target and off target sequence: For both on and off target sites, the proto-spacer sequence of the 20 nucleotide gRNA target domain plus the PAM sequence and primer sequences Target-specific PCRs (left and right without additional Illumina sequences) were aligned to the human genome reference sequence (GRCh38 construct) using a BLAST search (version 2.2.29+, Altschul et al., J Mol Biol., 1990, 215 (3): 403-10, PMID: 2231712). Target and off-target sites with multiple genomic sites were signaled.
[0907] [0907] Step 2, unzipping the sequencer file: The FASTQ.GZ files generated by the lllumina sequencer were unzipped to FASTQ files using the gzip script (version
[0908] [0908] Step 3, sequence reading alignment and quality cut: The sequencing readings in the FASTQ files were aligned to the human genome reference sequence (GRCh38 construct) using the BIVA-MEM aligner (version 0.7.4-r385, Li and Durbin, Bioinformatics, 2009, 25 (14): 1754-60, PMID: 19451168) using 'hard-clipping' to trim the 3 'ends of Illumina sequence readings and low-quality bases. The resulting aligned readings, in the BAM file format (Li et al, Bioinformatics, 2009 25 (16): 2078-9, PMID: 19505943), were converted to FASTQ files using the SAMtools script (version 0.1.19-44428cd, Li et al, Bioinformatics, 2009 25 (16): 2078-9, PMID: 19505943). The FASTQ files were then aligned again with the reference sequence of the human genome (construct GRCh38) using the BWVA-MEM aligner, this time without "hard-clipping".
[0909] [0909] Step 4, variant analysis (SNP and INDEL): The BAM files of aligned readings were processed using the variant caller
[0910] [0910] Step 5, dbSNP filtering and treated / untreated differential analysis: The identified variants were filtered for known variants (SNPs and indels) found in dbSNP (construct 142, Shery et al., Nucleic Acids Res. 2001, 29 ( 1): 308-11, PMID: 11125122). The variants in the treated samples were additionally filtered to exclude: 1) variants identified in the unedited control samples; 2) variants with a VarDict chain deviation of 2: 1 (where the forward and reverse read counts that support the reference sequence are balanced, but unbalanced for the so-called non-reference variant); 3) variants located> 5 bp on each side of the potential Cas9 cut site; 4) single nucleotide variants. Results:
[0911] [0911] Here we show the surprising result that the targeted disruption of specific sequences (for example, by creating indel in or near these regions) in the regions promoting
[0912] [0912] For efficient genome editing via programmable nuclease, Cas9, successful delivery of guide RNA (gRNA) and Cas9 protein to target cells and tissues is essential. Here, we show the delivery of pre-complex gRNA / Cas9 ribonucleoprotein (RNP) complexes by electroporation leads to efficient and specific genome editing almost immediately after delivery and are degraded in cells, reducing off-target effects. In contrast, the use of plasmid and viral vector systems used to deliver Cas9 results in prolonged enzyme expression, which can exacerbate the off-target effects associated with the system. In addition, delivery of RNPs to target cells does not require additional tools that greatly facilitate the translation of the genome edition for therapeutic purposes in the clinic.
[0913] [0913] Recombinant S. Pyogenes Cas9 protein (SEQ ID NO: 236) was purified from Escherichia coli and complexed with double synthetic gRNAs (dgRNA) consisting of crRNA and tracr to generate ribonucleoprotein (RNP) complexes. The list and sequences of gRNA target domains used in the study are shown in Table 1. Most gRNA sequences have perfect targets or only 1 or 2 mismatches in both the HBG1 and HBG2 promoter areas. This situation is due to the duplication of HBG genes in the human beta globin locus. RNP complexes were subjected to electroporation on CD34 + HSPC through electroporation as described in materials and methods. The cells were expanded before delivery of the RNP complexes. Without being linked to theory, active cell division can facilitate the capture of RNP complexes delivered by electroporation.
[0914] [0914] Table 4 List of gRNAs targeting the HBG1 and HBG2 promoter region used in the current study. All gRNA molecules were tested in duplicate in the dgRNA format, described above.
[0915] [0915] HSPC edited and not edited by genome was analyzed by flow cytometry for expression levels of fetal globin and the cell surface erythroid marker of the transferrin receptor (CD71) using antibodies conjugated to fluorescent dyes. Live cells were identified and blocked by excluding Live / Dead Violet. Genome editing did not adversely affect erythroid differentiation, since cultured cells showed percentages of CD71 + cells consistent with erythroblasts similar to unedited cells. The delivery of these RNPs gRNA to HSPCs resulted in an increase in the percentage of descending erythroid cells containing HbF (up to 62.75%) compared to simulated electroporated cells (16.9%) on day 7 after electroporation (Table 4). The additional assessment of HbF induction levels on day 14 confirmed high levels of induction for the best performing dgRNA sequences (table 4), although the levels of HbF detected in the controls were higher compared to day 7. Induction of HbF positive cells by dgRNA including the g8 target domain targeting BCL11A exon 2 was also observed in parallel, and several of the gRNAs for the HBG1 or HBG 2 regions resulted in higher levels of F cells than g8. Products from
[0916] [0916] The methods are as in Example 2.1, with the following modifications.
[0917] [0917] Human CD34 * cell culture. Human CD34 + cells were derived from bone marrow from healthy adult donors (Lonza t 2M-101D catalog). The cells were thawed and then expanded for 6 days.
[0918] [0918] Assembly of Cas9 and ribonucleoprotein (RNP) complexes of guide RNA, preparation of HSPC and electroporation of RNP in HSPC. For RNP formation using simple guide RNAs (sgaRNAs), 12 µg of each of saRNA, 12 µg of CAS9 protein and 1 µl of 10x CCE buffer (20 mM HEPES, 100 mM KCL, 5 mM MgCL2, 5% Glycerol and DTT Recently added 1 mM were combined in a total volume of 10 µL, incubated at 37 ° C for 5 min. For the condition No / control, vehicle instead of sagRNA was added. The cell density in P3 buffer + supplement was 3.9 x 10º / mL.
[0919] [0919] Erythropoiesis in vitro and FACS analysis for erythroid cells containing HbF. On day 11, the cells were transferred to EDM without additional supplements. On day 14 and day 18 the cells were pelleted and resuspended in fresh EDM without additional supplements. An aliquot of cells was taken for analysis of HbF expression on day 14 and day 21. The cells were stained similar to day 7, but surface markers were excluded from staining to prevent cell aggregation and anti-HbF antibody was used the 1:20 dilution.
[0920] [0920] Preparation of genomic DNA and next generation sequencing (NGS). Genomic DNA was prepared from edited and unedited HSPC, 3 days after electroporation, using Quick Extract DNA extraction solution (Epicenter Cat No. QE09050). The NGS analysis described below did not show significant editing in electroporated control samples with Cas9 alone.
[0921] [0921] The preparation of the NGS library and sequencing of the amplification products was carried out as described in Example 2.1. The analysis of the NGS QC and variant sequencing data was performed as described in Example 2.1. Results:
[0922] [0922] We show here that targeted disruption of specific sequences within the promoter regions of HBG1 or HBG2, leading to the production of F cells, can also be performed using sRNA format gRNA.
[0923] [0923] Table 5. List of selected gRNAs targeting the HBG1 and HBG2 promoter region used in the current study. All sgaRNA molecules were tested in duplicate in the dgRNA format, described above.
[0924] [0924] Adult bone marrow-derived HSPCs were electroporated with RNP complexes formed from recombinant protein Cas9 from S. Pyogenes (SEQ ID NO: 236) and the indicated gRNA of the saRNA format. The resulting edited and unedited HSPC by genome was analyzed by flow cytometry for expression levels of fetal globin and the cell surface erythroid marker of the transferrin receptor (CD71) using antibodies conjugated to fluorescent dyes. Live cells were identified and blocked by excluding Live / Dead Violet. The delivery of these RNPs gRNA to HSPCs resulted in an increase in the percentage of erythroid cells containing HbF (up to 74.6%) compared to simulated electroporated cells (39.0%) on day 7 after electroporation (Table 5). Additional assessment of HbF induction levels on day 14 and day 21 confirmed high levels of induction for the best performing SsaRNA sequences (Table 5). PCR products of genomic DNA from the HBG1 and HBG2 promoter region isolated on day 3 after electroporation were also subjected to next generation sequencing (NGS) to determine the percentage of alleles edited in the cell population. High percentages of genome editing (excluding large deletions) in the HBG1 and HBG2 promoter region were observed in many cell cultures electroporated with RNPs containing Cas9 and sgRNA from the given target domain (Table 5), but not in control cells without delivered saRNA (only Cas9).
[0925] [0925] Targeting sites selected with> 17% increase in HbF + erythroid cells on day 7 in Example 2.1 were included in this study. The experimental design differed in two significant ways from that previously described in Example 2.1: the gRNA format (sgRNA instead of dgRNA) and the HSPC source (different donors and bone marrow derivatives instead of mobilized peripheral blood derivatives). Without being bound by theory, differences in baseline percentages of HbF + cells in unedited cell cultures between studies can be caused by inherent differences between HSPC sources. Despite these differences, all targeting sites, with the exception of GCR-47, remained associated with a> 17% increase in HbF + erythroid cells on day 7 (Figure 7). This increase was maintained until the 21st for GCR-0067 (Figure 7). Additionally, the formation of indel>
[0926] [0926] The methods are as in Example 2.1, with the following modifications.
[0927] [0927] Human CD34 * cell culture. Human CD34 + cells were derived from bone marrow from healthy adult donors (Lonza tt2M-101D catalog and Hemacare tt catalog (BM34-C). The cells were thawed and then expanded for 6 days.
[0928] [0928] Assembly of Cas9 and ribonucleoprotein (RNP) complexes of guide RNA, preparation of HSPC and electroporation of RNP in HSPC. For the formation of RNP using simple guide RNAs (sgRNAs),
[0929] [0929] Erythropoiesis in vitro and FACS analysis for erythroid cells containing HbF. On day 11, the cells were transferred to EDM without additional supplements. On day 14 and day 18 the cells were pelleted and resuspended in fresh EDM without additional supplements. An aliquot of cells was taken for analysis of HbF expression on day 14 and day 21. The cells were stained similar to day 7, but surface markers were excluded from staining to prevent cell aggregation and anti-HbF antibody was used the 1:20 dilution.
[0930] [0930] Preparation of genomic DNA and next generation sequencing (NGS). Genomic DNA was prepared from edited and unedited HSPC, 3 days after electroporation, using Quick Extract DNA extraction solution (Epicenter Cat No. QE09050). The NGS analysis described below did not show significant editing in electroporated control samples with Cas9 alone.
[0931] [0931] The preparation of the NGS library and sequencing of the amplification products was carried out as described in Example 2.1. The analysis of the NGS QC and variant sequencing data was performed as described in Example 2.1.
[0932] [0932] Non-quantitative PCR for detecting inversions and large deletions. The following sets of primers were used to amplify the gODNA in the HBG1 and HBG2 region. P1: 5'- TGCTGAGATGAAACAGGCGT-3 'direct primer (SEQ ID NO: 257), 5- TTAGGCATCCACAAGGGCTG-3' reverse primer (SEQ ID NO: 258), expected product of —2.8 kb for deletion between target sites / off target HBG1 and HBG2, expected product of —7.7 kb for inversion between target sites / off target HBG1 and HBG2, expected product of —7.7 kb for no deletion or large inversion (unedited indels or less at the target / off-target sites of HBG1 and / or HBG2, individually). P2: direct primer 5- GCTCTACAAATGGAACCCAACC-3 '(SEQ ID NO: 259), reverse primer 5-- CTGCTCTGATCTCTAACACCTCA-3' (SEQ ID NO: 260), no products expected for deletion between target sites / outside HBG1 target and HBG2, no product expected for inversion between target / non-target sites for HBG1 and HBG2, expected product of —3.8 kb for no major deletion or inversion (unedited or smaller indels at target / off target sites) HBG1 and / or HBG 2, individually). P3: forward primer 5- GAAGATACAGCTTGCCTCCGA -3 '(SEQ ID NO: 261), reverse primer 5- TIGCTGAGATGAAACAGGCGT -3' (SEQ ID NO: 262), no products expected for deletion between target / off-site HBG1 and HBG2, expected product of 1.75 kb for inversion between HBG1 and HBG2 target / out-of-target sites, no expected product for any large deletion or inversion (unedited or smaller indels in HBG1 target / out-of-target sites) and / or HBG 2, individually). The PCR products were visualized on an agarose gel along with a reference ladder containing bands of 0.5, 1, 1.5, 2, 3.4, 5, 6, 8 and 10 kb (L; New England Biolabs ft N3232L). The selected products were isolated as indicated and subjected to NGS as described in Example 2.1.
[0933] [0933] Quantification of large deletions. Large deletions between targeting sites in HBG1 and HBG2 promoters were quantified by digital drop PCR (ddPCR) in a non-competitive assay format to determine the number of copies according to the manufacturer's recommendations. Briefly, the gDNA was combined with ddPCR SuperMix for Probes (without dUTPs) (BioRad Cat 41863024), Hindlll-HF restriction enzyme (NEB Cat Z & R3104S) and each primer probe mixture, transferred to a DG8 cartridge together with the Droplet Generation for Probes (BioRad Cat tt1863005) and droplets generated with a QX200 droplet generator (BioRad). The droplets were subjected to POR in a Touch C1000 Thermocycler with Deep Well Reaction Module-96 (BioRad), followed by detection with QX200 droplet reader (BioRad). The copies per ul were determined by analysis with the QuantaSoft software (BioRad). A set of custom initiator probes (Life Technologies Cat tHAPZW76R, PN4331348, Direct Initiator: ACGGATAAGTAGATATTGAGGTAAGC (SEQ | D NO: 263), Reverse Initiator: GTCTCTTTCAGTTAGCAGTGG (SEQ ID NO: 264), FAM Probe: SEGGTGA 265)) used to amplify the gDNA within the HBG1- HBG2 intergenic region. A human TaqMan Copy Number Reference Assay, RNase P (Thermo Fisher cat f4403326) was used as the reference amplicon. Copies per ul for the HBG1-HBG2 and RNase P amplicons were within the manufacturer's linear range. Percentage of deletion has been reported as 100% times | minus the copy ratio per ul for HBG1-HBG2 and amplicons RNase P. For unedited control samples, the percentage of deletions of 2.4% was calculated, which may reflect the background of the assay.
[0934] [0934] Here we show that the targeted disruption of specific sequences within the HBG1 and HBG2 promoter regions leading to the production of F cells is associated with both indels on the HBG1 or HBG2 target or off-site, as well as deletions and inversions of the intervening region. The NGS analysis of the amplicons confirms this observation.
[0935] [0935] Table 6 List of selected gRNAs targeting the HBG1 and HBG2 promoter region used in the current study to edit cells from the first independent donor. All SgRNA molecules were tested in duplicate in the saRNA format, described above. Domain ID [%%%%%%%%%% | HbF + | HbF + | HbF + | HbF + | HbF + | HbF + | HBG1 | HBG1 | HBG2 | HBG2 nio- (cell | (cell | (cell | | cell | (cell | (cell | edited | edited | edited | edited by target F),) [as F)) as F),) las FP), [as F),) [the FP) | the, of, the, the, of the mean | deviate | average | deviate | average | deviate | Average | deviate | Average | divert the RNA from the guide replica | pattern | replica | pattern | replica | pattern | replica | pattern | replica | standard day [the day / day the day day the day | as o o 7 7 14 14 21 21 None | 37.6 17.1 na na na na na / C ontrol and GCR- | 61.3 75.1 59.1 1.0 0001 GCR- 2.1 67.3 0008 GCR- 75.0 0010 GCR- 43.8 73.5 80.4 0048 GCR- | 61.9 49.7 1n7 70.1 86.4 0051 GCR- | 72.2 58.1 1n7 774 68.8 12 77.5 0067
[0936] [0936] Table 7 List of selected gRNAs targeting the HBG1 and HBG2 promoter region used in the current study to edit cells from the second independent donor. All SARNA molecules were tested in duplicate in the saRNA format, described above.
[0937] [0937] Adult HSPC bone marrow derived from 2 independent donors were electroporated with RNP complexes formed from the recombinant protein Cas9 from S. Pyogenes (SEQ ID NO: 236) and the indicated gRNA of the saRNA format. evaluated saRNAs had targeting sequences outside the known areas of promoter function in the HBG1 and HBG2 genes (for example, map to chr11: 5,250,094-5,250,237; hg38 and chr11: 5,255,022-5,255,164; ho38 (Figure 4 and Figure 5) respectively). The resulting edited and unedited HSPC by genome was analyzed by flow cytometry for expression levels of fetal globin and the cell surface erythroid marker of the transferrin receptor (CD71) using antibodies conjugated to fluorescent dyes. Live cells were identified and blocked by excluding Live / Dead Violet. The delivery of these RNPs gRNA to HSPCs resulted in an increase in the percentage of descending erythroid cells containing HbF compared to simulated electroporated cells on days 7, 14 and 21 after electroporation (Tables 6 and 7). All targeting sites included were associated with an> 17% increase in HbF + erythroid cells from both donors at all times - days 7, 14 and 21 (Figure 8). PCR products of genomic DNA from the HBG1 and HBG promoter region isolates on day 3 after electroporation were also subjected to next generation sequencing (NGS) to determine the percentage of alleles edited in the cell population. High percentages of genome editing in the promoter region of HBG1 and HBG 2 (53% to 92% indels) were observed in cell cultures from both donors electroporated with RNPs containing Cas9 and saRNAs with the target domains GCR-0008, GCR -0048, GCR-0051 and GCR-0067 (Tables 6 and 7), but not in control cells without delivered saRNA (Cas9 only) The GCR-0010 target domain was associated with low but considerable editing percentages in the HBG1 promoter region and HBG2 (17% to 28% indels) in cell cultures from both donors; while GCR-0001 was associated with efficient and selective editing in HBG1 (59% and 62%) compared to HBG2 (1% and 1%) in the two donors (Tables 6 and 7).
[0938] [0938] The target domains GCR-0048 and GCR-0067 are present in both HBG1 and HBG2, and the other target domains are present in HBG1 (GCR-0001, GCR-0008 and GCR-0010) or HBG2 (GCR -0051) with an incompatible potential outside the target site on the other promoter. Simultaneous cleavage at both HBG1 and HBG2 target / non-target sites has the potential to result in deletion and / or inversion of the 4.9 kb intervening genomic sequence; here, sometimes also referred to as '4.9 kb' or 'HBG1-HBG2' or 'large' inversion or deletion. Thus, a set of three different PCR reactions was used to detect the presence of genomes with deletion or inversion in that region. The P2 reaction was designed so that genomic sequences without the HBG1-HBG2 deletion or inversion would be amplified with a 3.8 kb resulting product, while the sequences with the HBG1-HBG2 deletion or inversion would not be amplified.
[0939] [0939] For further characterization of the region sequence covering the HBG1 and HBG2 editing sites, a fourth long-range PCR condition (P4) was developed to amplify a 6192 bp region encompassing the HBG1 and HBG2 editing sites for gRNA GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067. The amplification of edited samples in the genome results in two sizes of amplicons (1.2 kb and 6.2 kb), while unedited samples generate only one size of amplicon (6.2 kb). Sequence analysis of the 6.2 kb and 1.2 kb amplicons shows that the 1.2 kb amplicons are the result of 4.9 kb deletions between the HBG1 and HBG2 cut sites, while the 6.2 kb amplicons kb consists of indelible and wild type alleles at the HBG1 and HBG2 editing sites. The sequencing analysis also shows a low level of inversions between the HBG1 and HBG2 cut sites where the sequence excised between the two sites was reincorporated into the genome in the opposite direction. This analysis is not quantitative, but inversion is probably very rare, as it is only detected in a small percentage (<1%) of sequencing readings covering the HBG1 and HBG2 cut-off sites.
[0940] [0940] Together, these results suggest that, in addition to indels at the target / non-target site of HBG1 or HBG2, cultures electroporated with RNPs containing Cas9 and saRNAs with the indicated target domains contain alleles edited with deletion or inversion of intervening HBG1-HBG 2 region. Thus, a given post-editing allele could be an inversion of HBG1-HBG1, a deletion of HBG1-HBG2, an indel located at the HBG1 site, an indel located at the HBG2 site or an indel located at the HBG1 site along with a located at the HBG2 site without modification of the intervening region. The most common editing repair pattern variants on the target generated by editing with the gCRNAs GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067 are shown in Table 7-2. The variants shown are the localized indels generated at the HBG1 and HBG2 loci and large deletions of 4.9 kb caused by the excision of the sequence between the HBG1 and HBG2 cut sites. Localized indels were characterized using PCR and NGS analysis as described in Example 2.1. A quantitative ddPCR assay was developed to determine the frequency of alleles with the large 4.9 kb deletion of the intervening region HBG1 to HBG2 (Figure 10). Briefly, the copy number of the region upstream of the HBG1 promoter (between the 5.2 kb Fwd and Rev primers shown in Figure 10) was defined in relation to the copy number at the RPPH1 locus as described in the methods. The allelic frequencies for the smaller indels located are not related to the total frequency of indel, because the amplification of the HBG1 or HBG2 site for NGS would not occur in alleles containing the 4.9 kb inversion or deletion. For example, for the GCR-0001 gRNA the deletion frequency of 4.9kb is 35.2%, with the remaining 64.8% (100% - 35.2%, ignoring inversions) being a mixture of wild type indels. and located minors. Therefore, the 9% deletion of the single base pair A would be 5.8% of the total frequency of the indel, that is, 9% of 64.8%. The allele frequencies shown in Table 7-2 vary slightly between experiments and should not be considered as absolute values.
[0941] [0941] Table 7-2. Variants of the superior editing repair pattern on the target (collectively for any gRNA molecule, also referred to here as "indelible pattern"), generated by editing the first donor cells with gRNAs GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067. The size of the variant, the type of variant (Ins = insertion, Del = deletion), the reference allele, the variant allele, the start and end position of the variant relative to the hg38 construct of the chromosome 11 reference genome and the allelic frequency They are shown. The HBG2 locus edited with GCR-0001 gRNA does not show significant localized evidence, but based on the qPCR results, it results in a 4.9 kb deletion. * HBG1 / 2 target site with incompatibilities. Name of Ta- Type Allele of Allele Position of | Frequency gRNA reference hand variant beginning and allelic (bp) end of variant GCR-0001 No No 5250172- o, 5250171- o, 5250172- o, 5250172- 5250172- 5250172- GCR-0008 No No 5250150- 5250150- 5250151- 5250150- 5250151- 5250150- GCR- No No 5250150- (HBG2) 5255078- 1 T TA 5255079 55.8% CCCTTTA
[0942] [0942] Elevated global editing frequencies at the gamma globin locus, including high frequencies of 4.9 kb large deletions between the targeting sites of the HBG1 and HBG2 promoter, were observed after electroporation of cells with RNPs containing the domain-RNA indicated target. The target editing patterns shown were identified in cells that generated increased F cells after erythroid differentiation, as described. In embodiments, the indelible pattern for any gRNA molecule described herein includes 1, 2, 3,4, 5o0u6 most frequent indels detected at the HBG1 locus. In embodiments, the indelible standard for any gRNA molecule described herein includes 1, 2, 3, 4, 5 or 6 most frequent indels detected at the HBG2 locus. In embodiments, the indelible standard for any gRNA molecule described here includes the most frequent 1, 2, 3,4, 5 or 6 indels detected at the HBG1 locus and the most frequent 1,2, 3,4, 5 or 6 indels detected in the HBG2 locus, for example, as described in Table 7-2 (but not counting twice the deletion of approximately 4.9 kb). Example 2.4: Exemplary editing patterns in isolated subpopulations of HSPCs after editing the gamma globin promoting region Methods:
[0943] [0943] The methods are as in Example 2.1, with the following modifications.
[0944] [0944] Human CD34 * cell culture. Human CD34 + cells were isolated from mobilized peripheral blood G-CSF from adult donors (Hemacare catalog tMO01F-GCSF-3) using immunoselection (Miltenyi) according to the manufacturer's instructions and expanded for 2 days before electroporation with the complexes of RNP.
[0945] [0945] Assembly of Cas9 and ribonucleoprotein (RNP) complexes of guide RNA, preparation of HSPC and electroporation of RNP in HSPC. For RNP formation using simple guide RNAs (SgRNAs), 12 µg of sgRNA each, 12 µg of CAS9 protein and 1 µl of 10x CCE buffer (20 mM HEPES, 100 mM KCL, 5 mM MgCL2, 5% Glycerol and DTT Recently added 1 mM were combined in a total volume of 10 µl, incubated at 37 ° C for 5 min. The cell density in P3 buffer + supplement was 1.3 x 10 ° / ml. Seven replications of electroporations were performed using GCR-0067 and cells were - combined after electroporation. For condition None / control, cells were transferred directly from buffer P3 to expansion medium without electroporation or addition of Cas9. After electroporation with RNP complexes, cells were expanded for another 3 days before cell separation flow cytometry.
[0946] [0946] Cell separation by flow cytometry analysis of editing efficiency in stem subpopulations and hematopoietic progenitors. Cell cultures edited at 3 days after electroporation were harvested and incubated with anti-CD34 (BD Biosciences, Cat * t 348057), anti-CD38 (BD Biosciences, Catit 560677), anti-cCD90 (BD Biosciences, Cat 559869), anti -CD45RA (BD Biosciences, Cat 563963), anti-CD49f (BD Biosciences, Catft 562598) in FACS staining buffer consisting of HBSS (GE Life
[0947] [0947] Preparation of genomic DNA and next generation sequencing (NGS). Genomic DNA was prepared from unedited HSPC and separate subpopulations of HSPC edited in 3 days post-electroporation using the DNeasy Blood & Tissue Kit (Qiagen Cat 69504). The NGS analysis described below did not show significant editing in unedited control samples.
[0948] [0948] The preparation of the NGS library and amplicon sequencing was carried out as described in Example 2.1. The analysis of the NGS QC and variant sequencing data was performed as described in Example 2.1.
[0949] [0949] Quantification of major deletions. Large deletions between targeting sites in HBG1 and HBG2 promoters were quantified by digital drop PCR (ddPCR) in a non-competitive assay format to determine the number of copies according to the manufacturer's recommendations. Briefly, the gDNA was combined with ddPCR SuperMix for Probes (without dUTPs) (BioRad Cat 41863024), Hindlll-HF restriction enzyme (NEB Cat% & R3104S) and each primer probe mixture, transferred to a DG8 cartridge along with Oil Droplet Generation System (BioRad Cat tt1863005) and droplets generated with a QX200 droplet generator (BioRad). The droplets were subjected to PCR in a Touch C1000 Thermocycler with Deep Well Reaction Module-96 (BioRad), followed by detection with QX200 droplet reader (BioRad). The copies per ul were determined by analysis with the QuantaSoft software (BioRad). A set of custom initiator probes (Life Technologies Cat tHAPZW76R, PN4331348, Direct Launcher: ACGGATAAGTAGATATTGAGGTAAGC (SEQ | D NO: 270), Reverse Launcher: GTICTCTTTCAGTTAGCAGTGG (SEQ ID NO: 271), FAM Probe: SEAT IDTGG 272)) used to amplify gONA within the HBG1- HBG2 intergenic region. A human TaqMan Copy Number Reference Assay, RNase P (Thermo Fisher cat f4403326) was used as the reference amplicon. Copies per uL for the amplicons HBG1-HBG2 and RNase P were within the manufacturer's linear range. Percentage of deletion has been reported as 100% times | minus the copy ratio per ul for HBG1-HBG2 and RNase P amplicons. For unedited control samples, the percentage of deletions up to 13% was calculated, which may reflect the assay background.
[0950] [0950] Without being limited by theory, the HSPC CD34 + population is believed to contain cells of various potentials for grafting, self-renewal and cell fate, with additional markers further enriching cells with shared properties, including stem cell grafting long-term hematopoietic (Notta, Science, July 8, 2011; 333 (6039) 218-21. doi
[0951] [0951] The methods are as in Example 2.1, with the following modifications.
[0952] [0952] Human CD34 * cell culture. Human CD34 + cells were derived from bone marrow from healthy adult donors (catalog Lonza% & 2M-101D). The cells were thawed, then expanded for 2 days before electroporation with RNP complexes.
[0953] [0953] Assembly of Cas9 and ribonucleoprotein (RNP) complexes of guide RNA, preparation of HSPC and electroporation of RNP in HSPC. For RNP formation using simple guide RNAs (sgRNAs), 12 µg each of saRNA, 12 µg CAS9 protein and 1 µl 10x CCE buffer (20 mM HEPES, 100 mM KCL, 5 mM MgCL2, 5% Glycerol and DTT Recently added 1 mM were combined in a total volume of 10 μL, incubated at 37 ºC for 5 min. The cell density in P3 buffer + supplement was 6.64 x 10 º / mL. Two electroporation replicates were performed with cells from each one of two independent donors For the condition None / control, the vehicle instead of the sgRNA was added.After electroporation with RNP complexes, the cells returned to the expansion medium.
[0954] [0954] Preparation of genomic DNA and next generation sequencing (NGS). Genomic DNA was prepared from unedited HSPC and separate subpopulations of HSPC edited in 3 days post-electroporation using the DNeasy Blood & Tissue Kit (Qiagen Cat 69504). The NGS analysis described below did not show significant editing in electroporated control samples with Cas9 alone.
[0955] [0955] The preparation of the NGS library and sequencing of the amplification products was carried out as described in Example 2.1. The analysis of the NGS QC and variant sequencing data was performed as described in Example 2.1.
[0956] [0956] Quantification of major deletions. Large deletions between targeting sites in HBG1 and HBG2 promoters were quantified by digital drop PCR (ddPCR) in a non-competitive assay format to determine the number of copies according to the manufacturer's recommendations. Briefly, the gDNA was combined with ddPCR SuperMix for Probes (without dUTPs) (BioRad Cat 41863024), Hindlll-HF restriction enzyme (NEB Cat HR3104S) and each primer probe mixture, transferred to a DG8 cartridge together with the Droplet Generation for Probes (BioRad Cat tt1863005) and droplets generated with a QX200 droplet generator (BioRad). The droplets were subjected to PCR in a Touch C1000 Thermocycler with Deep Well Reaction Module-96 (BioRad), followed by detection with QX200 droplet reader (BioRad). Copies per ul were determined by analysis with the QuantaSoft software
[0957] [0957] Unit test of colony forming cell. Two days after delivery of RNP, viable cells were enumerated by flow cytometry on a LSR Fortessa (BD Biosciences) using TruCount tubes (BD Biosciences Cat% 340334) according to the manufacturer's recommendations. Unviable cells were broken down using DAP! (4 ', 6-diamidino-2-phenylindole). The analysis was performed using the FlowJo software (Tree Star). For the colony forming unit (CFU) assay, cells and 1x antibiotic / antimycotic (Gibco, Cat. 10378-016) were added to the MethoCult H4034 Optimum methylcellulose medium (Stemcell Technologies) and 1 mL was plated in triplicate on SmartDish plates (StemCell Technologies). The culture plates were incubated in a humidified incubator at 37 ºC. Cultures were visualized on day 14 post-plating using a StemVision (Stemcell Technologies). Colonies were marked manually using the Colony Marker software (Stemcell Technologies). The number of colonies per well (average of three wells) was divided by the number of cells plated per mL of Methocult (ranged from 226 to 318) and multiplied by 1000 to obtain the frequency of CFU per 1000 cells. Results:
[0958] [0958] Here we show that HSPCs function in the colony formation assay after targeted disruption of specific sequences within the HBG1 and HBG2 promoter regions associated with the production of edited and unedited F. HSPC cells in the genome were evaluated for the composition of progenitor cells and differentiation potential using a single colony formation assay. Colonies were counted and classified as derived from erythroid progenitor cells (CFU-erythroid [CFU-E] and rupture-forming erythroid unit [BFU-E]), granulocyte and / or macrophage progenitor cells (CFU-granulocytes, macrophages [CFU- GM]; CFU-granulocytes [CFU-G]; and CFU-macrophages [CFU-M]), or progenitor cells with multiple potentials (CFU-granulocytes, erythrocytes, macrophages, megakaryocytes [CFU-GEMM]). Table 8 List of selected gRNAs targeting the HBG1 and HBG2 promoter region used in the current study to edit cells from the first independent donor. Dd ID%%% him /% edilBFU- JCFU- [raw | Total mine - from HBG1 | HBG2 | tion from | tion EICFU- | GIM / GM | GEMM | lap directs edited | edited | HBG1- approx. And why | per minute per HBG2 min. 1000 1000 1000 1000 RNA guide cells | cell phone cell E Fr E Ee and Control [screen for [7 fes qe dm dm Rr Tm) [screen 349 [180 [25 [so la mo fa [1 [Goro jet Jeso aos fons 2 10 fo 168 | [Goroost jsto f7o lies mouth x 1 2 1 | [Goroo0sT seo hay 513 [oa [2 fm [5 [15
[0959] [0959] Table 9. List of selected gRNAs targeting the HBG1 and HBG2 promoter region used in the current study to edit cells from the second independent donor.
[0960] [0960] Both donors electroporated with RNPs with the indicated gRNAs had efficient editing, both indels located in the target / non-target sites of the HBG1 and HBG2 promoter, as well as deletions from the intervening region (Tables 8 and 9). The edited cultures were associated with a decrease in the general capacity of colony formation (Tables 8 and 9), possibly indicating a decrease in the fitness of the cells submitted to editing. Despite this reduction in the total number of colonies, all erythroid, granulocyte / macrophage and multi-potential colonies were all observed in at least one donor (Tables 8 and 9). In addition, there were minimal differences in the proportion of colony types between unedited and edited samples (Figure 14), indicating that the cell cultures edited at these target sites had no distorted differentiation capacity. Example 2.6: HSPC cell proliferation in vitro after editing the gamma globin promoter region Methods:
[0961] [0961] The methods are as in Example 2.1, with the following modifications.
[0962] [0962] Human CD34 * cell culture. Human CD34 + cells were derived from bone marrow from healthy adult donors (Lonza catalog tt2M-101D and Hemacare catalog tBM34-C). The cells were thawed, then expanded for 2 days before electroporation with RNP complexes.
[0963] [0963] Assembly of Cas9 and ribonucleoprotein (RNP) complexes of guide RNA, preparation of HSPC and electroporation of RNP in HSPC. For RNP formation using simple guide RNAs (saRNAs), 12 µg of each of saRNA, 12 µg of CAS9 protein and 1 µl of 10x CCE buffer (20 mM HEPES, 100 mM KCL, 5 mM MgCL2, 5% Glycerol and DTT Recently added 1 mM) were combined in a total volume of 10 µL, incubated at 37 ° C for 5 min. Cell density in P3 buffer + supplement was 1 x 107 to 2.5 x 10 ”/ mL. An electroporation replica was performed with cells from each of the three independent donors. For the None / control condition, the vehicle instead of sagRNA was added. After electroporation with RNP complexes, the cells returned to the expansion medium.
[0964] [0964] Preparation of genomic DNA and next generation sequencing (NGS). Genomic DNA was prepared from unedited HSPC and edited 3 days after electroporation using Quick Extract DNA Extract Solution (Epicenter Cattt! QE09050) or the NGS analysis of the DNeasy Blood & Tissue Kit (Qiagen Catitf 69504) described below, did not show significant editing in the electroporated control samples with Cas9 alone.
[0965] [0965] The preparation of the NGS library and sequencing of the amplification products was carried out as described in Example 2.1. The analysis of the NGS QC and variant sequencing data was performed as described in Example 2.1.
[0966] [0966] Cell proliferation and phenotyping. Two days after delivery of RNP, viable cells were enumerated by flow cytometry in an LSR Fortessa (BD Biosciences) using TruCount tubes (BD Biosciences Catft 340334) according to the manufacturer's recommendations.
[0967] [0967] Here we show that HSPCs can expand with typical cell composition in vitro after targeted disruption of specific sequences within the HBG1 and HBG2 promoter regions associated with the production of F. HSPC cells edited and not edited by the genome were evaluated for how much to the capacity of proliferation and cell composition in culture conditions that promote the expansion of HSPC. Independent donor replicates electroporated with RNPs containing GCR-0067 were efficiently edited (Table 9-2). Table 9-2. Percentage of edited amplicons from the HBG1 or HBG2 promoter region in cells from three independent donors using the indicated gRNA. Domain ID-% HBG1 | % HBG2 /% HBG1 |% HBG2 |% HBG1 | /% HBG2 grandfather of edited, edited, edited, edited, edited, edited, GCR- EE EEEF
[0968] [0968] Edited cultures were associated with a drop in the capacity for global proliferation, possibly indicating a decrease in the ability of the cells being edited, although this did not reach significance for three independent cell donors (Figure 15). Similar reductions were observed within the CD34 + CD90 + population enriched in hematopoietic stem cells, as in the total CD34 + population, indicating that this population is not differentially affected (Figure 15). Under these culture conditions, both HSPC and the differentiated progeny are expected to be present, thus, the cell composition was further analyzed for the expression of a more comprehensive panel of cell surface markers. The discrimination of these cell populations is exemplified in Figure 16. The cell composition was similar between edited and unedited cultures in the genome through three independent cell donors, with no significant difference in a given population by the unpaired t test (Figure 17) . The large error bars for the CD33 + population result from a single donor with insignificant CD33 + cells in the edited and unedited cultures in the genome (Figure 17). Example 3: Evaluation of Cas9 Variants Evaluation on CD34 + hematopoietic stem cells
[0969] [0969] 14 purified Streptococcus pyogenes (SPyCas9) Cas9 proteins were evaluated, measuring their inactivation efficiency of the beta-2-microglobulin (B2M) gene in primary human hematopoietic stem cells (HSCs). These proteins were divided into 3 groups: the first group consisted of SPyCas9 variants with improved selectivity (Slaymaker et al. 2015, Science 351: 84 (e1.0, e1.1 and K855A); Kleinstiver et al. 2016, Nature 529: 490 (HF)). The second group consisted of wild type SPyCas9 with different numbers and / or positions of the SV40 nuclear localization signal (NLS) and the 6xHistidine marker (His6) (SEQ ID NO: 247) or 8xHistidine (His8)
[0970] [0970] Defrost and grow the cells following Lonza's recommendations, add medium every 2 or 3 days. On day 5, pellet the cells at 200 x g for 15 min, wash once with PBS,
[0971] [0971] FACS: remove 250 μl of cells from each well of the 24-well plate, into a 96-well U-bottom plate and pellet the cells. Wash once with 2% FCS (fetal calf serum) -PBS. Add 50 ul FACS blocking buffer to the cells and incubate on ice for 10 minutes, add 1 ul of FITC-labeled B2M antibody and incubate for 30 minutes. Wash with 150 µl of FACS wash buffer once followed by again with 200 µl of FACS wash buffer once. The cells were resuspended in 200 µL of FACS buffer from FACS analysis.
[0972] [0972] Preparation of the NGS sample: transfer 250 μl of cell suspension from each well of the 24-well plate to a 1.5 ml Eppendorf tube, add 1 ml of PBS and pellet the cells. Add 100 μl of Chelex suspension, incubate at 99 ºC for 8 minutes and shake for 10 seconds, followed by incubation at 99 ºC for 8 minutes, vortex for 10 seconds. Sediment the resin by centrifugation at
[0973] [0973] The preparation of the NGS library and sequencing of the amplification products was carried out as described in Example 2.1. The analysis of the NGS QC and variant sequencing data was performed as described in Example 2.1.
[0974] [0974] Statistics: The percentage of B2M KO cells per FACS and the percentage of indels per NGS are used to assess the effectiveness of CAS 9 cleavage. The experiment was created with Cas9 as a fixed effect. Each experiment is embedded within donors, as embedded random effects. Therefore, the mixed linear model was applied for the analysis of the FACS and NGS data. Results
[0975] [0975] In order to normalize the experimental and donor variations, we graphically represent the relative activity of each protein for iProt105026, the original creation with two SV40 NLS flanking the wild type SPyCas9 and the His6 marker (SEQ ID NO: 247) at the terminal Protein C (Figure 6). Statistical analysis shows that compared to the reference Cas9 protein iProt105026, iProt106331, iProt106518, iProt106520 and iProti06521 are not significantly different in B2M inactivation in HSCs, while the other tested variants (PID426303, iProt106519, iProt106522, iProt 106545, iProt1066 iProt106745, iProt106746, iProt106747, iProt 106884) are highly significantly different from the reference iProt 105026 in inactivating B2M in HSCs. We found that moving the His6 marker (SEQ ID NO: 247) from the C terminal to the N terminal
[0976] [0976] After editing HSC genes, in addition to capturing fetal hemoglobin production by flow cytometry, changes in individual hemoglobin subunits in erythroid cells were also measured by capillary electrophoresis mass spectrometry (CE-MS). CD34 + cells derived from peripheral blood from sickle cell patients were either not edited in the presence of the Cas9 protein (with two NLS and a His marker, iProt106331) and without guide RNA, or edited by genes in the presence of the Cas9 protein and guide RNA sg0067 , and differentiated into erythroid lineage in culture as previously described. On day 14 of erythroid differentiation, the same number of cells from each condition were stained with FITC-conjugated antibody recognizing fetal hemoglobin (HbF), and the cells were subjected to flow cytometry to quantify HbF induction after genetic editing.
[0977] [0977] To assess stem cell function of HSCs edited by genes, edited human HSCs were transplanted into immunocompromised mice to examine long-term hematopoietic regeneration. Five hundred thousand human CD34 + cells derived from bone marrow were thawed on day 0, electroporated on day 3 with a simulated RNP complex (Cas9 alone and without gRNA) or RNP complex formed with Cas9 and sagRNA comprising the target domain of CRO01128 (sometimes referred to stops here at sg1128) AUCAGAGGCCAAACCCUUCCGUUUUAGAGCUAGAAAUAGCAAGU
[0978] [0978] In a separate study, we evaluated the function of HSC stem cells edited with gRNAs from the gamma globin promoter region (sg-G0008, sg-G0051, sg-G0010, sg-G0048, sg-G0067 in Table 11 ) compared to gRNA of the erythroid-specific enhancer region of the BCL11A gene (sg-G1128). Human HSCs edited with these gRNAs were transplanted into immunocompromised NSG mice to examine hematopoietic regeneration (Figure 22). Table 11 Guide RNAs used in the transplant study, designed to target the gamma globin promoter region. Name of | ID of | ID of | Domain string | 100mer sa sequence / RNA gRNA Domain-crRNA of target gRNA target sg-Go008 | GCR-0008 | CROO5821 | GEAGAAGGAAACUA | GGAGAAGGAAACUAGCUAA GCUAAA (SEQ ID NO: | AGUULUUAGAGCUAGAAAUA 8) GCAAGUUAAAAUAAGGCUA GUCCGUUAUCAACUUGAAA
[0979] [0979] Bone marrow CD34 + cells were thawed and cultured. Each vial of 1 million bone marrow CD34 + cells was removed from liquid nitrogen, sprayed with 70% ethanol and quickly thawed in a 37ºC water bath until a small ice sediment was left. The flask was sprayed with 70% ethanol and rubbed. Using a 5 mL pipette, the contents of the flask were transferred to a 50 mL falcon tube. The cryotube was washed once with 1 ml of pre-heated IMDM, 10% FBS and added dropwise to the 50 ml falcon tube; 25 mL of preheated IMDM, 10% FBS was slowly added over 2 min to the cells, shaking gently to mix. The cells were centrifuged at 300g for 10min. Twenty-eight million CD34 + cells were cultured in StemSpan SFEM + 100 ng / ml SCF / IL6 / FIt3L / TPO + 500 nM Compound 4 + 1X Pen / Strep at 0.2-0.5 x 10e6 cells / ml.
[0980] [0980] Forty-eight hours after thawing, the cells were electroporated with 1) only Cas9; or 2) Cas9 RNP complex containing sg1128 (as described above), or one of the saRNAs in Table
[0981] [0981] Twenty-four hours after electroporation, 500 K equivalent of starting cells were transplanted per mouse. 30,000-100,000 cells were used for erythroid differentiation to assess post-editing of HbF induction. The remaining cells were left in culture for an additional 24 hours (48 hours after total electroporation) for analysis of NGS editing frequency. Transplant
[0982] [0982] Ten NSG / pathology mice were irradiated 4-24 h before transplantation with 200 Rad using the RadSource X-ray irradiator, or 2 Gray using a cesium 138 irradiator. Five hundred thousand equivalent cell initials were transplanted per mouse via injection into the tail vein. After the transplant, the mice were placed on an antibiotic regimen for 4-8 weeks. The mice were treated according to the animal care procedures of the institutes and following the approved IACUC protocol. At 4, 8, 12, 16 and 20 weeks, peripheral blood was collected for grafting and lineage analysis through the caudal vein. At 8-9 weeks and 20 weeks, bone marrow was collected for analysis (flow cytometry, Tagman qPCR and NGS as described in previous protocols, for example, Example 2.1) as well as for hoCD45 + CD34 + cell separation for erythroid differentiation as described in the previous protocols. At week 20, hoD45 + CD34 + cell separation and erythroid differentiation were performed.
[0983] [0983] Figure 22 shows the schematic diagram of the transplant study to assess the function of HSCs stem cells edited with gamma globin promoter region SsgRNAs (sg-G0008, sg-GO0051, sg-G0010, sg-G0048, sg-G0067) compared to the erythroid-specific enhancer region gRNA of the BCL11A gene (sg-G1128; also referred to as sg1128). Five hundred thousand human CD34 + cells were thawed on day O, electroporated with a simulated RNP complex (Cas9 only and without gRNA) or with the RNP complex formed with Cas9 (NLS-Cas9-NLS-His6 ("His6" disclosed as SEQ ID NO: 247)) and several gRNAs on day 3. On day 6, all cells from each condition were harvested and transplanted into NOD.Cg-Prkdec * “N2rg!" WM / SzJ (NSG) mice sub-lethally irradiated with 2 Gy ( Figure 22) The mice were bled at 4, 8, 12, 16 and 20 weeks after transplantation At 8 and 20 weeks after transplantation, the animals' bone marrow cells were also harvested to examine the human cell graft in the bone marrow Results HSC editing was able to graft in the long term and support the differentiation of multiple strains in NSG mice
[0984] [0984] The results showed that sgRNAs in the gamma globin promoter region reached, on average, 20-40% bone marrow graft at 8 weeks after transplantation, comparable to the sg-G1128. Although the graft at initial time points may be contributed by short-lived hematopoietic progenitor cells, the graft at longer time points (20 weeks) is proof of reconstitution by long-term HSCs. The tested sgRNAs showed 5-22% bone marrow graft 20 weeks after transplantation (Figure 23A-F and 24A and 24B). It is important to note that we injected only -500,000 simulated or genomic CD34 + cells into each mouse to achieve this level of graft. This level of graft was comparable to other studies that transplanted 1 million CD34 + cells edited by the genome, indicating a highly efficient graft of the instantaneous cells. In addition, we observed normal recovery of myeloid, B lymphoid and T lymphoid cells at 4, 8, 12, 16, 20 weeks after transplantation (Figure 23A-F and 25) when we compared all groups edited by genes with the edited simulated control . These data demonstrate that, by targeting the promoter region of gamma globin with a gRNA that does not map to any known HPFH, the genome editing strategy does not affect the long-term grafting capacity of HSCs, nor does it alter its reconstitution function. multi-lineage when transplanted to a new host. In contrast, we can achieve a robust graft by injecting 50% fewer cells compared to other reported strategies. In summary, the HSCs edited by these RNAs are capable of long-term grafting with robust reconstitution of multiple strains in the niche of hematopoietic stem cells to support hematopoiesis in the long term.
[0985] [0985] We examined the editing efficiency of the saRNAs tested by NGS as described in the experimental procedure. Three days after the editing gene, 100,000 of the human CD34 + cells edited into the gene, along with simulated edited CD34 + cells were subjected to NGS analysis. The remaining cells were transplanted to NSG receptors, as previously described. At 8 weeks and 20 weeks after transplantation, bone marrow cells were harvested from transplanted NSG mice and 100,000 human CD45 + cells were subjected to NGS to measure editing efficiency. The results show that saRNA sg-G1128, sg-G0048 and sg-GO0067 reached 80-90% editing, while sg-G0010 demonstrated 45% editing (Figure 26). When comparing the efficiency of pre-transplant editing with after 8 or 20 weeks of transplantation, the results showed that events edited in populations of stem cells and hematopoietic progenitors were maintained over the long term throughout the transplant period. These data demonstrate the durability of the edited cells in the transplanted individual. We even observed an increase in the editing efficiency of the sg-G1128 at 20 weeks after transplantation, which implies that the edited cells were selected by the bone marrow microenvironment or have a survival advantage after transplantation.
[0986] [0986] In another transplant-independent repeat, NGS analysis revealed an editing range achieved with the tested gRNAs, with sg-G1128 resulting in more than 90% editing two days after electroporation (Fig 8A). Editing was also detected in human CD34 + cells isolated at 9 and 20 weeks after transplantation (Fig. 27B and 27C). Gene editing and long-term grafted HSCs increased fetal hemoglobin production
[0987] [0987] NSG mice do not support erythroid development, since the mouse does not have the correct human cytokine to allow erythroid maturation. However, grafted human HSCs can be harvested from mouse bone marrow, placed in erythroid differentiation medium (as described elsewhere in these examples) to induce erythroid differentiation. The data show that the 20-week HSCs, edited by genes, were able to produce a higher level of HbF, compared to edited and transplanted HSCs (Figure 28). Example 7. Indel Out-of-Target Pattern Analysis In silico identification of potential loci outside the gRNA target
[0988] [0988] Potential off-target loci for the GGB-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067 gRNAs in the HGB1 / HBG2 region were identified as follows. For each gRNA, the 20-nucleotide gRNA target domain sequence was aligned to the human genome reference sequence (GRCh38 construction) using the BFAST sequence aligner (version 0.6.4f, Homer et al, PLoS One, 2009, 4 (11), e7767, PMID: 19907642) using standard parameters allowing up to 5 nucleotide mismatches. The identified loci were filtered to contain only sites that are 5 'adjacent to the canonical PAM sequence 5'-NGG-
[0989] [0989] Genomic DNA was isolated from bone marrow-derived HSPC CD34 +, edited and unedited with 3-day RNP, using the Quick-DNA Miniprep kit (Zymo Research) following the manufacturer's recommendations. Creation of PCR primers for targeted amplification of potential off-target sites
[0990] [0990] PCR amplicons targeting potential off-target loci with 0-3 mismatches (and the target locus) identified for HBG1 / HBG2 region gRNAs (GCR-0001, GCR-0008, GCR- 0010, GCR- 0048, GCR-0051 and GCR-0067) were designed using Primer3 (version 2.3.6, Untergasser et al, Nucleic Acids Res., 2012 40 (15): 2115, PMID: 22730293) using predefined parameters targeting a size range of amplicon approximately 160-300 base pairs in length with the sequence of the gRNA target domain located in the center of the amplicon. Resulting pairs of PCR primers and amplicon sequences were checked for uniqueness by the BLAST search (version 2.2.19, Altschul et al, J Mol Biol., 1990, 215 (3): 403-10, PMID: 2231712) for sequences against the reference sequence of the human genome (construct GRCh38). The primer pairs that resulted in more than one sequence of amplicons were discarded and re-created. Table 3 shows the counts of pairs of successfully designed PCR primers. Preparation, quantification and sequencing of the Illumina sequencing library
[0991] [0991] HSPC genomic DNA edited (2 replicates per gRNA) or unedited (2 replicates per gRNA) with RNP was quantified using the Quant-iT PicoGreen dsDNA kit (Thermo Fisher, Cat & P7581) using the manufacturer's recommendations . Illuminating sequencing libraries targeted to individual off-target loci (and the target locus) were generated for each sample using two sequential PCR reactions. The first PCR amplified the target locus using target-specific PCR primers (designed above) that were tailed with compatible universal Illumina sequencing sequences. The second PCR added additional Illumina compatible sequences to the first PCR amplicon,
[0992] [0992] The methods are as in Example 2.1, with the following modifications. For Stage 5 of the analysis, sites with a combined indelible frequency of> 2% (editing in more than approximately 10 cells) were considered and potential active editing sites were examined at the reading alignment level using the Integrative Genome Viewer (version IGV 2.3, Robinson et al, Nat Biotechnol. 2011, 9 (1): 24-6, PMID: 21221095) that allows visual inspection of the reading alignments for the genome reference sequence. Quantification of large deletions.
[0993] [0993] Large deletions between targeting sites in HBG1 and HBG2 promoters were quantified by digital drop PCR (ddPCR) in a non-competitive assay format to determine the number of copies in accordance with the manufacturer's recommendations. Briefly, the gDNA was combined with ddPCR SuperMix for Probes (without dUTPs) (BioRad Cat 41863024), Hindl! 1-HF restriction enzyme (NEB Cat tt / R3104S) and each primer probe mixture, transferred to a DG8 cartridge together with Droplet Generation Oil for Probes (BioRad Cat% 1863005) and droplets generated with a QX200 droplet generator (BioRad). The droplets were subjected to PCR in a Touch C1000 Thermocycler with Deep Well Reaction Module-96 (BioRad), followed by detection with QX200 droplet reader (BioRad). The copies per ul were determined by analysis with the QuantaSoft software (BioRad). A set of custom initiator probes (Life Technologies Cat HAPZW7GR, PN4331348, Direct Initiator: ACGGATAAGTAGATATTGAGGTAAGC (SEQ ID NO: 285), Reverse Initiator: GTCTCTTTCAGTTAGCAGTGG (SEQ ID NO: 286), FAM Probe: ACTGGT: ACTGG )) used to amplify the gDNA within the HBG1- HBG2 intergenic region. A human TaqMan Copy Number Reference Assay, RNase P (Thermo Fisher cat f4403326) was used as the reference amplicon. Copies per uL for the amplicons HBG1-HBG2 and RNase P were within the manufacturer's linear range. Deletion percentage was reported as 100% times 1 minus the copy ratio per ul for HBG1-HBG2 and RNase P amplicons. For unedited control samples, the percentage of deletions up to 2% was calculated, which may reflect the background of the test. Analysis of results of the HBG1 / HBG2 region in silico outside the target
[0994] [0994] Table 13 shows the number of off-target sites successfully characterized. The uncharacterized sites failed to design the PCR primer or amplify by PCR and remain under evaluation.
[0995] [0995] gRNA GCR-0001: The HBG1 site on the target showed robust localized editing with an average frequency of INDEL of approximately 58%, while the homologous target site of HBG2 with two mismatches (here and below, shown in lower case) in Regarding the target domain sequence of gRNA (5'- AGTCCTGGTATCtTCTATGA - PAM-3 ', PAM = TGG (SEQ ID NO: 288)) showed minimal localized edition. However, the 4.9 kb deletion ddPCR analysis showed that it occurs at a frequency of approximately 34%. Further analysis identified a positive off-target site with an average INDEL frequency of approximately 26% in both replicates. The site has 3 mismatches in relation to the sequence of the gRNA target domain (5'-ALTCCcaGTATCCTCTATGA-PAM-3 ', PAM = TGG (SEQ ID NO: 289)) and is located in an intergenic on the Y chromosome in the pair position of bases 21,470,475-21,470,497. It is not clear whether editing on this off-target site has any detrimental effect on gene expression or cell viability, further analysis is needed.
[0996] [0996] gRNA GCR-0008: The HBG1 site on the target showed robust localized editing with an average INDEL frequency of approximately 88%. The homologous target site of HBG2 with a mismatch with respect to the sequence of the gRNA target domain
[0997] [0997] gRNA GCR-0010: The HBG1 site on the target showed robust localized editing with an average INDEL frequency of approximately 32%. The homologous target site of HBG2 with a mismatch with respect to the sequence of the gRNA target domain (5- GGGAGAAGaAAACTAGCTAA-PAM-3 ', PAM = AGG (SEQ ID NO: 291)) showed robust localized editing with an average frequency of Approximately 27%. The ddPCR analysis of the 4.9 kb deletion showed that it occurs at a frequency of approximately 33%. No other site showed editing.
[0998] [0998] gRNA GCR-0048: Sites on target HBG1 and HBG2 showed robust localized editing with an average INDEL frequency of approximately 86% for both sites. The ddPCR analysis of the 4.9 kb deletion showed that they occur at a frequency of approximately 45%. No other site showed editing.
[0999] [0999] gRNA GCR-0051: The HBG2 site on the target showed robust localized editing with an average INDEL frequency of approximately 88%. The homologous target site of HBG1 with a mismatch in relation to the sequence of the gRNA target domain (5- GGAGAAGgAAACTAGCTAAA-PAM-3 ', PAM = GGG (SEQ ID NO: 292)) showed robust localized editing with an average frequency of Approximately 59%. The ddPCR analysis of the 4.9 kb deletion showed that it occurs at a frequency of approximately 40%. No other site showed editing.
[1000] [1000] gRNA GCR-0067: Target sites HBG1 and HBG2 showed robust localized editing with an average frequency of
[1001] [1001] The localized INDEL frequencies described above are not relative to the total indel frequency and do not take into account the frequency of the 4.9 kb large deletion. Table 13. Silicon counts of 0-3 unpaired off-target sites, identified for the HBG1 / HBG2 region gRNAs GCR-0001, GCR-0008, GCR-0010, GCR-0048, GCR-0051 and GCR-0067, counts of sites successfully characterized in HSPC edited by genome and the counts of sites displaying editing are shown. Number of 0-3 Name of sites outside the target Number of sites in | Number of in silico sites characterized | incompatible off-target silica [ee | | QU "[er | QT Impartial Target Analysis
[1002] [1002] An oligo insertion-based assay (see, for example, Tsai et al., Nature Biotechnology. 33, 187-197; 2015) was used to determine potential off-target genomic sites cleaved by Cas9 targeting HBG1 and / or HBG2. In these experiments, HEK293 cells expressing Cas9GFP (HEK-293 Cas9GFP) were transfected with gRNAs (15 nM crRNA: tracr) and insertion oligo (10 nM) using Lipofectamine & RNAIMAX. The assay is based on the identification of the oligo incorporated in double breaks in the genome, which may or may not result from Cas9 cleavage.
[1003] [1003] In one experiment, gRNAs (double guide RNAs comprising the target domain indicated in Figure 29) targeting HBG1 and / or HBG2 were screened in HEK-293 Cas9GFP cells, and the results are plotted in Figure 29. In one separate experiment, the same methodology was used to screen some of them, as well as additional gRNAs (including double guide and single guide RNAs comprising the target domain indicated in Figure 30) targeting HBG1 and / or HBG 2 in HEK-293 cells Cas9GFP and the results are shown in Figure 30. The experiment with the data presented in Figure 30 used an alternative oligo insertion primer with a balanced content in G / C and minimized complementarity with the human genome, as well as other modifications for the steps of PCR, compared to the published Tsai et al. methods, which improved sensitivity and decreased false positives. In both experiments, the trial detected high-efficiency editing on the expected target sequences and one or more potential off-target sites.
[1004] [1004] While the detection of the insertion oligo at sites in the genome other than the target site identifies potential off-target effects from Cas9, targeted deep sequencing of the potential off-target sites can be used to determine whether the potential sites are indeed , off-target sites cleaved by Cas9. For this purpose, an experiment was carried out in which HEK-293 Cas9GFP cells were similarly transfected with the gRNAs (at 25 nM crRNA: tracr) used in the two previous experiments, but without the oligo insertion. Deep sequencing of the fragment for each of the potential off-target sites represented in Figures 29 and 30 was used to identify whether the indels (indicative of Cas9 cleavage events) were present at the potential off-target sites on the HEK-293 cell line Cas9GFP. The results of this experiment demonstrated that most of the potential off-target sites identified by the oligo insertion assay do not have detectable indels after transfection of the gRNAs, with some exceptions, as provided in Table 14 below. It is important to note that the HEK-293 Cas9GFP cells used in these experiments to detect possible off-target Cas9 overexpress, probably leading to a greater number of possible off-target "hits" compared to a transient delivery modality (for example, delivery of RNP) in various types of cells of interest (for example, CD34 + HSCs).
[1005] [1005] Table 14. Off-target sites validated on the HEK-293 Cas9GFP cell line.
[1006] [1006] Using the methods described here, potential off-target sites were examined on CD34 + HSPCs edited with SARNA / Cas9 and showed no editing on the CD34 + cell type. Example 8 Experimental Procedure
[1007] [1007] CD34 + cells harvested from mobilized peripheral blood (MPB) from healthy donors were cultured and gene-edited under the same conditions as described above in Example 6. The cells were characterized for their biological function using the same methods described in previous paragraphs.
[1008] [1008] CD34 + cells derived from mobilized peripheral blood maintain their CD34 + cell count, expandability and viability after editing
[1009] [1009] The number of CD34 + cells transplanted to the patient is directly correlated with the success of a bone marrow transplant. Therefore, we enumerated the percentage of CD34 + cells over a period of 10 days after editing genes using a clinically acceptable method, ISHAGE. The results show that neither sg1128 nor sg0067 editing had an impact on the CD34 + cell count (Figure 31) when compared to the simulated edit control. For the total duration of our cell process, in which only the cells were kept in culture for 3 days after electroporation, the percentage of CD34 + cells was maintained at approximately 90%. (Figure 31A). Gene editing also had no impact on the expansion capacity of CD34 + cells (Figure 31B) and its post-electroporation viability varied between 70-90% (Figure 31C). We observed a cell expansion -2 times on day 3 after electroporation, expansion of —10 times on day 7 after electroporation and 15-25 times expansion on day 10 after electroporation (Figure 31B). CD34 + cells derived from peripheral blood mobilized from healthy individuals demonstrate similar editing efficiencies compared to the source of bone marrow cells or CD34 + cells from patients with sickle cell disease
[1010] [1010] Sg1128 demonstrated about 70-75% editing efficiency in mPB-derived CD34 + cells from healthy donors, while sg0067 showed> 95% total editing efficiency (including large 5 kb deletion and small indels) (Figure 32 ). The editing efficiencies of these saRNA were similar in cells from different sources, including CD34 + cells derived from bone marrow from healthy donors or CD34 + cells derived from the peripheral blood of patients with sickle cell disease (see other examples). It is important to note that both the editing efficiency and the editing pattern of each sgRNA were highly consistent across all donors tested. CRISPR silencing of BCL11A or mutation of the g-globin gene group increases g-globin transcription and F-cell production
[1011] [1011] Silencing BCL11A by sg1128, or mutating the potential BCL11A binding site in the g-globin gene group by sgo0067, both significantly increased g-globin transcripts (Figure 33) leading to a 15-20% increase production of F cells compared to the simulated edited control (Figure 34). In summary, sg 1128 and sg0067 can edit CD34 + cells with high efficiency and generate highly consistent editing patterns. Edited cells maintain approximately 90% CD34 + cell count at the end of our cell processing procedure for 6 days. The expansion capacity and the viability of the cells were not impaired by the gene editing process. Edited CD34 + cells, when differentiated into erythrocytes, expressed a significantly higher level of g-globin transcripts, translating into an increase in the number of F cell production. This improvement in hemoglobin expression and the number of F cells can rescue the hematological characteristics of sickle cell disease. Example 9: In vivo grafting and characterization of HSPCs edited by genes derived from patients with sickle cell disease Experimental Procedure
[1012] [1012] CD34 + cells from patients with sickle cell disease were cultured and gene-edited under the same conditions as previously described in Example 8. The cells were characterized for their biological function using the same methods described in the previous Examples.
[1013] [1013] Hematopoietic stem cells and progenitors (HSPCs), which maintain their primitive cellular state, must express CD34. This is one of the most important clinical markers associated with grafted hematopoietic stem cells after transplantation, and the success of a transplant is directly correlated with the number of CD34 + cells transplanted. Therefore, we enumerate the number of CD34 + cells obtained from peripheral blood from sickle cell individuals after editing using a clinically acceptable method, ISHAGE. The results show that neither sg1128 nor sg0067 editing had an impact on CD34 + cell count (Figure 35A and Figure 35B) when compared to the simulated edit control. The percentage of CD34 + cells was maintained at -80% on day 3 after editing (Figure 35B). Gene editing also had no impact on the expansion capacity of patient-derived CD34 + cells (Figure 35C) and its post-electroporation viability (DO electroporation) was greater than 75% at all times measured over a period of days (Figure 35D). CD34 + cells derived from individuals with sickle cell disease demonstrate similar editing efficiencies compared to CD34 + cells from healthy donors
[1014] [1014] Sg1128 demonstrated 65% editing efficiency in CD34 + cells derived from patients with sickle cell anemia, while sgo0067 showed 80-95% editing efficiency in patient samples (Figure 36). The level of editing efficiency is similar to that obtained using CD34 + cells derived from bone marrow or derived from peripheral blood from healthy donors. The editing pattern for each guide RNA measured by NGS (as described in Example 2.1) was also highly consistent across different patients.
[1015] [1015] Progenitor cells and hematopoietic stem cells edited into genes were completely able to differentiate into erythroid, granulocytic, monocytic and megakaryocytic strains, as measured by colony-forming unit assays (Figure 37). CRISPR silencing of BCL11A or mutation of the g-globin gene group increases g-globin transcription, F-cell production and fetal hemoglobin expression
[1016] [1016] Both silencing BCL11A by sg1128, and creating indels / deletions in the g-globin group by s9g0067, significantly increased g-globin transcripts (Figure 38) and increased the number of F cells as measured by cytometry flow (Figure 39). In addition, the intensity of F cell fetal hemoglobin expression was also improved per cell, measured by flow cytometry (Figure 40). Editing patient-derived CD34 + cell genes led to a significant reduction in sickle cell count and an increase in the number of normal cells
[1017] [1017] Finally, to understand whether gene editing can rescue sickle cell morphology, we edit with CRISP CD34 + cells derived from patients with sickle cell anemia, differentiate these cells into red blood cells and subject these cells to a hypoxia chamber for 4 days to induce sickle cell morphology. The cells were co-stained with anti-HbF-FITC antibody, fixed inside the chamber, and subjected to flow cytometry by imaging to capture a single image per cell. Single cell imaging flow cytometry can distinguish sickle cells versus normal cells based on cell length and high yield HbF expression (40,000 images of individual cells from each patient were used for data analysis). The results show that editing genes with sg1128 or sg0067 was able to decrease the sickle cell count by approximately 40% (Figure 41A) and, at the same time, increase the normal cell count by 1.2 times (Figure 41B). The compound's effect of decreasing sickle cell counts and simultaneously increasing normal red blood cell counts will greatly benefit patients when they are translated into the clinic.
[1018] [1018] In summary, sg1128 and sgo0067 can edit CD34 + cells from sickle cell disease patients with high efficiency and generate highly consistent editing patterns. The percentage of CD34 + cells, the cell's expansion capacity and cell viability were not affected by the gene editing process when comparing the edited cells to the edited simulated control group. Edited CD34 + cells, when differentiated into erythrocytes, expressed a significantly higher level of g-globin transcripts. This translates into an increase in the number of F cells and also an increased expression of HbF per cell. The increase in the number of F cells expressing HbF and the reduction in the number of sickle red blood cells was reflected in our single cell flow cytometry analysis. We observed a decrease of 50% of sickle cells in the group edited by genes and, simultaneously, an increase of 1.2 times in the normal red blood cells expressing HbF in edited groups, when compared to the modified control in simulation. This effect of the compound in reducing the number of sickle cells with a concomitant increase in normal red blood cells expressing elevated HbF after gene editing should significantly benefit patients when translated into the clinic. Together, these data support the development of CRISPR / Cas-mediated genome editing as a means of cell therapy to treat b-globinopathies.
[1019] [1019] To the extent that there is any discrepancy between any sequence listing and any sequence cited in the specification, the sequence cited in the specification should be considered the correct sequence. Unless otherwise indicated, all genomic locations are in accordance with hg38. INCORPORATION BY REFERENCE
[1020] [1020] All publications, patents and patent applications mentioned herein are hereby incorporated by reference in their entirety, as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict, this request, including any settings here, will control. EQUIVALENTS
[1021] [1021] Those skilled in the art will recognize, or be able to determine using, no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Although this invention has been disclosed with reference to specific aspects, it is evident that other aspects and variations of this invention can be devised by other persons skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be interpreted to include all of these aspects and equivalent variations.

权利要求:
Claims (93)
[1]
1. gRNA molecule, characterized by comprising a cr »RNA tracre, in which the crRNA comprises a target domain that: a) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,250,094-5,250,237, - chain, hg38; b) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,255,022-5,255,164, - strand, hg38; c) is complementary to a target sequence of a non-deleting HFPH region (for example, a non-human HPFH region); d) is complementary to a target sequence within the genomic nucleic acid sequence at Chr11: 5,249,833 to Chr11: 5,250,237, - strand, hg38; e) is complementary to a target sequence in the genomic nucleic acid sequence in Chr11: 5,254,738 to Chr11: 5,255,164, - strand, hg38; f) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,249,833-5,249,927, - strand, hog38; g) is complementary with a target sequence within the genomic nucleic acid sequence in Chr11: 5,254,738-5,254,851, - strand, hog38; h) is complementary to a target sequence within the genomic nucleic acid sequence in Chr11: 5,250,139-5,250,237, - strand, hg38; or i) their combinations.
[2]
2. The gRNA molecule according to claim 1, characterized by the fact that the target domain comprises, for example, any of SEQ ID NO: 1 to SEQ ID NO: 72, or a fragment thereof.
[3]
3. The gRNA molecule according to claim 2, characterized by the fact that the target domain comprises, for example, any of SEQ ID NO: 67, SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO: 63, or a fragment thereof.
[4]
4. The gRNA molecule according to claim 2, characterized by the fact that the target domain comprises, for example, any of the a) SEQ ID NO: 67, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 48, SEQ ID NO: 51, or a fragment thereof; or b) SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 54, or a fragment thereof.
[5]
A gRNA molecule according to any one of claims 2 to 4, characterized in that the target domain comprises, for example, consecutive nucleic acids 17, 18, 19 or any of the aforementioned target domain sequences .
[6]
6. A gRNA molecule according to claim 5, characterized in that the consecutive nucleic acids 17, 18, 19 or 20 of any of the target domain sequences recited are the consecutive nucleic acids 17, 18, 19 or 20 arranged at the 3 'end of the recited target domain sequence.
[7]
7. The gRNA molecule according to claim 5, characterized by the fact that the consecutive nucleic acids 17, 18, 19 or 20 of any of the target domain sequences recited are the consecutive nucleic acids 17, 18, 19 or 20 arranged at the 5 'end of the recited target domain sequence.
[8]
8. A gRNA molecule according to claim 5, characterized in that the consecutive nucleic acids 17, 18, 19 or 20 of any of the target domain sequences recited do not comprise the 5 or 3 ”nucleic acid of the sequence of target domain recited.
[9]
A gRNA molecule according to any one of claims 2 to 8, characterized in that the target domain consists of the recited target domain sequence.
[10]
A gRNA molecule according to any one of claims 1 to 9, characterized in that the target domain comprises, for example, SEQ ID NO: 67.
[11]
11. The gRNA molecule according to any one of claims 1 to 10, characterized by the fact that the gRNA molecule is a dual guide RNA molecule.
[12]
12. The gRNA molecule according to any one of claims 1 to 10, characterized by the fact that the gRNA molecule is a single guide RNA molecule.
[13]
13. The gRNA molecule according to claim 12, characterized by the fact that it comprises: (a) SEQ ID NO: 195; (b) SEQ ID NO: 231; or (c) any (a) or (b), above, further comprising, at the 3 'end, 1, 2, 3, 4, 5, 6 or 7 uracil nucleotides (U); wherein the sequence of any (a) to (c) is arranged 3 ', optionally immediately 3', to the target domain.
[14]
14. The gRNA molecule according to claim 1, characterized by the fact that it comprises, for example, the sequence: (a) SEQ ID NO: 174;
(b) SEQ ID NO: 175; or (c) SEQ ID NO: 176.
[15]
A gRNA molecule according to claim 1, characterized in that it comprises, for example: (a) a cr »RNA comprising, for example SEQ ID NO: 177 and a tracr comprising, for example, SEQ ID NO : 224; (b) a cr »RNA comprising, for example, SEQ ID NO: 177 and a tracer comprising, for example, SEQ ID NO: 73; (c) a cr »RNA comprising, for example, SEQ ID NO: 178 and a tracer comprising, for example, SEQ ID NO: 224; or (d) a cr »RNA comprising, for example, SEQ ID NO: 178 and a tracer comprising, for example, SEQ ID NO: 73.
[16]
16. A gRNA molecule according to any one of claims 1 to 15, characterized by the fact that a) when a CRISPR system (for example, an RNP as described here) comprising a gRNA molecule is introduced into a cell, a indel is formed at or near the target sequence complementary to the target domain of the gRNA molecule; and / or b) when a CRISPR system (for example, an RNP as described herein) comprising the gRNA molecule is introduced into a cell, a deletion is created comprising the sequence, for example, comprising substantially the entire sequence, between a sequence complementary to the gRNA target domain (for example, at least 90% complementary to the gRNA target domain, for example, fully complementary to the gRNA target domain) in the HBG1 promoter region and a complementary sequence to the gRNA target domain ( for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region.
[17]
17. The gRNA molecule according to claim 16,
characterized by the fact that the indel does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, - strand (hg38), optionally where the indel does not comprise a nucleotide from a non-deletable HPFH or transcription factor binding site.
[18]
18. The gRNA molecule according to any one of claims 1 to 17, characterized by the fact that when a CRISPR system (for example, an RNP as described here) comprising the gRNA molecule is introduced into a population of cells, a indel is formed at or near the target sequence complementary to the target domain of the gRNA molecule by at least about 15%, for example, at least about 17%, for example, at least about 20%, for example, at least about 30%, for example, at least about 40%, for example, at least about 50%, for example, at least about 55%, for example, at least about 60%, for example, at least about 70%, for example, at least about 75%, of the population cells.
[19]
19. The gRNA molecule according to any one of claims 1 to 18, characterized by the fact that when a CRISPR system (for example, an RNP as described here) comprising a gRNA molecule is introduced into a cell (for example , a population of cells): (a) fetal hemoglobin expression is increased in said cell or in its progeny, for example, its erythroid progeny, for example, its red blood cell progeny, optionally in which said hemoglobin expression fetal growth is increased by at least about 15%, for example, at least about 17%, for example, at least about 20%, for example, at least about 25%, for example, at least about 30%, for example, at least about 35%, for example, at least 40% relative to the level of fetal hemoglobin expression in a cell population to which the gRNA molecule has not been introduced or a population of its progeny, for example, their erythroid descent, po for example, their red blood cell progeny; (b) said cell or cell population, or its offspring, for example, its offspring, for example, its erythroid offspring, for example, its offspring of red blood cells, produces at least about 6 picograms (for example, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or from about 8 to about 9 picograms or about 9 to about picograms) of fetal hemoglobin per cell; (c) off-target indels do not form in said cell, for example, off-target indels do not form outside the HBG1 and / or HBG2 promoter regions, for example, as detectable by next generation sequencing and / or insertion assay nucleotides; and / or (d) indel off-target is not detected, for example, indel off-target outside the HBG1 and / or HBG2 promoter regions, by more than about 5%, for example, more than about 1% , for example, more than about 0.1%, for example, more than about 0.01%, of cells in the cell population, for example, as detectable by next generation sequencing and / or an assay of nucleotide insertion.
[20]
20. A gRNA molecule according to any one of claims 16 to 19, characterized in that the cell is (or the population of cells comprises) a mammalian, primate or human cell, for example, it is a human cell, optionally wherein said cell is obtained from a patient suffering from hemoglobinopathy, for example, sickle cell disease or thalassemia, for example, beta-thalassemia.
[21]
21. The gRNA molecule according to claim 20, characterized in that the cell is (or population of cells comprises) an HSPC, optionally an HSPC CD34 +, optionally an HSPC CD34 + CD90 +.
[22]
22. The gRNA molecule according to any one of claims 16 to 21, characterized in that the cell is autologous or allogeneic with respect to a patient to be administered with said cell.
[23]
23. Composition characterized by the fact that it comprises: 1) one or more gRNA molecules (including a first gRNA molecule), as defined in any one of claims 1a 22, and a Cas9 molecule; 2) one or more gRNA molecules (including a first gRNA molecule), as defined in any one of claims 1 to 22, and nucleic acid encoding a Cas9 molecule; 3) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule), as defined in any one of claims 1 to 22, and a Cas9 molecule; 4) nucleic acid encoding one or more gRNA molecules (including a first gRNA molecule), as defined in any one of claims 1 to 22, and nucleic acid encoding a Cas9 molecule; or 5) any one of 1) to 4), above, and a template nucleic acid; or 6) any one of 1) to 4) above, and nucleic acid comprising a sequence encoding a template nucleic acid.
[24]
24. Composition characterized by the fact that it comprises a first gRNA molecule, as defined in any one of claims 1 to 22, further comprising one of Cas9, optionally, wherein the Cas9 molecule is an active or inactive Cas9 of s. pyogenes, optionally wherein the Cas9 molecule comprises SEQ ID NO: 205 or a sequence with at least 95%, 96%, 97%, 98%, or 99% sequence homology therewith.
[25]
25. Composition according to claim 23 or 24, characterized in that the Cas9 molecule comprises, for example: (a) SEQ ID NO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e) SEQ ID NO: 237; (f) SEQ ID NO: 238; (9) SEQ ID NO: 239; (h) SEQ ID NO: 240; (i) SEQ ID NO: 241; (]) SEQ ID NO: 242; (k) SEQ ID NO: 243; or (1) SEQ ID NO: 244.
[26]
26. Composition according to any one of claims 23 to 25, characterized in that the first gRNA molecule and the Cas9 molecule are present in a ribonuclear protein (RNP) complex.
[27]
27. Composition according to any one of claims 23 to 26, characterized in that it is formulated in a suitable medium for electroporation.
[28]
28. Composition according to any one of claims 23 to 27, characterized by the fact that each of said gRNA molecules is in an RNP with a Cas9 molecule described here, and in which each of said RNP is in a concentration of less than about 10 µM, for example, less than about 3 µM, for example, less than about 1 µM, for example, less than about 0.5 µM, for example, less than that about 0.3 µM, for example, less than about 0.1 µM, optionally, wherein the concentration of said RNP is about 2 µM or is about 1 µM, optionally, where the composition comprises still a population of cells, for example, HSPCs.
[29]
29. Nucleic acid sequence, characterized by the fact that it encodes one or more gRNA molecules, as defined in any one of claims 1 to 22.
[30]
30. Vector, characterized by the fact that it comprises nucleic acid, as defined in claim 29, optionally in which the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector (AAV), a vector of the herpes simplex virus (HSV), a plasmid, a minicircle, a nanoplasmid and an RNA vector.
[31]
31. Method of altering a cell (for example, a population of cells), (for example, changing the structure (for example, nucleic acid sequence)) at or near a target sequence within that cell, characterized by fact that it comprises contact of (for example, introduction into) said cell (for example, population of cells) with: 1) one or more gRNA molecules, as defined in any of claims 1 to 22, and a Cas9 molecule ; 2) one or more gRNA molecules, as defined in any one of claims 1 to 22, and nucleic acid encoding a Cas9 molecule; 3) nucleic acid encoding one or more gRNA molecules, as defined in any one of claims 1 to 22, and a Cas9 molecule; 4) nucleic acid encoding one or more gRNA molecules, as defined in any one of claims 1 to 22, and nucleic acid encoding a Cas9 molecule; 5) any one of 1) to 4), above, and a template nucleic acid; 6) any one of 1) to 4) above, and nucleic acid comprising a sequence encoding a template nucleic acid; 7) the composition, as defined in any one of claims 23 to 28; or 8) the vector, as defined in claim 30.
[32]
32. The method of claim 31, characterized in that the cell is an animal cell, for example, a mammalian, primate or human cell, for example, is a human cell; optionally wherein said cell is obtained from a patient suffering from hemoglobinopathy, for example, sickle cell disease or thalassemia, for example, beta-thalassemia.
[33]
33. Method according to claim 31 or 32, characterized in that the cell is an HSPC, optionally an HSPC CD34 +, optionally an HSPC CD34 + CD90 +.
[34]
34. Method according to any one of claims 31 to 33, characterized in that the cell is arranged in a composition comprising a population of cells that has been enriched for CD34 + cells.
[35]
35. Method according to any of claims 31 to 34, characterized in that the cell (eg cell population) has been isolated from the bone marrow, peripheral blood (eg mobilized peripheral blood) or cord blood umbilical.
[36]
36. Method according to any one of claims 31 to 35, characterized in that the cell is autologous or allogeneic with respect to a patient to be administered with said cell.
[37]
37. Method according to any of the claims
31 to 36, characterized by the fact that: (a) the change results in an indelible at or close to a complementary genomic DNA sequence with the target domain of one or more gRNA molecules; and / or b) the change results in a deletion comprising a sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of one or more gRNA molecules (for example, at least 90% complementary to the target domain of gRNA, for example, completely complementary to the gRNA target domain) in the HBG1 promoter region and a sequence complementary to the target domain of one or more gRNA molecules (for example, at least 90% complementary to the gRNA target domain , for example, completely complementary to the target domain of gRNA) in the HBG2 promoter region, optionally, where the deletion does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, - strand (h9g38).
[38]
38. Method according to any one of claims 31 to 37, characterized in that: (a) the method results in a population of cells in which at least about 15%, for example, at least about 17% , for example, at least about 20%, for example, at least about 30%, for example, at least about 40%, for example, at least about 50%, for example, at least about 55% , for example, at least about 60%, for example, at least about 70%, for example, at least about 75% of the population has changed, for example, comprise an indel, optionally where the indel selected from one indel listed in Table 2-7, optionally where the population cells do not comprise a deletion of a nucleotide arranged between 5,250,092 and 5,249,833, - strand (hg38); (b) the change results in a cell (for example,
cell population) that is able to differentiate into a differentiated cell from an erythroid lineage (for example, a red blood cell) and in which that differentiated cell exhibits an increased level of fetal hemoglobin, for example, in relation to a cell ( eg cell population) unchanged; (c) the change results in a population of cells that is able to differentiate into a population of differentiated cells, for example, a population of cells of an erythroid lineage (for example, a population of red blood cells) and in which said differentiated cell population has an increased percentage of F cells (for example, at least about 15%, at least about 20%, at least about 25%, at least about 30% or at least about 40% percentage higher F cells), for example, in relation to a population of unchanged cells; and / or (d) the change results in a cell (for example, a population of cells) that is able to differentiate into a differentiated cell, for example, a cell of an erythroid lineage (for example, a red blood cell) and wherein said differentiated cell produces at least about 6 picograms (for example, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or about 8 to about 9 picograms or about 9 to about 10 picograms) of fetal hemoglobin per cell.
[39]
39. Cell, characterized by the fact that it is altered by the method, as defined in any of claims 31 to 38, or a cell obtainable by the method, as defined in any of claims 31 to 38.
[40]
40. Cell, characterized by the fact that it comprises an indel described in Table 7-2, optionally, in which the cell does not comprise a deletion of a nucleotide arranged between 5,250,092 and 5,249,833, - chain (hg38).
[41]
41. A cell, characterized by the fact that it comprises a first gRNA cell, as defined in any of claims 1 or 22, or a composition, as defined in any of claims 23 to 28, a nucleic acid, as defined in claim 29, or a vector, as defined in claim 30.
[42]
42. Cell according to any one of claims 39 to 41, characterized by the fact that the cell is capable of differentiating into a differentiated cell, for example, a cell of an erythroid lineage (for example, a red blood cell), and wherein said differentiated cell exhibits an increase in the level of fetal hemoglobin, for example, relative to a cell of the same type that has not been modified to comprise a gRNA molecule, optionally in which the differentiated cell (for example, cell of a erythroid lineage, eg red blood cells) produces at least about 6 picograms (for example, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms, or from about 8 to about 9 picograms, or from about 9 to about 10 picograms) of fetal hemoglobin, for example, relative to a differentiated cell of the same type that has not been modified to comprise a gRNA molecule.
[43]
43. Cell according to any one of claims 39 to 42, characterized by the fact that the cell has been placed in contact with a stem cell expander.
[44]
44. Cell according to claim 43, characterized by the fact that the stem cell expander is: a) (1r, A4r) -N '- (2-benzyl-7- (2-methyl-2H-tetrazole -5-i1) -9H-pyrimido [4,5-b] indol-4-yl) cyclohexane-1,4-diamine; b) 4- (3-piperidin-1-ylpropylamine) -9H-pyrimido [4,5-b] indole-7-
methyl carboxylate; Cc) 4- (2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol; d) (S) -2- (6- (2- (IH-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) - 9H-purin-9-yl) propan-l- ol; or e) their combinations (for example, a combination of (1r, 4r) -N '- (2-benzyl-7- (2-methyl-2H-tetrazol-5-yl) - 9H-pyrimido [4,5- b] indol-4-yl) cyclohexane-1,4-diamine and (S) -2- (6- (2- (IH-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3 -yl) -9H-purin-9-yl) propan-1-ol).
[45]
45. A cell according to any one of claims 39 to 44, characterized by the fact that it comprises: a) an indel in or near a genomic DNA sequence complementary to the target domain of a gRNA molecule, as defined in any of claims 1 to 22; and / or b) a deletion comprising sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of a gRNA molecule, as defined in any one of claims 1 to 22, (for example, at least 90 % complementary to the target domain of gRNA, for example, completely complementary to the target domain of gRNA) in the HBG1 promoter region and a sequence complementary to the target domain of a gRNA molecule, as defined in any one of claims 1 to 22 , (for example, at least 90% complementary to the gRNA target domain, for example, completely complementary to the gRNA target domain) in the HBG2 promoter region, optionally, where the deletion does not comprise a nucleotide arranged between
5,250,092 and 5,249,833, - chain (hg38).
[46]
46. Cell according to any one of claims 39 to 45, characterized in that the cell is an animal cell, for example, a mammalian, primate or human cell, for example,
it is a human cell; optionally wherein said cell is obtained from a patient suffering from hemoglobinopathy, for example, sickle cell disease or thalassemia, for example, beta-thalassemia.
[47]
47. Cell according to any one of claims 39 to 46, characterized in that the cell is an HSPC, optionally an HSPC CD34 +, optionally an HSPC CD34 + CD90 +.
[48]
48. Cell according to any one of claims 39 to 47, characterized by the fact that the cell (eg cell population) has been isolated from bone marrow, peripheral blood (eg mobilized peripheral blood) or cord blood umbilical.
[49]
49. Cell according to any one of claims 39 to 48, characterized by the fact that the cell is autologous or allogeneic with respect to a patient to be administered with said cell.
[50]
50. Cell population, characterized by the fact that it comprises the cell, as defined in any one of claims 39 to 49, optionally in which at least about 50%, for example, at least about 60%, for example, at least at least about 70%, for example, at least about 80%, for example, at least about 90% (for example, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%) of the cells in the population are a cell, as defined in any one of claims 39 to 49.
[51]
51. Cell population according to claim 50, characterized by the fact that the cell population is able to differentiate into a differentiated cell population, for example, a cell population of an erythroid lineage (for example, a population of red blood cells), and where said population of differentiated cells has an increased percentage of F cells
(for example, at least about 15%, at least about 17%, at least about 20%, at least about 25%, at least about 30%, or at least about 40% higher percentage of cells F), for example, in relation to a population of unmodified cells of the same type; optionally, where the F cells of the differentiated cell population produce an average of at least 6 picograms (for example, at least 7 picograms, at least 8 picograms, at least 9 picograms, at least 10 picograms, or approximately 8 to about 9 picograms, or about 9 to about 10 picograms) of fetal hemoglobin per cell.
[52]
52. Cell population according to claim 50 or 51, characterized by the fact that it comprises: 1) at least 1 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 2) at least 2 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 3) at least 3 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; 4) at least 4 and 6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered; or 5) from 2e6 to 10e6 CD34 + cells / kg of body weight of the patient to whom the cells are to be administered.
[53]
53. Cell population according to any one of claims 50 to 52, characterized by the fact that at least about 40%, for example, at least about 50%, (for example, at least about 60%, at least about 70%, at least about 80%, or less than about 90%) of the population's cells are CD34 + cells, optionally at least about 10%, for example, at least about 15%, for example , at least 20%, for example, at least about 30% of the population's cells are cells
CD34 + CD90 +.
[54]
54. Cell population according to any one of claims 50 to 53, characterized by the fact that the cell population is derived from umbilical cord blood, peripheral blood (for example, mobilized peripheral blood) or bone marrow, for example , is derived from the bone marrow.
[55]
55. Cell population according to any one of claims 50 to 54, characterized in that the cell population comprises, for example, mammalian cells, for example, human cells, optionally, in which the cell population is obtained from a patient suffering from hemoglobinopathy, for example, sickle cell disease or thalassemia, for example, beta-thalassemia.
[56]
56. Cell population according to any of claims 50 to 55, characterized by the fact that the cell population is (i) autologous in relation to a patient to whom it is to be administered, or (ii) allogeneic in relation to to a patient who is to be administered.
[57]
57. Cell population (for example, CD34 + cells), for example, according to any one of claims 50 to 56, characterized in that it comprises an indelible pattern as described in Table 7-2, optionally in which the indels of the indelible pattern described in Table 7-2 are detectable in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the population's cells.
[58]
58. Composition, characterized by the fact that it comprises the cell or cell population, as defined in any one of claims 39 to 57, optionally comprising a pharmaceutically acceptable medium, for example, a pharmaceutically acceptable medium suitable for cryopreservation.
[59]
59. Use of a cell or population of cells, as defined in any of claims 39 to 57, or of a composition, as defined in claim 58, characterized by the fact that it is for the preparation of a medicament for the treatment of a hemoglobinopathy in a patient.
[60]
60. Use of a cell or population of cells, as defined in any of claims 39 to 57, or of a composition, as defined in claim 58, characterized by the fact that it is for the preparation of a medicament to increase the expression of fetal hemoglobin in a mammal.
[61]
61. Use according to claim 60, characterized by the fact that hemoglobinopathy is beta-thalassemia or sickle cell disease.
[62]
62. Method of preparing a cell (for example, a population of cells) characterized by the fact that it comprises: (a) providing a cell (for example, a population of cells) (for example, an HSPC (for example, a population of HSPCs)); (b) culturing said cell (e.g., said cell population) ex vivo in a cell culture medium comprising a stem cell expander; and (c) introducing into said cell a first gRNA molecule, as defined in any one of claims 1 to 22, a nucleic acid molecule encoding a first gRNA molecule, as defined in any one of claims 1 to 22, a The composition, as defined in any one of claims 23 to 28, a nucleic acid, as defined in claim 29, or a vector, as defined in claim 30.
[63]
63. Method according to claim 62, characterized by the fact that, after said introduction of step (c), said cell (eg cell population) is able to differentiate into a differentiated cell (eg , differentiated cell population), for example, a cell of an erythroid lineage (for example, a cell population of an erythroid lineage), for example, a red blood cell (for example, a population of red blood cells) and in which said differentiated cell (eg differentiated cell population) produces increased fetal hemoglobin, for example, in relation to the same cell that was not subjected to step (c).
[64]
64. Method according to claim 62 or 63, characterized in that the stem cell expander is: a) (1r, 4r) -N1- (2-benzyl-7- (2-methyl-2H- tetrazol-5-i1) -9H-pyrimido [4,5-b] indol-4-yl) cyclohexane-1,4-diamine; b) methyl 4- (3-piperidin-1-ylpropylamine) -9H-pyrimido [4,5-b] indole-7-carboxylate; Cc) 4- (2- (2- (benzo [b] thiophen-3-i1) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol; d) (S) -2- (6- (2- (IH-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) - 9H-purin-9-yl) propan-1- o1; or e) their combinations (for example, a combination of (1r, 4r) -N1- (2-benzyl-7- (2-methyl-2H-tetrazol-5-i1) -9H-pyrimido [4,5-b ] indol- 4-yl) cyclohexane-1,4-diamine and (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3- yl) -9H-purin-9-yl) propan-1-ol).
[65]
65. Method according to any one of claims 62 to 64, characterized by the fact that the cell culture medium comprises thrombopoietin (TPO), FIt3 ligand (FIt-3L), and human stem cell factor (SCF ), optionally, wherein the cell culture medium further comprises human interleukin-6 (IL-6); optionally, in which the cell culture medium comprises thrombopoietin (Tpo), FIt3 ligand (FIt-3L), human stem cell factor (SCF) and, if present, human IL-6, each in a concentration that varies from about 10 ng / ml to 1000 ng / ml, optionally each at a concentration of about 50 ng / ml, for example, at a concentration of 50 ng / ml.
[66]
66. Method according to any one of claims 62 to 65, characterized in that the cell culture medium comprises a stem cell expander, at a concentration ranging from about 1 nM to about 1 MM, optionally, at a concentration ranging from about 1 µM to about 100 nM, optionally at a concentration ranging from about 500 nM to about 750 nM, optionally at a concentration of about 500 nM, for example, at a concentration of 500 nM , or at a concentration of about 750 nM, for example, at a concentration of 750 nM.
[67]
67. Method according to any one of claims 62 to 66, characterized in that the culture of step (b) comprises a culture period before the introduction of step (c), optionally, in which the culture period before the introduction of step (c) is at least 12 hours, for example, it is for a period of about 1 day to about 12 days, for example, it is for a period of about 1 day to about 6 days, for example, it is for a period of about 1 day to about 3 days, for example, for a period of about 1 day to about 2 days, for example, it is for a period of about 2 days.
[68]
68. Method according to any one of claims 62 to 67, characterized in that the culture of step (b) comprises a culture period after the introduction of step (c), optionally, in which the culture period after the introduction of step (c) is at least 12 hours, for example, it is for a period of about 1 day to about 12 days, for example, it is for a period of about 1 day to about 6 days, for example, for a period of about 2 days to about 4 days, for example, it is for a period of about 2 days or it is for a period of about 3 days or it is for a period of about 4 days.
[69]
69. Method according to any one of claims 62 to 68, characterized in that the cell population is expanded ex-vivo at least 3 times, for example, at least 4 times, for example, at least 5 times, for example, at least 10 times.
[70]
70. Method according to any one of claims 62 to 69, characterized in that the introduction of step (c) comprises an electroporation.
[71]
71. Method according to any one of claims 62 to 70, characterized in that the cell (e.g., cell population) provided in step (a) is a human cell (e.g., a population of human cells) .
[72]
72. Method according to claim 71, characterized by the fact that the cell (e.g., cell population) provided in step (a) is isolated from the bone marrow, peripheral blood (e.g., mobilized peripheral blood) or blood umbilical cord.
[73]
73. Method according to claim 72, characterized by the fact that (1) the cell (eg, cell population) provided in step (a) is isolated from the bone marrow, for example, it is isolated from the bone marrow of a patient suffering from hemoglobinopathy, optionally where hemoglobinopathy is sickle cell disease or thalassemia, optionally, where thalassemia is beta-thalassemia; or (ii) the cell (e.g., cell population) provided in step (a) is isolated from peripheral blood, for example, it is isolated from the peripheral blood of a patient suffering from hemoglobinopathy, optionally in which hemoglobinopathy is sickle cell disease or thalassemia, optionally, where thalassemia is beta-thalassemia; optionally in which peripheral blood is mobilized peripheral blood, optionally in which peripheral blood is mobilized using Plerixafor, G-CSF or a combination of these.
[74]
74. Method according to any one of claims 62 to 73, characterized in that the cell population provided in step (a) is enriched for CD34 + cells.
[75]
75. Method according to any one of claims 62 to 74, characterized by the fact that subsequent to the introduction of step (c)) the cell (e.g., cell population) is cryopreserved.
[76]
76. The method of any one of claims 62 to 75, characterized by the fact that subsequent to the introduction of step (c), the cell (for example, cell population) comprises: (a) an indel at or near a genomic DNA sequence complementary to the target domain of the first gRNA molecule; and / or b) deletion comprising sequence, for example, substantially the entire sequence, between a sequence complementary to the target domain of the first gRNA molecule (e.g., at least 90% complementary to the target domain of gRNA, for example, completely complementary to the target domain of gRNA) in the HBG1 promoter region and a sequence complementary to the target domain of the first gRNA molecule (for example, at least 90% complementary to the target domain of gRNA, for example, completely complementary to the targeting gRNA) in the HBG2 promoter region, optionally, where the indel, for example, the deletion, does not comprise a nucleotide arranged between 5,250,092 and 5,249,833, -
chain (h9g38).
[77]
77. Method according to any one of claims 62 to 76, characterized in that: (a) after the introduction of step (c), at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cells in the cell population comprise an indel at or near a genomic DNA sequence complementary to the target domain of the first gRNA molecule, optionally where the indel is selected from an indel listed in Table 2 -7, optionally, in which no cell of the population comprises a deletion of a nucleotide arranged between 5,250,092 and 5,249,833, - chain (hg38); (b) after the introduction of step (c), the cell (for example, cell population) is able to differentiate into a cell differentiated from an erythroid lineage (for example, a red blood cell) and in which said cell differentiated exhibits an increased level of fetal hemoglobin, for example, relative to an unchanged cell (e.g., cell population); (c) after the introduction of step (c), the cell population is able to differentiate into a population of differentiated cells, for example, a population of cells of an erythroid lineage (for example, a population of red blood cells) and wherein said population of differentiated cells has an increased percentage of F cells (for example, at least about 15%, at least about 20%, at least about 25%, at least about 30% or at least about 40% higher percentage of F cells), for example, in relation to a population of unchanged cells; (d) after the introduction of step (c), the cell (for example,
a population of cells) is able to differentiate into a differentiated cell, for example, a cell of an erythroid lineage (for example, a red blood cell) and in which said differentiated cell (for example, a population of differentiated cells) produces at least about 6 picograms (for example, at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or from about 8 to about 9 picograms or about from 9 to about 10 picograms) of fetal hemoglobin per cell; (e) after the introduction of step (c), no off-target indels are formed in said cell, for example, no off-target indels are formed outside the HBG1 and / or HBG2 promoter regions, for example, as detectable by next generation sequencing and / or nucleotide insertion assay; and / or (f) after the introduction of step (c), indelible is not detected outside the target, for example, indelible is not detected outside the target indelible outside the HBG1 and / or HBG2 promoter regions, in more than about 5%, for example, more than about 1%, for example, more than about 0.1%, for example, more than about 0.01%, of the cells in the cell population, for example, as detectable by next generation sequencing and / or a nucleotide insertion assay.
[78]
78. Cell (for example, cell population), characterized by the fact that it is obtainable by the method, as defined in any of claims 62 to 77.
[79]
79. Cell, for example, an altered cell, for example, a cell, as defined in claim 78, characterized by the fact that: (a) at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% about 95%, at least about 96%, at least about 97%, at least about 98% or at least at least about 99% of the cells in the cell population comprise an indel in or near a genomic DNA sequence complementary to the target domain of the gRNA molecule, as defined in any one of claims 1 to 22, optionally, where the indel is selected from an indel listed in Table 2-7, optionally, in which no cell in the population comprises a deletion of a nucleotide arranged between 5,250,092 and 5,249,833, - strand (h9g38);
(b) the cell (eg, cell population) is able to differentiate into a differentiated cell from an erythroid lineage (eg, a red blood cell) and in which said differentiated cell exhibits an increased level of fetal hemoglobin, for example example, in relation to an unchanged cell (eg cell population);
(c) the population of cells is able to differentiate into a population of differentiated cells, for example, a population of cells of an erythroid lineage (for example, a population of red blood cells) and in which said population of differentiated cells has an increased percentage of F cells (for example, at least about 15%, at least about 20%, at least about 25%, at least about 30% or at least about 40% higher percentage of F cells ), for example, for a population of unchanged cells;
(d) the cell (for example, a population of cells) is able to differentiate into a differentiated cell, for example, a cell of an erythroid lineage (for example, a red blood cell) and in which said differentiated cell (for example, example, a population of differentiated cells) produces at least about 6 picograms (for example,
at least about 7 picograms, at least about 8 picograms, at least about 9 picograms, at least about 10 picograms or from about 8 to about 9 picograms or about 9 to about picograms) of fetal hemoglobin per cell; (e) off-target indels do not form in said cell, for example, off-target indels do not form outside the HBG1 and HBG2 promoter regions, for example, as detectable by next generation sequencing and / or insertion assay nucleotides; (f) indel is not detected outside the target, for example, indel is not detected outside the target indel outside the HBG1 and / or HBG2 promoter regions, in more than about 5%, for example, more than about 1 %, for example, more than about 0.1%, for example, more than about 0.01%, of the cells in the cell population, for example, as detectable by next generation sequencing and / or an assay nucleotide insertion; and / or (g) said cell or its progeny is detectable in a patient to whom it is transplanted more than 16 weeks, more than weeks or more than 24 weeks after the transplant, optionally detected by detecting an indel in one or close to a genomic DNA sequence complementary to the target domain of a gRNA molecule, as defined in any one of claims 1 to 22, optionally wherein the indel is selected from an indel listed in Table 2-7.
[80]
80. Cell according to claim 78 or 79, characterized in that the cell is an animal cell, for example, a mammalian, primate or human cell, for example, it is a human cell; optionally wherein said cell is obtained from a patient suffering from hemoglobinopathy, for example, sickle cell disease or thalassemia, for example, beta-thalassemia.
[81]
81. Cell according to any one of claims 78 to 80, characterized in that the cell is an HSPC, optionally an HSPC CD34 +, optionally an HSPC CD34 + CD90 +.
[82]
82. Cell according to any one of claims 78 to 81, characterized by the fact that the cell (eg cell population) has been isolated from bone marrow, peripheral blood (eg mobilized peripheral blood) or cord blood umbilical.
[83]
83. Cell according to any one of claims 78 to 82, characterized by the fact that the cell is autologous or allogeneic with respect to a patient to be administered with said cell.
[84]
84. Use of a cell or population of cells, as defined in any of claims 39 to 57 or 78 to 83, characterized by the fact that it is for the preparation of a medicament for the treatment of a hemoglobinopathy in a human patient.
[85]
85. Use of a cell or population of cells, as defined in any of claims 39 to 57 or 78 to 83, characterized by the fact that it is for the preparation of a medicament to increase fetal hemoglobin expression in a human patient.
[86]
86. Use according to claim 84, characterized by the fact that hemoglobinopathy is beta-thalassemia or sickle cell disease.
[87]
87. Use according to any of claims 84 to 86, characterized in that the human patient is administered a composition comprising at least about 2 and 6 cells, as defined in any of claims 39 to 57 or 78 to 83, per kg of human patient's body weight, for example, at least about 2 and 6 CD34 + cells, as defined in any one of claims 39 to 57, or 78 to 83, per kg of human patient's body weight.
[88]
88. Use according to any of claims 84 to 87, characterized by the fact that the cell or cell population, or its progeny, is detectable in the human patient for more than 16 weeks, more than 20 weeks, or more than 24 weeks after administration, optionally, as detected by the detection of an indel at or near a genomic DNA sequence complementary to the target domain of a gRNA molecule, as defined in any of claims 1 to 22, optionally where the indel is selected from an indel listed in Table 2-7; optionally, where the level of detection of indel in a population of reference cells (eg CD34 + cells) more than 16 weeks, more than 20 weeks or more than 24 weeks after administration is reduced by not more than 50%, not more than 40%, not more than 30%, not more than 20%, not more than 10%, not more than 5% or not more than 1%, in relation to the level of detection of the indel in the cell population immediately before administration.
[89]
89. The gRNA molecule according to any one of claims 1 to 22, a composition, as defined in any one of claims 23 to 28 or 58, a nucleic acid, as defined in claim 29, a vector, as defined in claim 30, a cell or population of cells, as defined in any one of claims 39 to 57, or 78 to 83, characterized by the fact that it is for use as a medicine.
[90]
90. Use of a gRNA molecule according to any one of claims 1 to 22, a composition, as defined in any one of claims 23 to 28 or 58, a nucleic acid, as defined in claim 29, a vector, as defined in claim 30, a cell or population of cells, as defined in any one of claims 39 to 57, or 78 to 83, characterized by the fact that it is in the manufacture of a medicament.
[91]
91. Use of a gRNA molecule, as defined in any of claims 1 to 22, a composition, as defined in any of claims 23 to 28 or 58, a nucleic acid, as defined in claim 29, a vector, as defined in claim 30, a cell or population of cells, as defined in any of claims 39 to 57, or 78 to 83, characterized by the fact that it is in the manufacture of a medicament for the treatment of a disease.
[92]
92. Use of a gRNA molecule, as defined in any of claims 1 to 22, a composition, as defined in any of claims 23 to 28 or 58, a nucleic acid, as defined in claim 29, a vector, as defined in claim 30, a cell or population of cells, as defined in any of claims 39 to 57, or 78 to 83, characterized by the fact that it is in the manufacture of a medicament for the treatment of a disease, wherein the disease it is a hemodglobinopathy, optionally in which hemoglobinopathy is sickle cell disease or thalassemia (for example, beta-thalassemia).
[93]
93. Invention, characterized by any of its embodiments or categories of claim encompassed by the material initially disclosed in the patent application or in its examples presented here.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US20200102561A1|2020-04-02|Compositions and methods for the treatment of hemoglobinopathies

---

AU2016381313B2|2020-11-26|Compositions and methods for the treatment of hemoglobinopathies

---

EP3274454B1|2021-08-25|Crispr/cas-related methods, compositions and components

---

AU2016261358B2|2021-09-16|Optimized CRISPR/Cas9 systems and methods for gene editing in stem cells

---

US20190136230A1|2019-05-09|Genetically engineered cells and methods of making the same

---

US20160281111A1|2016-09-29|Crispr/cas-mediated gene conversion

---

AU2016262521A1|2017-12-14|CRISPR/CAS-related methods and compositions for treating HIV infection and AIDS

---

WO2017053729A1|2017-03-30|Nuclease-mediated genome editing of primary cells and enrichment thereof

---

AU2015236128A1|2016-11-10|CRISPR/CAS-related methods and compositions for treating HIV infection and AIDS

---

JP2019525759A|2019-09-12|BCL11A homing endonuclease variants, compositions and methods of use

---

CA3142521A1|2020-12-24|Compositions and methods for editing beta-globin for treatment of hemaglobinopathies

---

JP7030522B2|2022-03-07|Optimized CRISPR / CAS9 system and method for gene editing in stem cells

---

WO2021123920A1|2021-06-24|Compositions and methods for the treatment of hemoglobinopathies

---

WO2019106522A1|2019-06-06|Pooled crispr/cas9 screening in primary cells using guide swap technology

同族专利:

---

公开号 | 公开日

---

AU2018215726A1|2019-08-22|

---

AR110962A1|2019-05-22|

---

TW201839136A|2018-11-01|

---

AU2018215726B2|2021-11-04|

---

CN110546262A|2019-12-06|

---

EA201991862A1|2020-02-04|

---

PH12019501812A1|2020-09-21|

---

RU2019127919A|2021-03-09|

---

WO2018142364A1|2018-08-09|

---

EP3577223A1|2019-12-11|

---

CA3052275A1|2018-08-09|

---

SG11201907056XA|2019-08-27|

---

JP2020505934A|2020-02-27|

---

RU2019127919A3|2021-11-03|

---

US20200102561A1|2020-04-02|

---

IL268406D0|2019-09-26|

---

KR20190121319A|2019-10-25|

---

MX2019009361A|2020-01-30|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US5800815A|1903-05-05|1998-09-01|Cytel Corporation|Antibodies to P-selectin and their uses|

---

US6309639B1|1991-02-05|2001-10-30|The Board Of Regents Of The University Of Oklahoma|Method for inhibiting an inflammatory response using antibodies to P-selectin glycoprotein ligand|

---

NZ252542A|1992-05-05|1997-08-22|Cytel Corp|Compositions of blocking p-selectin antibodies and their use in treating inflammatory or thrombotic disease|

---

US5622701A|1994-06-14|1997-04-22|Protein Design Labs, Inc.|Cross-reacting monoclonal antibodies specific for E- and P-selectin|

---

JP5059411B2|2003-12-19|2012-10-24|ノバルティスバクシンズアンドダイアグノスティックス，インコーポレーテッド|Cell transfection formulations of short interfering RNA, related compositions and methods of making and uses|

---

ES2403055T3|2004-04-13|2013-05-13|F. Hoffmann-La Roche Ag|Anti-P-selectin antibodies|

---

US8945565B2|2006-12-01|2015-02-03|Selexys Pharmaceuticals Corporation|Methods of treating inflammatory or thrombotic conditions with anti-P-selectin antibodies|

---

US20110212096A1|2006-12-01|2011-09-01|Scott Rollins|Anti-p-selectin antibodies and methods of their use and identification|

---

PL2662091T3|2006-12-01|2019-05-31|Novartis Ag|Anti-P-selectin antibodies and methods of using the same to treat inflammatory diseases|

---

PE20100362A1|2008-10-30|2010-05-27|Irm Llc|PURINE DERIVATIVES THAT EXPAND HEMATOPOYETIC STEM CELLS|

---

WO2011076807A2|2009-12-23|2011-06-30|Novartis Ag|Lipids, lipid compositions, and methods of using them|

---

CA2804396C|2010-07-06|2021-06-29|Novartis Ag|Liposomes with lipids having an advantageous pka-value for rna delivery|

---

EP2611420B1|2010-08-31|2019-03-27|GlaxoSmithKline Biologicals SA|Lipids suitable for liposomal delivery of protein-coding rna|

---

MX350764B|2011-07-06|2017-09-18|Novartis Ag|Liposomes having useful n:p ratio for delivery of rna molecules.|

---

RS59898B1|2011-10-17|2020-03-31|Massachusetts Inst Technology|Intracellular delivery|

---

US9394547B2|2012-01-03|2016-07-19|City University Of Hong Kong|Method and apparatus for delivery of molecules to cells|

---

WO2013103467A1|2012-01-06|2013-07-11|Alcon Research, Ltd.|Interfering rna delivery system and uses thereof|

---

PT2807165T|2012-01-27|2019-07-12|Univ Montreal|Pyrimido[4,5-b]indole derivatives and use thereof in the expansion of hematopoietic stem cells|

---

AU2013222170B2|2012-02-24|2017-01-05|Fred Hutchinson Cancer Research Center|Compositions and methods for the treatment of hemoglobinopathies|

---

EP2958996B1|2013-02-25|2019-10-16|Sangamo Therapeutics, Inc.|Methods and compositions for enhancing nuclease-mediated gene disruption|

---

EP3608308B1|2013-03-08|2021-07-21|Novartis AG|Lipids and lipid compositions for the delivery of active agents|

---

DK2970948T3|2013-03-15|2019-04-08|Glaxosmithkline Biologicals Sa|METHODS OF RNA PURIFICATION|

---

CA2902709A1|2013-03-15|2014-09-25|Global Blood Therapeutics, Inc.|Compositions and methods for the modulation of hemoglobin |

---

PL3033184T3|2013-08-16|2021-09-20|Massachusetts Institute Of Technology|Selective delivery of material to cells|

---

EP3080271B1|2013-12-12|2020-02-12|The Broad Institute, Inc.|Systems, methods and compositions for sequence manipulation with optimized functional crispr-cas systems|

---

US10426737B2|2013-12-19|2019-10-01|Novartis Ag|Lipids and lipid compositions for the delivery of active agents|

---

PL3083556T3|2013-12-19|2020-06-29|Novartis Ag|Lipids and lipid compositions for the delivery of active agents|

---

WO2016103233A2|2014-12-24|2016-06-30|Dana-Farber Cancer Institute, Inc.|Systems and methods for genome modification and regulation|

---

CN107532182A|2015-02-23|2018-01-02|克里斯珀医疗股份公司|Treat the material and method of hemoglobinopathy|

---

EP3430142A1|2016-03-14|2019-01-23|Editas Medicine, Inc.|Crispr/cas-related methods and compositions for treating beta hemoglobinopathies|US10704021B2|2012-03-15|2020-07-07|Flodesign Sonics, Inc.|Acoustic perfusion devices|

---

US9725710B2|2014-01-08|2017-08-08|Flodesign Sonics, Inc.|Acoustophoresis device with dual acoustophoretic chamber|

---

WO2015148863A2|2014-03-26|2015-10-01|Editas Medicine, Inc.|Crispr/cas-related methods and compositions for treating sickle cell disease|

---

US11214789B2|2016-05-03|2022-01-04|Flodesign Sonics, Inc.|Concentration and washing of particles with acoustics|

---

US11268082B2|2017-03-23|2022-03-08|President And Fellows Of Harvard College|Nucleobase editors comprising nucleic acid programmable DNA binding proteins|

---

EP3645721A1|2017-06-30|2020-05-06|Novartis AG|Methods for the treatment of disease with gene editing systems|

---

EP3697906A1|2017-10-16|2020-08-26|The Broad Institute, Inc.|Uses of adenosine base editors|

---

EP3704245A1|2017-11-01|2020-09-09|Novartis AG|Synthetic rnas and methods of use|

---

KR20200089334A|2017-12-14|2020-07-24|프로디자인 소닉스, 인크.|Acoustic transducer driver and controller|

---

US20220047637A1|2018-11-29|2022-02-17|Editas Medicine, Inc.|Systems and methods for the treatment of hemoglobinopathies|

---

WO2020112195A1|2018-11-30|2020-06-04|Yale University|Compositions, technologies and methods of using plerixafor to enhance gene editing|

---

AU2020223060A1|2019-02-13|2021-07-29|Beam Therapeutics Inc.|Compositions and methods for treating hemoglobinopathies|

---

CN113785059A|2019-04-30|2021-12-10|博雅辑因（北京）生物科技有限公司|Method for predicting treatment effectiveness of hemoglobinopathy|

---

CN111575306A|2020-06-11|2020-08-25|南方医科大学|Method for activating gamma globin gene expression in erythrocyte|

法律状态:
2021-11-03| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

---

US201762455464P| true| 2017-02-06|2017-02-06|

---

US62/455,464|2017-02-06|

---

PCT/IB2018/050712|WO2018142364A1|2017-02-06|2018-02-05|Compositions and methods for the treatment of hemoglobinopathies|

---