aadc polynucleotides for the treatment of parkinson's disease

专利摘要:
the invention relates to compositions and methods for the preparation, production and therapeutic use of polynucleotides encoding aadc for the treatment of parkinson's disease.
公开号:BR112019025122A2
申请号:R112019025122-7
申请日:2018-06-14
公开日:2020-07-28
发明作者:Maria Scheel；Bernard Ravina
申请人:Voyager Therapeutics, Inc.；
IPC主号:

专利说明:

[001] [001] The present application is being filed together with a sequence listing in electronic format. The Sequence Listing file, titled 14482-022-228 Sequence Listing.txt, was created on June 10, 2018, and has a size of 6,461,106 bytes. The information in the electronic format of the Sequence Listing is hereby incorporated in its entirety by way of reference. Field of Invention
[002] [002] The invention relates to compositions, particularly nucleic acid molecules, for example polynucleotides encoding AADC, for use in the treatment of Parkinson's disease. In some embodiments such AADC polynucleotides may be encoded by recombinant adeno-associated viruses or in recombinant adeno-associated viruses (AAVs). Background of the Invention
[003] [003] Aromatic L-amino acid decarboxylase (AADC) is a pyridoxal phosphate-dependent homodimeric enzyme responsible for the synthesis of dopamine and serotonin. The encoded protein catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA or levodopa) to dopamine; L-5-hydroxytryptophan to serotonin; and L-tryptophan into tryptamine. Defects in this gene are the cause of aromatic L-amino acid decarboxylase (AADCD) deficiency, which is a congenital defect in neurotransmitter metabolism that leads to a combined deficiency in serotonin and catecholamine that results in severe motor and autonomic dysfunction.
[004] [004] Parkinson's disease (PD) is a progressive neurodegenerative disease of the central nervous system (CNS) that produces sensory and motor symptoms. Dopamine replacement (i.e., levodopa) has been the traditional pharmacotherapy! for motor impairment in PD. However, the benefit of dopamine therapy is becoming less pronounced due, in part, to the "progressive death of dopamine-generating cells and corresponding loss of AADC activity. In addition, systemic administration of dopamine at high doses is complicated by side effects." , such as fluctuations in motor performance, dyskinesias, and hallucinations, resulting from dopaminergic stimulation of the mesolimbric system. One strategy to restore dopaminergic function and minimize side effects is the use of gene therapy to deliver AADC directly to a target region of the CNS.
[005] [005] The adeno-associated virus (AAV) has emerged as an attractive vector for gene therapy due to its long-term gene expression, the inability to autonomously replicate without a helper virus, the ability to translate dividing and non-dividing cells, and the lack of pathogenicity arising from wild-type infections (see, for example, Hadaczek et al. Mol. Ther. 18(8), 1458-1461, Aug. 2010). AAV is a helper-dependent DNA parvovirus that belongs to the Dependovirus genus.
[006] [006] The present invention provides such improved nucleic acid constructs, e.g. polynucleotides, for use with AAV-derived vectors comprising the dopa carboxylase ("DDC") gene sequence encoding the full-length AADC protein intended for gene therapy. in the treatment of Parkinson's disease.
[007] [007] The nucleic acid constructs described in this application comprise at least a 5"-ITR and a 3'-ITR, each or both of which may be derived from an AAV, positioned about a DDC gene sequence, as well as components necessary for gene expression and clone selection Summary of the Invention
[008] [008] This application describes compositions, methods, processes, kits and devices for the design, preparation, production and/or formulation of AADC polynucleotides.
[009] [009] In some embodiments such AADC polynucleotides may be encoded or contained in plasmids or vectors or recombinant adeno-associated viruses (AAV).
[0010] [0010] Details of the various embodiments of the invention are presented in the description below. Other features, objects, and advantages of the invention will become apparent from the description and claims. Brief Description of Drawings
[0011] [0011] These and other objects, features and advantages above will become apparent from the following description of particular embodiments of the invention as illustrated in the accompanying drawings. The drawings are not necessarily to scale, rather the illustration of the principles of various embodiments of the invention is emphasized.
[0012] [0012] Figure 1 is a schematic representation of a viral genome of the invention. Detailed Description Compositions of the Invention Adeno-associated viruses (AAVs) and AAV particles
[0013] [0013] The viruses of the family Parvoviridae are small, non-enveloped icosahedral enveloped viruses characterized by a single-stranded DNA genome. Parvoviridae viruses consist of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Due to its relatively simple structure, easily manipulated using traditional molecular biology techniques, this family of viruses is useful as a biological tool. The virus genome can be modified to contain a minimum of components for assembling a functional recombinant virus, or viral particle, that is loaded with a desired payload or genetically engineered to express or deliver a desired payload, which can be delivered. to a target cell, tissue, organ, or organism.
[0014] [0014] Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth |. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996), the entirety of which is incorporated herein by reference.
[0015] [0015] The Parvoviridae family comprises the Dependovirus genus which includes adeno-associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.
[0016] [0016] The vector genome is a linear single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. The AAV viral genome may comprise a payload region and at least one inverted terminal region (ITR) or ITR region. ITRs traditionally flank nucleotide sequences—coding for nonstructural proteins (encoded by Rep genes) and structural proteins (encoded by capsular genes or Cap genes). Although we do not wish to be bound by theory, an AAV viral genome typically comprises two ITR sequences. The vector genome comprises a characteristic T-shaped hairpin structure defined by the 145 nt self-complementary terminals of the 5' and 3' ends of the sSDNA that form an energetically stable double stranded region. Double stranded hair clip structures comprise multiple functions including, but not limited to, acting as an origin for DNA replication by functioning as primers for the endogenous DNA polymerase complex of the replicating host viral cell.
[0017] [0017] In addition to the encoded heterologous payload, the AAV particles may comprise the viral genome, in whole or in part, of any naturally occurring and/or recombinant AAV serotype nucleotide sequence or variant. AAV variants can have significant sequence homology at the nucleic acid (genome or capsid) level and at the amino acid (capsid) level, to produce constructs that are generally physical and functional equivalents, replicate by similar mechanisms, and come together by similar mechanisms. similar. Chiorini et al., J. Vir. 71: 6823-33(1997); Srivastava et al., J. Vir. 45:555-64 (1983); Chiorini et al, J. Vir. 73:1309-1319 (1999); Rutledge et al., J. Vir. 72:309-319 (1998); and Wu et al., J. Vir. 74: 8635-47 (2000), the entirety of each being incorporated herein by reference.
[0018] [0018] In one embodiment, the AAV particles of the present invention are recombinant AAV particles that are replication defective, lacking sequences encoding functional proteins Rep and Cap in their viral genome. These defective AAV particles may lack most or all of the parental coding sequences and essentially carry only one or two AAV ITR sequences and the nucleic acid of interest for delivery to a cell, tissue, organ, or organism.
[0019] [0019] In one embodiment, the viral genome of the AAV particles of the present invention comprises at least one control element that provides for the replication, transcription and translation of a coding sequence encoded therein. Not all control elements need always be present as long as the coding sequence is capable of being replicated, transcribed and/or translated into an appropriate host cell. Non-limiting examples of expression control elements include transcription initiation and/or termination sequences, promoter and/or enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic MRNA, sequences that improve translation efficiency (e.g., Kozak consensus sequence), sequences that improve protein stability, and/or sequences that improve protein processing and/or secretion.
[0020] [0020] In accordance with the present invention, AAV particles for use in therapy and/or diagnosis comprise a virus that has been distilled or reduced to the minimum components necessary for transduction of a nucleic acid payload or payload of interest. In this way, AAV particles are constructed as vehicles for specific delivery without exhibiting the harmful replication and/or integration characteristics found in wild-type viruses.
[0021] [0021] The AAV particles of the present invention can be produced recombinantly and can be based on parental or reference sequences from adeno-associated virus (AAV). As used in this application, a "vector" is any molecule or moiety that transports, transduces or otherwise acts as a carrier for a heterologous molecule such as the nucleic acids described in this application.
[0022] [0022] In addition to single stranded AAV particles (e.g. ssAAVs), the present invention also features self-complementary AAV particles (scAAVs). The scAAV particles contain strands of DNA that combine to form double-stranded fna.
[0023] [0023] In one embodiment, the AAV particle of the present invention is an SscAAV.
[0024] [0024] In one embodiment, the AAV particle of the present invention is an ssAAV.
[0025] [0025] Methods for producing and/or modifying AAV particles are already disclosed in the art such as pseudotified AAV particles (PCT Patent Publication No.* WO200028004; WO200123001; WOZ2004112727, WO 2005005610 and WO 2005072364, the entirety of each one of which is incorporated herein by reference).
[0026] [0026] AAV particles can be modified to improve distribution efficiency. Such modified AAV particles can be efficiently packaged and used to successfully infect target cells at high frequency and with minimal toxicity. In some embodiments the capsids of AAV particles are constructed according to the methods described in US Publication Number US 20130195801, the entirety of which is incorporated herein by reference.
[0027] [0027] In one embodiment, AAV particles comprising a payload region encoding the polypeptides of the invention can be introduced into mammalian cells. AAV serotype
[0028] [0028] The AAV particles of the present invention may comprise or be derived from any natural or recombinant AAV serotype. In accordance with the present invention, AAV particles may utilize or be based on a serotype selected from any of the following serotypes PHP.B, PHP.A, AAV1I, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAVA4-,
[0029] [0029] In some embodiments, the AAV serotype may be, or have a modification as described in US Publication No. US 20160361439, the entirety of which is incorporated herein by reference, such as, but not limited to, Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F of the wild-type AAV1, AAV2, AAV3, AAVA4, AAVS, AAV6, AAV7, AAV8, AAV9, AAV1O , AAV11, AAV12, and hybrids thereof.
[0030] [0030] In some embodiments, the AAV serotype may be, or have, the mutation as described in U.S. Patent No. US 9,546,112, the entirety of which is incorporated herein by reference, such as, but not limited to, at least two, but not all, of the F129L, D418E, K531E, L584F, V598A and H642N mutations in the AAV6 sequence (SEQ ID NO:4 of US 9546112), AAV1
[0031] [0031] In some embodiments, the AAV serotype may be, or have, a mutation in the AAV1 sequence, as described in U.S. Patent No. US 20130224836 , the entirety of which is incorporated herein by reference, such as, however, without limitation, at least one of the surface exposed tyrosine residues, preferably at positions 252, 273, 445, 701, 705 and 731 of AAV1 (SEQ ID NO: 2 of US 20130224836) is replaced by another amino acid, preferably by a phenylalanine residue. In one embodiment, the AAV serotype can be, or have, a mutation in the AAV9 sequence, such as, but not limited to, at least one of the surface-exposed tyrosine residues, preferably at positions 252, 272, 444, 500 , 700, 704 and 730 of AAV2 (SEQ ID NO: 4 of US 20130224836) substituted by another amino acid, preferably by a phenylalanine residue. In one embodiment, the tyrosine residue at position 446 of AAV9 (SEQ ID NO: 6 US 20130224836) is substituted with a phenylalanine residue.
[0032] [0032] In some embodiments, the serotype may be AAV2 or a variant thereof, as described in International Publication No. WOZ2016130589, the entirety of which is incorporated herein by reference. The AAV2 amino acid sequence may comprise mutations N587A, ES48A, or N708A. In one embodiment, the amino acid sequence of any AAV may comprise a V708K mutation.
[0033] [0033] In some embodiments, the AAV serotype may be, or have, a sequence as described in the North American Publication
[0034] [0034] In some embodiments, the AAV serotype may be, or have, a sequence as described in US Publication No. US20150159173, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV2 (SEQ ID Nos: 7 and 23 of US20150159173), rh20 (SEQ ID No: 1 of US20150159173), rh32/833 (SEQ |D No. 2 of US20150159173), rh39 (SEQ ID No: 3, 20 and 36 of document US20150159173), rh46 (SEQ ID No. 4 and 22 of US20150159173), rh/73 (SEQ |D No. 5 of document US20150159173), rh74 (SEQ ID No. 6 of document US20150159173) AAV6.1 (SEQ ID No. 29 of document
[0035] [0035] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent No. US 7,198,951, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV9 ( SEQ ID NO: 1-3 of US 7198951), AAV2 (SEQ ID NO: 4 of US 7198951), AAV1 (SEQ ID NO: 5 of US 7198951), AAV3 (SEQ ID NO: 6 of US 7198951 ), and AAV8 (SEQ ID NO: 7 of US7198951).
[0036] [0036] In some embodiments, the AAV serotype may be, or have, a mutation in the AAV9 sequence as described by N Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011 ), the entirety of which is incorporated herein by reference), such as, but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9 .24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.
[0037] [0037] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent No. US 6,156,303, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV3B ( SEQ ID NO: 1 and 10 of US 6156303), AAV6 (SEQ ID NO: 2, 7 and 11 of US 6156303), AAV2 (SEQ ID NO: 3 and 8 of US 6156303), AAV3A (SEQ ID NO : 4 and 9 of US 6156303), or derivatives thereof.
[0038] [0038] In some embodiments, the AAV serotype may be, or have, a sequence as described in US Publication No. US20140359799, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV8 (SEQ ID No: 1 of US20140359799), AAVDJ (SEQ ID No: 2 and 3 of US20140359799), or variants thereof.
[0039] [0039] In some embodiments, the serotype may be AAVDJ or a variant thereof, such as AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology 82(12): 5887-5911 (2008 ), the entirety of which is incorporated herein by reference). The AAVDJ8 amino acid sequence may comprise two or more mutations to remove the heparin binding domain (HBD). As a non-limiting example, the AAV-DJ sequence described as SEQ ID NO: 1 in US Patent No. 7,588,772, the entirety of which is incorporated herein by reference, may comprise two mutations: (1) R587Q where arginine (R; Arg) at amino acid 587 is exchanged for glutamine (Q; GIn) and (2) R590T where arginine (R; Arg) at amino acid 590 is exchanged for threonine (T; Thr). As another non-limiting example, it may comprise three mutations: (1) K406R where lysine (K; Lys) at amino acid 406 is exchanged for arginine (R; Arg), (2) R587Q where arginine (R; Arg) in amino acid 587 is exchanged for glutamine (Q; GIn) and (3) R590T where arginine (R; Arg) at amino acid 590 is exchanged for threonine (T; Thr).
[0040] [0040] In some embodiments, the AAV serotype may be, or have, an AAV4 sequence as described in International Publication No. WO1998011244, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV4 (SEQ ID No.: 1-20 of WO 1998011244).
[0041] [0041] In some embodiments, the AAV serotype may be, or have, a mutation in the AAV2 sequence to generate AAV2G9 as described in International Publication No. WO2014144229 and the entirety of which is incorporated herein by reference.
[0042] [0042] In some embodiments, the AAV serotype may be, or have, a sequence as described in International Publication No. WO2005033321, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV3-3 (SEQ ID No: 217 of WOZ2005033321), AAV1 (SEQ ID No: 219 and 202 of WOZ2005033321), AAV106.1/hu.37 (SEQ ID No: 10 of WO2005033321), AAV114.3/hu.40 (SEQ ID No.: 11 of WO2005033321), AAV127.2/hu.41 (SEQ ID Nos. 6 and 8 of WO2005033321), AAV128.3/hu.44 (SEQ ID No: 81 of WOZ2005033321), AAV130.4/hu. 48 (SEQ ID NO: 78 of WOZ2005033321), AAV145.1/hu.53 (SEQ ID NO: 176 and 177 of WO2005033321), AAV145.6/hu.56 (SEQ ID NO: 168 and 192 of WOZ2005033321 ), AAV16.12/hu.11 (SEQ ID NOS: 153 and 57 of WO2005033321), AAV16.8/hu.10 (SEQ ID NOS:: 156 and 56 of WOZ2005033321), AAV161.10/hu.60 (SEQ ID NO: 170 from WOZ2005033321), AAV161.6/hu.61 (SEQ ID NO: 174 from W O2005033321), AAV1-7/rh.48 (SEQ ID NO: 32 of WOZ2005033321), AAV1-8/rh.49 (SEQ ID NOs: 103 and 25 of WO2005033321), AAV2 (SEQ ID NOs: 211 and 221 from WO2005033321), AAV2-15/rh.62 (SEQ ID NO: 33 and 114 from WO2005033321), AAV2-3/rh.61 (SEQ ID NO: 21 from WOZ2005033321), AAV2-4/rh.50 (SEQ ID NOS: 23 and 108 of WO2005033321), AAV2-5/rh.51 (SEQ ID NOS: 104 and 22 of WO2005033321), AAV3.1/hu.6 (SEQ ID NOS: 5 and 84 of document WO2005033321), AAV3.1/hu.9 (SEQ ID NO: 155 and 58 of WOZ2005033321), AAV3-11/rh.53 (SEQ ID NO: 186 and 176 of WOZ2Z2005033321), AAV3-3 (SEQ ID NO: : 200 from WO2005033321 ), AAV33.12/hu.17 (SEQ ID NO.4 from WO2005033321), AAV33.4/hu.15 (SEQ ID NO: 50 from WO2005033321), AAV33.8/hu.16 (SEQ ID NO: 51 from WO2005033321), AAV3-9/rh.52 (SEQ ID NO: 96 and 18 from WO2005033321), AAV4-19/rh.55 (SEQ ID NO: 117 from WOZ2005033321), AAV4-4 (SEQ ID No.: 201 and 218 of the document nto WOZ2005033321), AAV4-9/rh.54 (SEQ ID NO: 116 of WOZ2005033321), AAV5 (SEQ ID NO: 199 and 216 of WOZ2005033321), AAV52.1/hu.20 (SEQ ID NO: 63 of WO2005033321), AAV52/hu.19 (SEQ ID NO: 133 from WO2005033321), AAV5-22/rh.58 (SEQ ID NO: 27 from WO2005033321), AAV5-3/rh.57 (SEQ ID NO:: 105 of WO2005033321), AAV5-3/rh.57 (SEQ ID NO: 26 of WO2005033321), AAV58.2/hu.25 (SEQ ID NO: 49 of WOZ2005033321), AAV6 (SEQ ID NO: 203 and 220 of WO2005033321), AAV7 (SEQ ID NO: 222 and 213 of WO2005033321), AAV7.3/hu.7 (SEQ |D NO: 55 of WOZ2005033321), AAV8 (SEQ ID NO: 223 and 214 of document WO2005033321), AAVH-1/hu.1 (SEQ ID NO: 46 of WO2005033321), AAVH-5/hu.3 (SEQ ID NO: 44 of WO2005033321), AAVhu.1 (SEQ ID NO: 144 of WOZ2005033321 ), AAVhu.10 (SEQ |D No. 156 of WOZ2005033321), AAVhu.11 (SEQ ID No. 153 of WOZ2005033321), AAVhu.12 (WO2005033321 SEQ |D No: 59 ), AAVhu.13 (SEQ ID NO: 129 of WO2005033321), AAVhu.14/AAV9 (SEQ ID NO: 123 and 3 of WO2005033321), AAVhu.15 (SEQ ID NO: 147 of WO2005033321), AAVhu. 16 (SEQ ID NO: 148 of WO2005033321), AAVhu.17 (SEQ ID NO: 83 of WO2005033321), AAVhu.18 (SEQ ID NO: 149 of WO2005033321), AAVhu.19 (SEQ ID NO: 133 of WO2005033321), AAVhu.2 (SEQ ID No: 143 of WO2005033321), AAVhu.20 (SEQ ID No. 134 of WO2005033321), AAVhu.21 (SEQ ID No. 135 of WO2005033321), AAVhu.22 (SEQ ID No. 138 of WO2005033321), AAVhu.23.2 (SEQ ID No: 137 of WO2005033321), AAVhu.24 (SEQ ID No. 136 of WO2005033321), AAVhu.25 (SEQ ID No. 146 of WO2005033321), AAVhu.27 (SEQ ID No. 140 of WO2005033321), AAVhu.29 (SEQ ID No. 132 of WO2005033321), AAVhu.3 (SEQ ID No: 145 of WOZ2005033321), AAVhu.31 (SEQ I|D No. 121 of WO2005033321 ), AAVhu.32 (SEQ |D No.: 122 of WO2 document 005033321), AAVhu.34 (SEQ ID No. 125 of WOZ2005033321), AAVhu.35 (SEQ I|D No. 164 of WO2005033321), AAVhu.37 (SEQ ID No. 88 of WOZ2005033321), AAVhu.39 (SEQ ID No. 102 of WOZ2005033321), AAVhu4 (SEQ ID No. 141 of WOZ2005033321), AAVhu40 (SEQ ID No. 87 of WOZ2005033321), AAVhu41 (SEQ ID No. 91 of WO2005033321), AAVhu42 (SEQ I|D No. 85 of document WOZ2005033321), AAVhu.43 (SEQ I|D No. 160 of WOZ2005033321), AAVhu.44 (SEQ |D No. 144 of WO2005033321), AAVhu45 (SEQ |D No: 127 of WOZ2005033321), AAVhu.46 (SEQ ID No. 159 of WOZ2005033321), AAVhu47 (SEQ |D No: 128 of WOZ2005033321), AAVhu48 (SEQ |D No. 157 of WO2005033321), AAVhu49 (SEQ ID No: 189 of WOZ2005033321), AAVhu.51 (SEQ ID No. 190 of WOZ2005033321), AAVhu.52 (SEQ I|D No. 191 of WOZ2005033321), AAVhu.53 (SEQ ID No. 186 of WO2005033321), AAVhu.54 (SEQ ID No. 188 of WOZ20050 33321), AAVhu.55 (SEQ ID No. 187 of WOZ2005033321), AAVhu.56 (SEQ I|D No. 192 of WO2005033321), AAVhu.57 (SEQ ID No: 193 of WOZ2005033321), AAVhu.58 (SEQ ID No. 194 of WOZ2005033321), AAVhu.6 (SEQ ID No. 84 of WO2005033321), AAVhu.60 (SEQ ID No. 184 of WOZ2005033321), AAVhu.61 (SEQ ID No: 185 of document
[0043] [0043] In some embodiments, the AAV serotype may be, or have, a sequence as described in International Publication No. WOZ2015168666, the entirety of which is incorporated herein by reference, such as, but not limited to, AAVrh8Δ,R ( SEQ ID NO: 9 from WOZ2015168666), AAVrh8R AS8GR mutant (SEQ ID NO: from WO2015168666), AAVrh8R R533A mutant (SEQ ID NO: 11 from WO2015168666), or variants thereof.
[0044] [0044] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent No. US9233131, the entirety of which is incorporated herein by reference, such as, but not limited to, AAVhE1.1 (SEQ ID NO:44 from US9233131), AAVhEr1.5 (SEQ ID NO:45 from US9233131), AAVRER1.14 (SEQ ID NO:46 from US9233131), AAVhEr1.8 (SEQ ID NO:47 from US9233131) , AAVhEr1.16 (SEQ ID NO:48 from US9233131), AAVhEr1.18 (SEQ ID
[0045] [0045] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent Publication No. US20150376607, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV -PAEC (SEQ ID NO:1 from US20150376607), AAV-LKO1 (SEQ ID NO:2 from US20150376607), AAV-LKO2 (SEQ ID NO:3 from US20150376607), AAV-LKO3 (SEQ I|D NO.4 from US20150376607), AAV-LKO4 (SEQ ID No.5 from US20150376607), AAV-LKO5S (SEQ ID No.6 from US20150376607), AAV-LKO6 (SEQ I|D No.7 from US20150376607), AAV-LKO7 (SEQ ID No.8 from US20150376607), AAV-LKO8 (SEQ ID No.9 from US20150376607), AAV-LKO9 (SEQ ID No.10 from US20150376607), AAV-LKI0 (SEQ I|D No.11 from US20150376607), AAV-LKI1 (SEQ ID No.12 from US20150376607), AAV-LK12 (SEQ ID No.13 from US20150376607), AAV-LKI13 (SEQ I|D No.14 from US20150376607), AAV-LKI4 (SEQ ID No.15 from US document 20150376607), AAV-LKI15 (SEQ ID No.%16 of the document
[0046] [0046] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent No. US9163261, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV-2 -pre-miRNA-101 (SEQ ID NO: 1 US9163261), or variants thereof.
[0047] [0047] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent Publication No. US20150376240, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV -8h (SEQ ID No: 6 from US20150376240), AAV-8b (SEQ ID No: 5 from US20150376240), AAV-h (SEQ ID No: 2 from US20150376240), AAV-b (SEQ ID No. document US20150376240), or variants thereof.
[0048] [0048] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent Publication No. US20160017295, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV SM 10-2 (SEQ ID NO: 22 from document US20160017295), AAV Shuffle 100-1
[0049] [0049] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent Publication No. US20150238550, the entirety of which is incorporated herein by reference, such as, but not limited to, BNP61 AAV (SEQ ID NO: 1 from US20150238550), BNP62 AAV (SEQ ID NO: 3 from US20150238550), BNP63 AAV (SEQ ID NO: 4 from US20150238550), or variants thereof.
[0050] [0050] In some embodiments, the AAV serotype may be or may have a sequence as described in US Patent Publication No. US20150315612, the entirety of which is incorporated herein by reference, such as, but not limited to, AAVrh. 50 (SEQ ID No: 108 of US20150315612), AAVrh.43 (SEQ ID No: 163 of US20150315612), AAVrh.62 (SEQ ID No: 114 of US20150315612), AAVrh.48 (SEQ ID No: 115 of US20150315612), AAVhu.19 (SEQ ID No: 133 of US20150315612), AAVhu.11 (SEQ ID No: 153 of US20150315612), AAVhu.53 (SEQ ID No: 186 of US20150315612), AAV4-8/ rh.64 (SEQ ID NO: 15 from US20150315612), AAVLG-9/hu.39 (SEQ ID NO: 24 from US20150315612), AAV54.5/hu.23 (SEQ ID NO: 60 from US20150315612), AAV54.2/hu.22 (SEQ ID NO: 67 from US20150315612), AAV54.7/hu.24 (SEQ ID NO: 66 from US20150315612), AAV54.1/hu.21 (SEQ ID NO: 65 from document US20150315612), AAV54.4R/hu.27 (SEQ ID NO: 64 of US20150315612), AAV46.2/hu.28 (SEQ ID NO: 68 of US20150315612), AAV46.6/hu.29 (SEQ ID NO: 69 of US20150315612), AAV128.1/hu.43 ( SEQ ID NO: 80 of US20150315612), or variants thereof.
[0051] [0051] In some embodiments, the AAV serotype may be, or have, a sequence as described in International Publication No. WOZ2015121501, the entirety of which is incorporated herein by reference, such as, but not limited to, true-type AAV ( ttAAV) (SEQ ID NO: 2 of WOZ2015121501), "UPenn AAV10" (SEQ ID NO: 8 of WOZ2015121501), "Japanese AAV10" (SEQ ID NO: 9 of WO2015121501), or variants thereof.
[0052] [0052] In accordance with the present invention, the selection or use of the AAV capsular serotype can be from a variety of species. In one embodiment, the AAV may be an avian AAV (AAAV). The AMAV serotype may be, or have, a sequence as described in U.S. Patent No. US 9,238,800, the entirety of which is incorporated herein by reference, such as, but not limited to, AAAV (SEQ ID NO: 1, 2 , 4, 6, 8, 10, 12, and 14 of US 9,238,800), or variants thereof.
[0053] [0053] In one embodiment, the AAV may be a bovine AAV (BAAV). The BAAV serotype may be, or have, a sequence as described in U.S. Patent No. US 9,193,769, the entirety of which is incorporated herein by reference, such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of US 9193769), or variants thereof. The BAAV serotype may be or have a sequence as described in US Patent No. US7427396, the entirety of which is incorporated herein by reference, such as, but not limited to, BAAV (SEQ ID NO: 5 and 6 of US7427396 ), or variants thereof.
[0054] [0054] In one embodiment, the AAV may be a goat AAV. The goat AAV serotype may be, or have, a sequence as described in U.S. Patent No. US7427396, the entirety of which is incorporated herein by reference, such as, but not limited to, goat AAV (SEQ ID NO: 3 of document US7427396), or variants thereof.
[0055] [0055] In other embodiments the AAV can be constructed as a hybrid AAV from two or more parental serotypes. In one embodiment, the AAV may be AAV2G9 which comprises sequences from AAV2 and AAV9. The AAV serotype AAV2G9 may be, or have, a sequence as described in US Patent Publication No. US20160017005, the entirety of which is incorporated herein by reference.
[0056] [0056] In one embodiment, the AAV may be a serotype generated by the capsid library of and AAV9 with mutations at amino acids 390-627 (numbering VP1) as described by Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011 ), the entirety of which is incorporated herein by reference. The serotype and corresponding nucleotide and amino acid substitutions may be, but are not limited to, AAV9.1 (G1594C; D532H) , AAV6.2 (T1418A and TI436X; V473D and 1479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A5S87V), AAV9.6 (T1231A; F4111)), AAV9.9 (G1203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13
[0057] [0057] In some embodiments, the AAV serotype may be, or have, a sequence as described in International Publication No. WO2016049230, the entirety of which is incorporated herein by reference, such as, but not limited to, AAVF1/HSC1 (SEQ ID Nos: 2 and 20 of WO2016049230), AAVF2/HSC2 (SEQ ID NO: 3 and 21 of WO2016049230), AAVF3/HSC3 (SEQ ID NO: 5 and 22 of WO2016049230), AAVF4/HSC4 (SEQ ID NO:: 6 and 23 of WO2016049230), AAVF5/HSC5 (SEQ ID NO: 11 and 25 of WOZ2016049230), AAVF6/HSC6 (SEQ ID NO: 7 and 24 of WOZ2016049230), AAVF7/HSC7 (SEQ ID NO: 8 and 27 of WO2016049230 ), AAVF8/HSC8 (SEQ ID NOS: 9 and 28 of WOZ2016049230), AAVF9/HSC9 (SEQ ID NOS: 10 and 29 of WO2016049230), AAVF11/HSC11 (SEQ ID NOS: 4 and 26 of WO2016049230), AAVF12/HSC12 (SEQ ID Nos: 12 and 30 of WO2016049230), AAVF13/HSC13 (SEQ ID Nos: 14 and 31 of WO2016049230), AAVF14/HSC14 (SEQ ID Nos: 15 and 32 of WO2016049230 ), AAVF15/HSC15 (SEQ ID NOS: 16 and 33 of WO2016049230), AAVF16/HSC16 (SEQ ID NOS: 17 and 34 of WO2016049230), AAVF17/HSC17 (SEQ ID NOS: 13 and 35 of WO2016049230), or variants or derivatives thereof.
[0058] [0058] In some embodiments, the AAV serotype may be, or have, a sequence as described in U.S. Patent No. US 8,734,809, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV CBr -E1 (SEQ ID NO: 13 and 87 of US8734809), AAV CBr-E2 (SEQ ID NO: 14 and 88 of US8734809), AAV CBr-E3 (SEQ ID NO: 15 and 89 of US8734809), AAV CBr-E4 (SEQ ID NO: 16 and 90 of US8734809), AAV CBr-E5 (SEQ ID NO: 17 and 91 of US8734809), AAV CBr-e5 (SEQ ID NO: 18 and 92 of US8734809), AAV CBr-E6 (SEQ ID NO: 19 and 93 from US8734809), AAV CBr-E7 (SEQ ID NO: 20 and 94 from US8734809), AAV CBr-E8 (SEQ ID NO: 21 and 95 from US8734809) , AAV CLv-D1 (SEQ ID NO: 22 and 96 of US8734809), AAV CLv-D2 (SEQ |D NO: 23 and 97 of US8734809), AAV CLv-D3 (SEQ ID NO: 24 and 98 of document US8734809), AAV CLv-D4 (SEQ ID NOS: 25 and 99 of document US8734809), AAV CLv-D5 (SEQ ID NOS: 26 and 100 of document No. US8734809), AAV CLv-D6 (SEQ ID NO: 27 and 101 of US8734809), AAV CLv-D7 (SEQ ID NO: 28 and 102 of US8734809), AAV CLv-D8 (SEQ ID NO: 29 and 103 from US8734809), AAV CLv-E1 (SEQ ID NO: 13 and 87 from US8734809), AAV CLv-R1 (SEQ ID NO: 30 and 104 from US8734809), AAV CLv-R2 (SEQ ID NO: 31 and 105 of US8734809), AAV CLv-R3 (SEQ ID NO: 32 and 106 of US8734809), AAV CLv-R4 (SEQ ID NO: 33 and 107 of US8734809), AAV CLv-R5 (SEQ ID NO: 34 and 108 of US8734809), AAV CLv-R6 (SEQ ID NO: 35 and 109 of US8734809), AAV CLv-R7 (SEQ ID NO: 36 and 110 of US8734809), AAV CLv-R8 (SEQ ID NO. and X from US8734809), AAV CLv-R9 (SEQ |D No. X and X from US8734809), AAV CLg-F1 (SEQ ID NO: 39 and 113 from US8734809), AAV CLg-F2 (SEQ ID NO:: 40 and 114 of US8734809), AAV CLg-F3 (SEQ ID NO: 41 and 115 of US8734809), AAV CLg-F4 (SEQ ID NO: 42 and 116 of US8734809), AAV CLg-F5 (SEQ ID NO. : 4 3 and 117 of US8734809), AAV CLg-F6 (SEQ ID NO: 43 and 117 of US8734809), AAV CLg-F7 (SEQ ID NO: 44 and 118 of US8734809), AAV CLg-F8 (SEQ ID NO. : 43 and 117 of US8734809), AAV CSp-1 (SEQ ID NO: 45 and 119 of US8734809), AAV CSp-10 (SEQ ID NO: 46 and 120 of US8734809), AAV CSp-11 (SEQ ID 47 and 121 of US8734809), AAV CSp-2 (SEQ ID NOS: 48 and 122 of US8734809), AAV CSp-3 (SEQ ID NOS: 49 and 123 of US8734809), AAV CSp-4 (SEQ ID NOS: 50 and 124 of US8734809), AAV CSp-6 (SEQ ID NOS: 51 and 125 of document US8734809), AAV CSp-7 (SEQ ID NOS: 52 and 126 of document US8734809), AAV CSp-8 ( SEQ ID NO: 53 and 127 of US8734809), AAV CSp-9 (SEQ ID NO: 54 and 128 of US8734809), AAV CHt-2 (SEQ ID NO: 55 and 129 of US8734809), AAV CHt-3 (SEQ ID NO: 56 and 130 of US8734809), AAV CKd-1 (SEQ ID NO: 57 and 131 of US8734809), AAV CKd-10 (SEQ ID NO: 58 and 132 of US8734809), AAV CKd- two (SEQ ID NO: 59 and 133 of US8734809), AAV CKd-3 (SEQ ID NO: 60 and 134 of US8734809), AAV CKd-4 (SEQ ID NO: 61 and 135 of US8734809), AAV CKd- 6 (SEQ ID NO: 62 and 136 of US8734809), AAV CKd-7 (SEQ ID NO: 63 and 137 of US8734809), AAV CKd-8 (SEQ ID NO: 64 and 138 of US8734809), AAV CLv -1 (SEQ ID NO: 35 and 139 of US8734809), AAV CLv-12 (SEQ ID NO: 66 and 140 of US8734809), AAV CLv-13 (SEQ ID NO: 67 and 141 of US8734809), AAV CLv-2 (SEQ ID NOS: 68 and 142 of US8734809), AAV CLv-3 (SEQ ID NOS: 69 and 143 of US8734809), AAV CLv-4 (SEQ ID NOS: 70 and 144 of US8734809), AAV CLv-6 (SEQ ID NOS: 71 and 145 from US8734809), AAV CLv-8 (SEQ ID NOS: 72 and 146 from US8734809), AAV CKd-B1 (SEQ ID NOS: 73 and 147 from US8734809) , AAV CKd-B2 (SEQ ID NO: 74 and 148 of US8734809), AAV CKd-B3 (SEQ ID NO: 75 and 149 of US8734809), AAV CKd-B4 (SEQ ID NO: 76 and 150 of US87348 09), AAV CKd-B5 (SEQ ID NO: 77 and 151 of US8734809), AAV CKd-B6 (SEQ ID NO: 78 and 152 of US8734809), AAV CKd-B7 (SEQ ID NO: 79 and 153 of US8734809), AAV CKd-B8 (SEQ ID NO: 80 and 154 of US8734809), AAV CKd-H1 (SEQ ID NO: 81 and 155 of US8734809), AAV CKd-H2 (SEQ ID NO: 82 and 156 from US8734809), AAV CKd-H3 (SEQ ID NO: 83 and 157 from US8734809), AAV CKd-H4 (SEQ ID NO: 84 and 158 from US8734809), AAV CKd-H5 (SEQ ID NO: 85 and 159 of US8734809), AAV CKd-H6 (SEQ ID NO: 77 and 151 of US8734809), AAV CHt-1 (SEQ ID NO: 86 and 160 of US8734809), AAV CLv1-1 (SEQ ID NO: 171 from US8734809), AAV CLvi-2 (SEQ ID No: 172 from US8734809), AAV CLv1i-3 (SEQ ID No. 173 from US8734809), AAV CLvIi4 (SEQ ID No. 174 from US8734809), AAV Clvi-r (SEQ ID NO: 175 of US8734809), AAV Clvi-8 (SEQ ID NO: 176 of US8734809), AAV Clvi-9 (SEQ ID NO: 177 of US8734809), AAV Clv 1-10 (SEQ ID NO: 178 of US8734809), AAV.VR-355 (SEQ ID NO: 181 of US8734809), AAV.hu48R3 (SEQ ID NO: 183 of US8734809), or variants or derivatives thereof.
[0059] [0059] In some embodiments, the AAV serotype may be, or have, a sequence as described in International Publication No. WO2016065001, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV CHt-P2 (SEQ ID No: 1 and 51 of WO2016065001), AAV CHt-P5 (SEQ ID No: 2 and 52 of WO2016065001), AAV CHt-P9 (SEQ ID No: 3 and 53 of WO2016065001), AAV CBr-7.1 ( SEQ ID NO: 4 and 54 of WO2016065001), AAV CBr-7.2 (SEQ ID NO: 5 and 55 of WOZ2016065001), AAV CBr-7.3 (SEQ ID NO: 6 and 56 of WO2016065001), AAV CBr-7.4 (SEQ ID NO: 7 and 57 of WO2016065001), AAV CBr-7.5 (SEQ ID NO: 8 and 58 of WOZ2016065001), AAV CBr-7.7 (SEQ ID NO: 9 and 59 of WO2016065001), AAV CBr- 7.8 (SEQ ID NO: 10 and 60 of WO2016065001), AAV CBr-7.10 (SEQ ID NO: 11 and 61 of WO2016065001), AAV CKd-N3 (SEQ ID NO: 12 and 62 of WO2016065001), AAV CKd -N4 (SEQ ID NO: 13 and 63 from WO2016065001) , AAV CKd-N9 (SEQ ID NOS: 14 and 64 of WO2016065001), AAV CLv-L4 (SEQ ID NOS: 15 and 65 of WO2016065001), AAV CLv-L5 (SEQ ID NOS: 16 and 66 of WO2016065001 ), AAV CLv-L6 (SEQ ID NO: 17 and 67 of WO2016065001), AAV CLv-K1 (SEQ ID NO: 18 and 68 of WO2016065001), AAV CLv-K3 (SEQ ID NO: 19 and 69 of document WO2016065001), AAV CLv-K6 (SEQ ID NOS: 20 and 70 of WO2016065001), AAV CLv-M1 (SEQ ID NOS: 21 and 71 of WO2016065001), AAV CLv-M11 (SEQ ID NOS: 22 and 72 of WO2016065001), AAV CLv-M2 (SEQ ID NOS: 23 and 73 from WO2016065001), AAV CLv-M5 (SEQ ID NOS: 24 and 74 from WO2016065001), AAV CLv-M6 (SEQ ID NOS: 25 and 75 from WO2016065001), AAV CLv-M7 (SEQ ID NO: 26 and 76 from WO2016065001), AAV CLv-M8 (SEQ ID NO: 27 and 77 from WO2016065001), AAV CLv-M9 (SEQ ID NO: 28 and 78 of WO2016065001), AAV CHt-P1 (SEQ ID NO: 29 and 79 of WO2016065001), AAV CHt-P6 (SEQ ID NO: 30 and 80 of WO2016065 001), AAV CHt-P8 (SEQ ID NO: 31 and 81 of WO2016065001), AAV CHt-6.1 (SEQ ID NO: 32 and 82 of WO2016065001), AAV CHt-6.10 (SEQ ID NO: 33 and 83 of WOZ2016065001), AAV CHt-6.5 (SEQ ID NO: 34 and 84 of WO2016065001), AAV CHt-6.6 (SEQ ID NO: 35 and 85 of WO2016065001), AAV CHt-6.7 (SEQ ID NO: 36 and 86 from WO2016065001), AAV CHt-6.8 (SEQ ID NO: 37 and 87 from WO2016065001), AAV CSp-8.10 (SEQ ID NO:38 and 88 from WO2016065001), AAV CSp-8.2 (SEQ ID NO: 39 and 89 of WO2016065001), AAV CSp-8.4 (SEQ ID NO: 40 and 90 of WO2016065001), AAV CSp-8.5 (SEQ ID NO: 41 and 91 of WOZ2016065001), AAV CSp-8.6 (SEQ ID NO: 42 and 92 of WO2016065001), AAV CSp-8.7 (SEQ ID NO: 43 and 93 of WOZ2016065001), AAV CSp-8.8 (SEQ ID NO: 44 and 94 of WOZ2016065001), AAV CSp-8.9 (SEQ ID NO:: 45 and 95 of WO2016065001 ), AAV CBr-B7.3 (SEQ ID NO: 46 and 96 of WO2016065001), AAV CBr-B7.4 (SEQ ID NO: 47 and 97 of WOZ2016065001), AAV3B (SEQ ID Nos: 48 and 98 of WO2016065001), AAV4 (SEQ ID Nos. 49 and 99 of WOZ2016065001), AAV5 (SEQ ID Nos: 50 and 100 of WO2016065001), or variants or derivatives thereof.
[0060] [0060] In some embodiments, the AAV serotype may be, or have a modification as described in US Publication No. US 20160361439, the entirety of which is incorporated herein by reference, such as, but not limited to, Y252F, Y272F, Y444F, Y500F, Y700F, Y704F, Y730F, Y275F, Y281F, Y508F, Y576F, Y612G, Y673F, and Y720F of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAVB, AAV9, AAV1IO, AAV1AVI1I wild-type, and hybrids thereof.
[0061] [0061] In some embodiments, the AAV serotype may be, or have, a mutation as described in U.S. Patent No. US 9,546,112, the entirety of which is incorporated herein by reference, such as, but not limited to, at least two, but not all, of the F129L, D418E, K531E, L584F, V598A and H642N mutations in the sequence of AAV6 (SEQ ID NO:4 of US 9546112), AAV1 (SEQ ID NO:6 of US 9546112), AAV2, AAV3 , AAVA, AAVS5, AAV7, AAV9, AAV1IO or AAV11 or derivatives thereof. In yet another embodiment, the AAV serotype can be, or have, an AAV6 sequence comprising the K531E mutation (SEQ ID NO:5 of US 9546112).
[0062] [0062] In some embodiments, the AAV serotype may be, or have, a mutation in the AAV1 sequence, as described in the Patent
[0063] [0063] In some embodiments, the serotype may be AAV2 or a variant thereof, as described in International Publication No. WOZ2016130589, the entirety of which is incorporated herein by reference. The AAV2 amino acid sequence can comprise mutations N587A, ES48A, or N708A. In one embodiment, the AAV amino acid amino acid sequence may comprise a V708K mutation.
[0064] [0064] In one embodiment, the AAV may be a serotype selected from any of those found in Table 1.
[0065] [0065] In one embodiment, the AAV may comprise a sequence, fragment or variant thereof, from among the sequences in Table 1.
[0066] [0066] In one embodiment, the AAV may be encoded by a sequence, fragment or variant described in Table 1.
[0067] [0067] Non-limiting examples of nucleotide sequences that can encode amino acid inserts include the following, GATGGGACTTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID No: 54 from WOZ2017100671; in this application SEQ ID No. 963), GATGGGACGTTGGCGGTGCCTTTTAAGGCACAG (SEQ ID No. in this application SEQ ID NO: 964), CAGGCGGTTAGGACGTCTTTG (SEQ ID NO: 56 of WO-2017100671; in this application SEQ ID NO: 965),
[0068] [0068] AGTTTGGGCACT (SEQ ID NO: 70 of WO-2017100671 wherein N may be A, C, T, or G; in this application SEQ ID NO: 974),
[0069] [0069] In one embodiment, the AAV serotype may be, or may have a sequence as described in U.S. Patent No. US 9,624,274, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV1 ( SEQ ID NO: 181 from US9624274), AAV6 (SEQ ID NO: 182 from US9624274), AAV2 (SEQ ID NO: 183 from US9624274), AAV3b (SEQ ID NO: 184 from US9624274), AAV7 (SEQ ID No: 185 of US9624274), AAV8 (SEQ ID No: 186 of US9624274), AAVIO (SEQ ID No: 187 of US9624274), AAVA4 (SEQ ID No: 188 of US9624274), AAV11 (SEQ ID No:: 189 of US9624274), baAV (SEQ ID NO: 190 of US9624274), AAV5 (SEQ ID NO: 191 of US9624274), GPV (SEQ ID NO: 192 of US9624274; in this application SEQ ID NO: 992), B19 (SEQ ID NO: 193 of US9624274; in this application SEQ ID NO: 993), MVM (SEQ ID NO: 194 of US9624274; in this application SEQ ID NO: 994), FPV (SEQ ID NO: 195 of US962 4274; in this application SEQ ID NO: 995),
[0070] [0070] In one embodiment, the AAV serotype may be, or may have a sequence as described in U.S. Patent No. US 9,475,845, the entirety of which is incorporated herein by reference, such as, but not limited to, capsular proteins of AAV comprising modifying one or more amino acids at amino acid positions 585 to 590 of the native AAV 2 capsular protein. In addition, modification may result in, but not limited to, the amino acid sequence RGNRQA (SEQ ID NO: 3 of US9475845; in this application SEQ ID NO: 1418), SSSTDP (SEQ ID NO: 4 of US9475845; in this application SEQ ID NO: 1419), SSNTAP (SEQ ID NO: 5 from US9475845; in this application SEQ ID NO: 1420), SNSNLP (SEQ ID NO: 6 from US9475845; in this application SEQ ID NO: 1421), SSTTAP (SEQ ID No: 7 of US9475845; in this application SEQ ID No: 1422), AANTAA (SEQ ID No: 8 of US9475845; in this application SEQ ID No: 1423), QQNTAP (SEQ ID No: 9 of US9475845; in this application SEQ ID NO: 1424), SAQAQA (SEQ ID NO: 10 from US9475845; in this application SEQ ID NO: 1425), QOANTGP (SEQ ID NO: 11 from US9475845; in this application SEQ ID NO: 1426), NATTAP (SEQ ID No.: 12 of US9475845; in this application SEQ ID No: 1427), SSTAGP (SEQ ID No: 13 and 20 of US9475845; in this application SEQ ID No: 1428), QQNTAA (SEQ ID No: 14 of US9475845; in this SEQ I order D NO: 1429), PSTAGP (SEQ ID NO: 15 from US9475845; in this application SEQ ID NO: 1430), NONTAP (SEQ ID NO: 16 of US9475845; in this application SEQ ID NO: 1431), QOAANAP (SEQ ID NO: 17 of US9475845; in this application SEQ ID NO: 1432), SIVGLP (SEQ ID NO: 18 from US9475845; in this application SEQ ID NO: 1433), AASTAA (SEQ ID NO: 19, and 27 from US9475845; in this application SEQ ID NO: 1434), SONTTA (SEQ ID NO: 21 from US9475845; in this application SEQ ID NO: 1435), QQDTAP (SEQ ID NO: 22 of US9475845; in this application SEQ ID NO: 1436), QTNTGP (SEQ ID NO: 23 of US9475845; in this application SEQ ID NO: 1437 ), QTNGAP (SEQ ID NO: 24 from US9475845; in this application SEQ ID NO: 1438), QQNAAP (SEQ ID NO: 25 from US9475845; in this application SEQ ID NO: 1439), or AANTQA (SEQ ID NO: 26 from US9475845; in this application SEQ ID NO: 1440). In one embodiment, the amino acid modification is a substitution at amino acid positions 262 to 265 in the native AAV2 capsular protein or at the corresponding position in the capsular protein of another AAV with a vectoring sequence.
[0071] [0071] In one embodiment, the AAV serotype may be, or may have a sequence as described in US Publication No. US 20160369298, the entirety of which is incorporated herein by reference, such as, but not limited to, capsular protein site-specific mutated AAV2 (SEQ |D NO: 97 of US 20160369298; in this application SEQ ID NO: 1560) or variants thereof, where the specific site is at least one site selected from sites R447, G453, S578, N587, N587+1, S662 of VP1 or fragments thereof.
[0072] [0072] In addition, any of the mutated sequences described in US 20160369298 may be or may have, but not limited to, any of the following SDSGASN sequences (SEQ ID NO: 1 and SEQ ID NO: 231 of US20160369298; in this application SEQ ID NO: 1561), SPSGASN (SEQ ID NO: 2 from US20160369298; in this application SEQ ID NO: 1562), SHSGASN (SEQ ID NO: 3 from US20160369298; in this application SEQ ID NO: 1563), SRSGASN (SEQ ID No: 4 of US20160369298; in this application SEQ ID No: 1564), SKSGASN (SEQ ID No: 5 of US20160369298; in this application SEQ ID No: 1565), SNSGASN (SEQ ID No: 6 of US20160369298; in this application SEQ ID NO: 1566), SGSGASN (SEQ ID NO: 7 from document
[0073] [0073] In some embodiments, the AAV serotype may comprise an eye cell vectoring peptide as described in International Patent Publication WO2016134375, the entirety of which is incorporated herein by reference, such as, but not limited to, SEQ ID NO: 9 , and SEQ ID NO:10 of WO-2016134375. Furthermore, any of the vector peptides or eye cell amino acids described in WO2016134375 can be inserted into any serotype of the parental AAV, such as, but not limited to, AAV2 (SEQ ID NO:8 of WOZ2016134375; in this application SEQ ID NO. : 1729), or AAV9 (SEQ ID NO: 11 of WO2016134375; in this application SEQ ID NO: 1730) In some embodiments, modifications, such as insertions, are made to AAV2 proteins at P34-A35, T138-A139, A139 -P140, G453-T454, N587-R588, and/or R588-Q0589. In certain embodiments, inserts are made in D384, G385, 1560, T561, N562, E563, ES564, ES65, N704, and/or Y705 of AAV9. The eye cell vector peptide can be, but not limited to, any of the following amino acid sequences, GSTPPPM (SEQ |D NO: 1 of WO2016134375; in this application SEQ ID NO: 1731), or GETRAPL (SEQ ID NO: 4 from WO2016134375; in this application SEQ ID NO: 1732).
[0074] [0074] In some embodiments, the AAV serotype may be modified in the manner described in the US Publication US
[0075] [0075] In some embodiments, the AAV serotype may be modified in the manner described in International Publication WO2017083722 the entirety of which is incorporated herein by reference. AAV serotypes may include AAV1 (YTO5+731F+T492V), —AAV2 (Y444+500+730F+T491V) AAV3 (Y7T05+731F), AAV5, AAV 5(Y436+693+719F), AAVG6 (VP3 variant YTOSF/ YT731F/T492V), AAV8 (Y7T33F), AAV9, AAVO (VP3 variant Y7T31F), and AAV1O (Y733F).
[0076] [0076] In some embodiments, the AAV serotype may comprise, as described in International Patent Publication WO2017015102, the entirety of which is incorporated herein by reference, a genetically engineered epitope comprising the amino acids SPAKFA (SEQ ID NO: 24 of WOZ2017015102 ; in this application SEQ ID NO: 1733) or NKDKLN (SEQ ID NO: 2 of WO2017015102 ; in this application SEQ ID NO: 1734). The epitope can be inserted in the region of amino acids 665 to 670 based on the numbering of the capsid VP1 of AAV8 (SEQ ID NO:3 of WO2017015102) and/or of residues 664 to 668 of AAV3B (SEQ ID NO:3).
[0077] [0077] In one embodiment, the AAV serotype may be, or may have a sequence as described in International Patent Publication WO2017058892, the entirety of which is incorporated herein by reference, such as, but not limited to, AAV variants with capsular proteins which may comprise a substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 262-268, 370-379, 451-459, 472-473, 493-500 , 528-534, 547-552, 588-597, 709-710, 716-722 of AAV1I, in any combination, or at the equivalent amino acid residues in AAV 2, AAV3, AAVA4, AAV5, AAV6, AAV7, AAV8, AAV9 , AAV1O, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV or avian AAV.
[0078] [0078] The AAV particles of the present invention comprise a viral genome with at least one ITR region and one payload region. In one embodiment, the viral genome has two ITRs. These two ITRs flank the payload region at the 5' and 3' ends. ITRs function as origins of replication comprising recognition sites for replication. ITRs comprise sequence regions that may be complementary and symmetrically arranged. The ITRs incorporated into the viral genomes of the invention can be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.
[0079] [0079] ITRs can be derived from the same serotype as the capsid, selected from any of the serotypes listed in Table 1, or a derivative thereof. The ITR may be of a different serotype than the capsid. In one embodiment, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a viral genome comprising two ITRs. In one embodiment, the ITRs are of the same serotype as each other. In another embodiment, the ITRs are of different serotypes. Non-limiting examples include zero, one or both ITRs having the same serotype as the capsid. In one embodiment, both ITRs of the viral genome of the AAV particle are AAV2 ITRs.
[0080] [0080] Regardless, each ITR can be about 100 to about 150 nucleotides in length. An ITR can be about 100-105 nucleotides in length, 106-110 nucleotides in length, 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131- 135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length, or 146-150 nucleotides in length. In one embodiment, the ITRs are 140-142 nucleotides in length. Non-limiting examples of ITR length are 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto. Component of the Viral Genome: Promoters
[0081] [0081] In one embodiment, the payload region of the viral genome comprises at least one element to enhance transgene target specificity and expression (see, for example, Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the entirety of which is incorporated by reference). Non-limiting examples of elements to improve target transgene specificity and expression include promoters, miRNAs, endogenous regulatory elements — post-transcriptional (PREs), polyadenylation signal sequences (PolyA) and upstream enhancers (USEs), enhancers of CMV and introns.
[0082] [0082] One skilled in the art should recognize that expression of the polypeptides of the invention in a target cell may require a specific promoter, including, but not limited to, a promoter that is tissue-specific, or cell cycle-specific inducible and tissue-specific. species, (Parr et al., Nat. Med.3:1145-9 (1997); the entirety of which is incorporated herein by reference).
[0083] [0083] In one embodiment, the promoter is considered efficient when it induces expression of the polypeptides encoded in the payload region of the viral genome of the AAV particle. As a non-limiting example, this polypeptide is AADC.
[0084] [0084] In one embodiment, the promoter is a promoter considered efficient when it induces in-cell expression in the cell being targeted.
[0085] [0085] In one embodiment, the promoter is a promoter that has a tropism for the cell being targeted.
[0086] [0086] In one embodiment, the promoter induces the expression of the payload over a period of time in the target tissues. Expression induced by a promoter can be a period of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week , 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years , 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more than 10 years. The expression can be for 1-5 hours, 1-12 hours, 1-2 days, 1-5 days, 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-2 months, 1-4 months, 1-6 months, 2-6 months, 3-6 months, 3-9 months, 4-8 months, 6-12 months, 1-2 years, 1-5 years, 2-5 years, 3-6 years, 3-8 years, 4-8 years or 5-10 years. As a non-limiting example, the promoter is a weak promoter for uninterrupted expression of a payload in nerve tissues.
[0087] [0087] In one embodiment, the promoter induces expression of the polypeptides of the invention for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, | year, 2 years, 3 years 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 21 years, 22 years, 23 years, 24 years, 25 years, 26 years, 26 years, 27 years, 28 years, 29 years, 30 years, 31 years, 32 years, 33 years, 34 years, 35 years, 36 years, 37 years, 38 years, 39 years, 40 years, 41 years, 42 years, 43 years, 44 years, 45 years, 46 years, 47 years, 48 years, 49 years, 50 years, 55 years , 60 years old, 65 years old, or over 65 years old.
[0088] [0088] Promoters can be naturally or unnaturally occurring. Non-limiting examples of promoters include viral promoters, plant promoters and mammalian promoters. In some embodiments, the promoters may be human promoters. In some embodiments, the promoter may be truncated.
[0089] [0089] Promoters that induce or promote expression in most tissues include, but are not limited to, human elongation factor 1a (EF1ag), booster and/or cytomegalovirus (CMV) immediate early promoter, chicken B-actin (CBA) and its derivative CAG, B-glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements can be used to restrict expression to certain cell types, such as, but not limited to, muscle-specific promoters, B cell promoters, monocyte promoters, leukocyte promoters, macrophage promoters, pancreatic acinar cells, endothelial cell promoters, lung tissue promoters, astrocyte promoters, or nervous system promoters that can be used to restrict expression to neurons, astrocytes, or oligodendrocytes.
[0090] [0090] Non-limiting examples of muscle-specific promoters include mammalian muscle creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, troponin promoter | (TNNI2) from mammal, and skeletal alpha-actin (ASKA) from mammal (see, for example, US Patent Publication US 20110212529 , the entirety of which is incorporated herein by reference).
[0091] [0091] Non-limiting examples of tissue-specific expression elements for neurons include the neuron-specific enolase (NSE) promoters, platelet-derived growth factor (PDGF), platelet-derived growth factor B-chain (PDGF-B) , synapsins (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2*t/calmodulin-dependent protein kinase II (CaMHKII), metabotropic glutamate receptor 2 (mMGIluR2), light (NFL) or heavy ( NFH), nfB2 B-globin minigene, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2). Non-limiting examples of tissue-specific expression elements for astrocytes include the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. a non-limiting example of a tissue-specific expression element for oligodendrocytes includes the myelin basic protein (MBP) promoter.
[0092] [0092] In one embodiment, the promoter may be less than 1 kb. The promoter can have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410 , 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530,
[0093] [0093] In one embodiment, the promoter can be a combination of two or more components from the same start or parent promoters or from different start or parent promoters such as, but not limited to, CMV and CBA. Each component can have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 381, 382, 383 384, 385, 386, 387, 388, 389, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570 , 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800 Each component can have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300- 800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800. In one embodiment, the promoter is a combination of a 382 nucleotide CMV booster sequence and a 260 nucleotide CBA promoter sequence.
[0094] [0094] In one embodiment, the viral genome comprises a ubiquitous promoter. Non-limiting examples of ubiquitous promoters include CMV, CBA (including derivatives CAG, CBh, etc.), EF-1a, PGK, UBC, GUSB (hGBp), and UCOE (HNRPA2B1-CBX3 promoter).
[0095] [0095] Yu et al. (Molecular Pain 2011, 7:63; the entirety of which is incorporated herein by reference) evaluated the expression of eGFP under the CAG, EFla, PGK and UBC promoters in rat DRG cells and primary DRG cells using lentiviral vectors and found that the
[0096] [0096] Any of the promoters taught by YU, Soderblom, Gill, Husain, Passini Xu, Drews or Raymond mentioned above can be used in the present invention.
[0097] [0097] In one embodiment, the promoter is not cell-specific.
[0098] [0098] In one embodiment, the promoter is a ubiquitin c (UBC) promoter. The UBC promoter can be 300-350 nucleotides in length. As a non-limiting example, the UBC promoter is 332 nucleotides.
[0099] [0099] In one embodiment, the promoter is a B-glucuronidase (GUSB) promoter. The GUSB promoter can be 350-400 nucleotides in length. As a non-limiting example, the GUSB promoter is 378 nucleotides long.
[00100] [00100] In one embodiment, the promoter is a neurofilament light (NFL) promoter. The NFL promoter can be 600-700 nucleotides in length. As a non-limiting example, the NFL promoter is 650 nucleotides long.
[00101] [00101] In one embodiment, the promoter is a neurofilament promoter (NFH). The NFH promoter can be 900-
[00102] [00102] In one embodiment, the promoter is an SCN8A promoter. The SCN8A promoter can be 450-500 nucleotides in length. As a non-limiting example, the SCN8A promoter is 470 nucleotides long.
[00103] [00103] In one embodiment, the promoter is a frataxin (FXN) promoter. The FXN promoter may also be called the FRDA promoter.
[00104] [00104] In one embodiment, the promoter is a phosphoglycerate kinase 1 (PGK) promoter.
[00105] [00105] In one embodiment, the promoter is a chicken B-actin (CBA) promoter.
[00106] [00106] In one embodiment, the promoter is a cytomegalovirus (CMV) promoter.
[00107] [00107] In one embodiment, the promoter is an H1 promoter.
[00108] [00108] In one embodiment, the promoter is a genetically engineered promoter.
[00109] [00109] In one embodiment, the promoter is a hepatic or skeletal muscle promoter. Non-limiting examples of hepatic promoters include human α-1-antitrypsin (hAAT) and thyroxine-binding globulin (TBG). Non-limiting examples of skeletal muscle promoters include Desmin, MCK or synthetic C5-12.
[00110] [00110] In one embodiment, the promoter is an RNA pol III promoter. As a non-limiting example, the RNA pol III promoter is U6. As a non-limiting example, the RNA pol III promoter is H1.
[00111] [00111] In one embodiment, the viral genome comprises two promoters. As a non-limiting example, the promoters are an EF1a promoter and a CMV promoter.
[00112] [00112] In one embodiment, the viral genome comprises a booster element, a promoter, and/or a SUTR intron. The booster element, in this application also called booster, can be, but not limited to, a CMV booster, the promoter can be, but not limited to, a CMV, CBA, UBC, GUSB, NSE, Synapsin, MeCP2, and GFAP and the 5S"'UTR/intron may be, but are not limited to, SV40, and CBA-MVM. As a non-limiting example, the booster, promoter, and/or intron used in combination may be: (1) CMV booster, CMV promoter, SV40 S'UTR intron; (2) CMV booster, CBA promoter, SV 40 5SUTR intron; (3) CMV booster, CBA promoter, CBA-MVM 5S'UTR intron; (4) promoter UBC; (5) GUSB promoter; (6) NSE promoter; (7) Synapsin promoter; (8) MeCP2 promoter and (9) GFAP promoter.
[00113] [00113] In one embodiment, the viral genome comprises a genetically engineered promoter.
[00114] [00114] In another embodiment, the viral genome comprises a promoter derived from a naturally expressed protein.
[00115] [00115] In one embodiment, a region is located approximately —5 kb upstream of the first exon of the payload to allow expression of the payload with the promoter. (See, for example, Puspasari et al. Long Range Regulation of Human FXN Gene Expression PLOS ONE, 2011; the entirety of which is incorporated herein by reference; a 17 bp region is located approximately 4.9 kb upstream of the first exon of the frataxin gene to allow expression with the FRDA promoter).
[00116] [00116] In one embodiment, the vector genome may comprise a promoter such as, but not limited to, CMV or U6. As a non-limiting example, the promoter for the AAV particles comprising the payload of the present invention is a CMV promoter. As another non-limiting example, the promoter for the AAV particles comprising the payload of the present invention is a U6 promoter.
[00117] [00117] In one embodiment, the vector genome may comprise a CMV promoter and a U6 promoter.
[00118] [00118] In one embodiment, the vector genome may comprise a CBA promoter. Viral Genome Component: Untranslated Regions (UTRs)
[00119] [00119] By definition, wild-type untranslated regions (UTRs) of a gene are transcribed but not translated. Generally speaking, the 5' UTR starts at the transcription initiation site and ends at the start codon and the 3' UTR starts immediately after the stop codon and continues until the termination signal for transcription.
[00120] [00120] Characteristics typically found in genes expressed in abundance from specific target organs can be genetically manipulated in UTRs to improve stability and protein production. As a non-limiting example, a 5' UTR of mRNA normally expressed in the liver (eg, albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII) can be used in the viral genome. of the AAV particles of the invention to enhance expression in liver cell lines or in the liver.
[00121] [00121] Even without wishing to be bound by theory, wild-type 5' untranslated regions (UTRs) include features that play roles in translation initiation. Kozak sequences, which are commonly known to be involved in the process by which the ribosome initiates the translation of many genes, are usually included in the 5' UTRs. The Kozak sequences have the consensus CCR(A/G)JCCAUGG, where R is a purine (adenine or guanine) three bases upstream from the start codon (ATG), which is followed by another 'G'".
[00122] [00122] In one embodiment, the S"UTR in the viral genome includes a Kozak sequence.
[00123] [00123] In one embodiment, the "UTR in the viral genome does not include a Kozak sequence.
[00124] [00124] Even without wanting to stick to theory, it is known that the 3' have stretches of Adenosines and Uridines embedded in them. These AU-rich signatures are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be separated into three classes (Chen et al, 1995, the entirety of which is incorporated herein by reference): ARES Class |, such as, but not limited to, c-Myc and MyoD, contain multiple scattered copies of an AUUUA motif in the U-rich regions. Class |1 AREs, such as, but not limited to, GM-CSF and TNF-a, have two or more UUAUUUVA nonamers (U/A)U/A) superimposed. Class II AREs, such as, but not limited to, c-Jun and Myogenin, are less well defined. These U-rich regions do not contain an AUUUA motif. Most proteins that bind to AREs are known to destabilize the messenger, while members of the ELAV family, most notably HuR, have been documented to increase MRNA stability. HuR binds to AREs of all three classes. Genetic manipulation of HuR-specific binding sites in the 3' UTR of nucleic acid molecules will lead to HuR binding and thus message stabilization in vivo.
[00125] [00125] The introduction, removal or modification of AU-rich elements (AREs) in the 3' UTR can be used to modulate the stability of polynucleotides. By genetically manipulating specific polynucleotides, for example, it is possible to introduce heavily loaded regions of viral genomes, one or more copies of an ARE to make the polynucleotides less stable and thus reduce translation and decrease production of the resulting protein. Likewise, AREs can be identified and removed or mutated to increase intracellular stability and thus increase translation and production of the resulting protein.
[00126] [00126] In one embodiment, the 3' UTR of the viral genome may include an oligo(dT) sequence for patterned addition of a poly-A tail. In one embodiment, the viral genome can include at least one seed, binding site, or complete sequence of miIRNA microRNAs (or miRNA or miR) are 19-25 nucleotide non-coding RNAs that bind to nucleic acid target sites and infra -regulate gene expression by reducing the stability of the nucleic acid molecule or inhibiting translation. A microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence displays perfect Watson-Crick complementarity to the target miRNA nucleic acid sequence.
[00127] [00127] In one embodiment, the viral genome can be genetically manipulated to include, alter or remove at least one binding site, sequence or seed region of miRNA Any UTR of any gene known in the art can be incorporated into the viral genome of the AAV particle. These UTRs, or portions thereof, may be placed in the same orientation as the gene from which they were selected, or they may have their orientation or location altered. In one embodiment, the UTR used in the viral genome of the AAV particle can be inverted, shortened, augmented, made with one or more other 5' UTRs or 3' UTRs known in the art. As used in this application, the term "altered" when referring to a UTR means that the UTR has been altered in some way with respect to a reference sequence, e.g. a 3' or 5' UTR may be altered with respect to a reference sequence. a wild-type or native UTR by change in orientation or location as taught above or may be altered by including additional nucleotides, deleting nucleotides, exchanging or transposing nucleotides.
[00128] [00128] In one embodiment, the viral genome of the AAV particle comprises at least one artificial UTR that is not a variant of a wild-type UTR.
[00129] [00129] In one embodiment, the viral genome of the AAV particle comprises UTRs that have been selected from a family of transcripts whose proteins share a common function, structure, feature, or property. Component of the Viral Genome: Polyadenylation Sequence
[00130] [00130] In one embodiment, the viral genome of the AAV particles of the present invention comprises at least one polyadenylation sequence.
[00131] [00131] The viral genome of the AAV particle may comprise a polyadenylation sequence between the 3' end of the payload coding sequence and the 5' end of the 3''TR.
[00132] [00132] In one embodiment, the polyadenylation sequence or "polyfA sequence" can range from absent to about 500 nucleotides in length.
[00133] [00133] The polyadenylation sequence may be, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 , 70, 71, 72.73, 74,75,786, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 I , 121, 122, 123, 124,
[00134] [00134] In one embodiment, the polyadenylation sequence is 50-100 nucleotides in length.
[00135] [00135] In one embodiment, the polyadenylation sequence is 50-150 nucleotides in length.
[00136] [00136] In one embodiment, the polyadenylation sequence is 50-160 nucleotides in length.
[00137] [00137] In one embodiment, the polyadenylation sequence is 50-200 nucleotides in length
[00138] [00138] In one embodiment, the polyadenylation sequence is 60-100 nucleotides in length.
[00139] [00139] In one embodiment, the polyadenylation sequence is 60-150 nucleotides in length.
[00140] [00140] In one embodiment, the polyadenylation sequence is 60-160 nucleotides in length.
[00141] [00141] In one embodiment, the polyadenylation sequence is 60-200 nucleotides in length
[00142] [00142] In one embodiment, the polyadenylation sequence is 70-100 nucleotides in length.
[00143] [00143] In one embodiment, the polyadenylation sequence is 70-150 nucleotides in length.
[00144] [00144] In one embodiment, the polyadenylation sequence is 70-160 nucleotides in length.
[00145] [00145] In one embodiment, the polyadenylation sequence is 70-200 nucleotides in length.
[00146] [00146] In one embodiment, the polyadenylation sequence is 80-100 nucleotides in length.
[00147] [00147] In one embodiment, the polyadenylation sequence is 80-150 nucleotides in length.
[00148] [00148] In one embodiment, the polyadenylation sequence is 80-160 nucleotides in length.
[00149] [00149] In one embodiment, the polyadenylation sequence is 80-200 nucleotides in length.
[00150] [00150] In one embodiment, the polyadenylation sequence is 90-100 nucleotides in length.
[00151] [00151] In one embodiment, the polyadenylation sequence is 90-150 nucleotides in length.
[00152] [00152] In one embodiment, the polyadenylation sequence is 90-160 nucleotides in length.
[00153] [00153] In one embodiment, the polyadenylation sequence is 90-200 nucleotides in length. Component of the Viral Genome: Introns
[00154] [00154] In one embodiment, the payload region comprises at least one expression enhancing element such as one or more introns or portions thereof. Non-limiting examples of introns include MVM (67-97 bps), F. IX truncated intron 1 (300 bps), SD B-globin/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/ immunoglobulin splice acceptor (500 bps), late splice donor/SV40 splice acceptor (198/16S) (180 bps) and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).
[00155] [00155] In one embodiment, the intron or intron portion may be 100-500 nucleotides in length. The intron can have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200 , 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 , 460, 470, 480, 490 or 500.
[00156] [00156] The intron can have a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80-350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-
[00157] [00157] In one embodiment, the vector genome comprises at least one element for improving transgene specificity and target expression (see, for example, Powell et al. Viral Expression Cassette Elements to Improve Transgene Target Specificity and Expression in Gene Therapy, 2015; the entirety of which is incorporated herein by reference) such as an intron. Non-limiting examples of introns include MVM (67-97 bps), F. IX truncated intron 1 (300 bps), SD B-globin/immunoglobulin heavy chain splice acceptor (250 bps), adenovirus splice donor/ immunoglobulin splice acceptor (500 bps), late splice donor/SV40 splice acceptor (198/16S) (180 bps), and hybrid adenovirus splice donor/IgG splice acceptor (230 bps).
[00158] [00158] In one embodiment, the intron may be 100-500 nucleotides in length. The intron can have a length of 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 200 , 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450 , 460, 470, 480, 490 or 500 the intron can have a length between 80-100, 80-120, 80-140, 80-160, 80-180, 80-200, 80-250, 80-300, 80 -350, 80-400, 80-450, 80-500, 200-300, 200-400, 200-500, 300-400, 300-500, or 400-500. Component of the Viral Genome: Load Sequence
[00159] [00159] In one embodiment, the viral genome comprises one or more payload sequences.
[00160] [00160] In one embodiment, the viral genome comprises one or more stuffer sequences so that the length of the viral genome is the optimal size for packaging. As a non-limiting example, the viral genome comprises at least one stuffer sequence so that the length of the viral genome is about 2.3 kb. As a non-limiting example, the viral genome comprises at least one stuffer sequence so that the length of the viral genome is about 4.6 kb.
[00161] [00161] In one embodiment, the viral genome is a single-stranded (ss) viral genome and comprises one or more stuffer sequences that are between about 0.1 kb - 3.8 kb in length, such as, but without limitation, 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1.1kb, 1.2kb, 1.3kb, 1.4kb, 1.5kb, 1.6kb, 1.7kb, 1.8kb, 1.9kb, 2kb, 2, 1kb, 2.2kb, 2.3kb, 2.4kb, 2.5kb, 2.6kb, 2.7kb, 2.8kb, 2.9kb, 3kb, 3.1kb , 3.2kb, 3.3kb, 3.4kb, 3.5kb, 3.6kb, 3.7kb, or 3.8kb. As a non-limiting example, the total length of the stuffer sequence in the vector genome is 3.1 kb. As a non-limiting example, the total length of the stuffer sequence in the vector genome is 2.7 kb. As a non-limiting example, the total length of the stuffer sequence in the vector genome is 0.8 kb. As a non-limiting example, the total length of the stuffer sequence in the vector genome is 0.4 kb. As a non-limiting example, the length of each stuffer sequence in the vector genome is 0.8 kb. As a non-limiting example, the length of each stuffer sequence in the vector genome is 0.4 kb.
[00162] [00162] In one embodiment, the viral genome is a self-complementary (sc) viral genome and comprises one or more stuffer sequences that are between about 0.1 kb — 1.5 kb in length, such as, but not limited to, 0.1kb, 0.2kb, 0.3kb, 0.4kb, 0.5kb, 0.6kb, 0.7kb, 0.8kb, 0.9kb, 1kb, 1, 1kb, 1.2kb, 1.3kb, 1.4kb, or 1.5kb. As a non-limiting example, the total length of the stuffer sequence in the vector genome is 0.8 kb. As a non-limiting example, the total length of the stuffer sequence in the vector genome is 0.4 kb. As a non-limiting example, the length of each stuffer sequence in the vector genome is 0.8 kb. As a non-limiting example, the length of each stuffer sequence in the vector genome is 0.4 kb.
[00163] [00163] In one embodiment, the viral genome comprises any portion of a payload sequence. The viral genome may comprise 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of a charge sequence.
[00164] [00164] In one embodiment, the viral genome is a single stranded (ss) viral genome and comprises one or more stuffer sequences so that the length of the viral genome is about 4.6 kb. As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 3' to the 5' ITR sequence. As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 5' to a promoter sequence.
[00165] [00165] As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 3' to the polyadenylation signal sequence.
[00166] [00166] As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 5' to the 3' ITR sequence.
[00167] [00167] As a non-limiting example, the viral genome comprises at least one cargo sequence, and the stuffer sequence is located between two intron sequences.
[00168] [00168] As a non-limiting example, the viral genome comprises at least one cargo sequence, and the stuffer sequence is located in an intron sequence.
[00169] [00169] As a non-limiting example, the viral genome comprises two cargo sequences, and the first stuffer sequence is located 3' to the 5' ITR sequence and the second stuffer sequence is located 3' to the signal sequence of polyadenylation.
[00170] [00170] As a non-limiting example, the viral genome comprises two cargo sequences, and the first stuffer sequence is located 5' to a promoter sequence and the second stuffer sequence is located 3' to the polyadenylation signal sequence.
[00171] [00171] As a non-limiting example, the viral genome comprises two cargo sequences, and the first stuffer sequence is located 3' to the 5' ITR sequence and the second stuffer sequence is located 5' to the 5' sequence ' ITR.
[00172] [00172] In one embodiment, the viral genome is a self-complementary (sc) viral genome and comprises one or more stuffer sequences so that the length of the viral genome is about 2.3 kb.
[00173] [00173] As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 3' to the 5' ITR sequence.
[00174] [00174] As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 5' to a promoter sequence.
[00175] [00175] As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 3' to the polyadenylation signal sequence.
[00176] [00176] As a non-limiting example, the viral genome comprises at least one stuffer sequence and the stuffer sequence is located 5' to the 3' ITR sequence.
[00177] [00177] As a non-limiting example, the viral genome comprises at least one cargo sequence, and the stuffer sequence is located between two intron sequences.
[00178] [00178] As a non-limiting example, the viral genome comprises at least one cargo sequence, and the stuffer sequence is located in an intron sequence.
[00179] [00179] As a non-limiting example, the viral genome comprises two cargo sequences, and the first stuffer sequence is located 3' to the 5' ITR sequence and the second stuffer sequence is located 3' to the signal sequence of polyadenylation.
[00180] [00180] As a non-limiting example, the viral genome comprises two cargo sequences, and the first stuffer sequence is located 5' to a promoter sequence and the second stuffer sequence is located 3' to the polyadenylation signal sequence.
[00181] [00181] As a non-limiting example, the viral genome comprises two cargo sequences, and the first stuffer sequence is located 3' to the 5' ITR sequence and the second stuffer sequence is located 5' to the 5' sequence ' ITR.
[00182] [00182] In one embodiment, the viral genome may comprise one or more stuffer sequences between one or more regions of the viral genome.
[00183] [00183] In one embodiment, the stuffing region may be located before a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, a region booster, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and/or an exon region.
[00184] [00184] In one embodiment, the stuffing region may be located after a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, a region booster, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and/or an exon region.
[00185] [00185] In one embodiment, the stuffing region may be located before and after a region such as, but not limited to, a payload region, an inverted terminal repeat (ITR), a promoter region, an intron region, a booster region, a polyadenylation signal sequence region, a multiple cloning site (MCS) region, and/or an exon region.
[00186] [00186] In one embodiment, the viral genome may comprise one or more stuffer sequences that bifurcate at least one region of the viral genome.
[00187] [00187] The forked region of the viral genome can comprise 1%, 2%, 3%, 4%, 5%, 6%, T%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from region to 5 ' of the load sequence region.
[00188] [00188] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 10% of the region is located 5' to the stuffing sequence and 90% of the region is located 3' to the loading sequence.
[00189] [00189] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 20% of the region is located 5' to the stuffing sequence and 80% of the region is located 3' to the loading sequence.
[00190] [00190] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 30% of the region is located 5' to the stuffing sequence and 70% of the region is located 3' to the loading sequence.
[00191] [00191] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 40% of the region is located 5' to the stuffing sequence and 60% of the region is located 3' to the loading sequence.
[00192] [00192] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 50% of the region is located 5' to the stuffing sequence and 50% of the region is located 3' to the loading sequence.
[00193] [00193] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 60% of the region is located 5' to the stuffing sequence and 40% of the region is located 3' to the loading sequence.
[00194] [00194] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 70% of the region is located 5' to the stuffing sequence and 30% of the region is located 3' to the loading sequence.
[00195] [00195] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 80% of the region is located 5' to the stuffing sequence and 20% of the region is located 3' to the loading sequence.
[00196] [00196] As a non-limiting example, the stuffing sequence may bifurcate at least one region such that 90% of the region is located 5' to the stuffing sequence and 10% of the region is located 3' to the loading sequence.
[00197] [00197] In one embodiment, the viral genome comprises a stuffer sequence after the 5' ITR.
[00198] [00198] In one embodiment, the viral genome comprises a stuffer sequence after the promoter region.
[00199] [00199] In one embodiment, the viral genome comprises a stuffer sequence after the payload region.
[00200] [00200] In one embodiment, the viral genome comprises a stuffer sequence after the intron region.
[00201] [00201] In one embodiment, the viral genome comprises a stuffer sequence after the booster region.
[00202] [00202] In one embodiment, the viral genome comprises a stuffer sequence following the polyadenylation signal sequence region.
[00203] [00203] In one embodiment, the viral genome comprises a stuffer sequence after the MCS region.
[00204] [00204] In one embodiment, the viral genome comprises a stuffer sequence after the exon region.
[00205] [00205] In one embodiment, the viral genome comprises a stuffer sequence before the promoter region.
[00206] [00206] In one embodiment, the viral genome comprises a stuffer sequence before the payload region.
[00207] [00207] In one embodiment, the viral genome comprises a stuffer sequence before the intron region.
[00208] [00208] In one embodiment, the viral genome comprises a stuffer sequence before the booster region.
[00209] [00209] In one embodiment, the viral genome comprises a stuffer sequence before the polyadenylation signal sequence region.
[00210] [00210] In one embodiment, the viral genome comprises the filler sequence before the MCS region.
[00211] [00211] In one embodiment, the viral genome comprises a stuffer sequence before the exon region.
[00212] [00212] In one embodiment, the viral genome comprises a stuffer sequence before the 3' ITR.
[00213] [00213] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, 5'
[00214] [00214] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the 5' ITR and the payload region.
[00215] [00215] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the 5' ITR and the intron region.
[00216] [00216] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the 5' ITR and the boost region.
[00217] [00217] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the 5' ITR and the polyadenylation signal sequence region.
[00218] [00218] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the 5' ITR and the MCS region.
[00219] [00219] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the 5' ITR and the exon region.
[00220] [00220] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the promoter region and the payload region.
[00221] [00221] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the promoter region and the intron region.
[00222] [00222] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the promoter region and the enhancer region.
[00223] [00223] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the promoter region and the polyadenylation signal sequence region.
[00224] [00224] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the promoter region and the MCS region.
[00225] [00225] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the promoter region and the exon region.
[00226] [00226] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the promoter region and the 3' ITR.
[00227] [00227] In one embodiment, a stuffing sequence can be located between two regions, such as, but not limited to, the payload region and the intron region.
[00228] [00228] In one embodiment, a fill sequence may be located between two regions, such as, but not limited to, the payload region and the reinforcement region.
[00229] [00229] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the payload region and the polyadenylation signal sequence region.
[00230] [00230] In one embodiment, a fill sequence may be located between two regions, such as, but not limited to, the payload region and the MCS region.
[00231] [00231] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the payload region and the exon region.
[00232] [00232] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the payload region and the 3' ITR.
[00233] [00233] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the intron region and the reinforcement region.
[00234] [00234] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the intron region and the polyadenylation signal sequence region.
[00235] [00235] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the intron region and the MCS region.
[00236] [00236] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the intron region and the exon region.
[00237] [00237] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the intron region and the 3' ITR.
[00238] [00238] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the boost region and the polyadenylation signal sequence region.
[00239] [00239] In one embodiment, a fill sequence may be located between two regions, such as, but not limited to, the boost region and the MCS region.
[00240] [00240] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the reinforcement region and the exon region.
[00241] [00241] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the boost region and the 3' ITR.
[00242] [00242] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the polyadenylation signal sequence region and the MCS region.
[00243] [00243] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the polyadenylation signal sequence region and the exon region.
[00244] [00244] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the polyadenylation signal sequence region and the 3' ITR.
[00245] [00245] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the MCS region and the exon region.
[00246] [00246] In one embodiment, a stuffing sequence may be located between two regions, such as, but not limited to, the MCS region and the 3' ITR.
[00247] [00247] In one embodiment, a stuffer sequence may be located between two regions, such as, but not limited to, the exon region and the 3' ITR.
[00248] [00248] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. payload region.
[00249] [00249] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. intron region.
[00250] [00250] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. reinforcement region.
[00251] [00251] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. polyadenylation signal sequence region.
[00252] [00252] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. MCS region.
[00253] [00253] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. exon region.
[00254] [00254] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the promoter region and the promoter region. 3' ITR.
[00255] [00255] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the payload region and the intron region.
[00256] [00256] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00257] [00257] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00258] [00258] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00259] [00259] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the payload region and the exon region.
[00260] [00260] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the payload region and the 3'ITR.
[00261] [00261] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the intron and the reinforcement region.
[00262] [00262] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the intron and the polyadenylation signal sequence region.
[00263] [00263] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the intron and the MCS region.
[00264] [00264] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the intron and the exon region.
[00265] [00265] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the intron region and the 3g3iITR .
[00266] [00266] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the enhancer region and the polyadenylation signal sequence region.
[00267] [00267] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the enhancer region and the MCS region.
[00268] [00268] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the enhancer region and the exon region.
[00269] [00269] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the enhancer region and at 3' ITR.
[00270] [00270] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the MCS region.
[00271] [00271] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the exon region.
[00272] [00272] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the 3' ITR.
[00273] [00273] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00274] [00274] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the MCS region and the 3'ITR.
[00275] [00275] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the promoter region, and the second stuffer sequence can be located between the exon region and the 3iITR .
[00276] [00276] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the payload region.
[00277] [00277] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the intron region.
[00278] [00278] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the reinforcement region.
[00279] [00279] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region.
[00280] [00280] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the MCS region.
[00281] [00281] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the exon region.
[00282] [00282] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the promoter region and the 3' ITR.
[00283] [00283] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. payload and intron region.
[00284] [00284] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. payload and the reinforcement region.
[00285] [00285] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. payload and the polyadenylation signal sequence region.
[00286] [00286] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. payload and the MCS region.
[00287] [00287] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. payload and the exon region.
[00288] [00288] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. payload and 3'ITR.
[00289] [00289] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. intron and the reinforcement region.
[00290] [00290] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. intron and the polyadenylation signal sequence region.
[00291] [00291] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. intron and the MCS region.
[00292] [00292] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. intron and exon region.
[00293] [00293] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the 5' ITR and payload region. intron ea3iITR.
[00294] [00294] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. booster and the polyadenylation signal sequence region.
[00295] [00295] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. reinforcement and the MCS region.
[00296] [00296] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. reinforcement and the exon region.
[00297] [00297] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. reinforcement and the 3' ITR.
[00298] [00298] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the MCS region.
[00299] [00299] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the exon region.
[00300] [00300] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the 3' ITR.
[00301] [00301] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00302] [00302] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the MCS region and the 3'ITR.
[00303] [00303] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the payload region, and the second stuffer sequence can be located between the payload region. easILTR exon.
[00304] [00304] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. the payload region.
[00305] [00305] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. the intron region.
[00306] [00306] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. the reinforcement region.
[00307] [00307] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. the polyadenylation signal sequence region.
[00308] [00308] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. the MCS region.
[00309] [00309] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. the exon region.
[00310] [00310] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the promoter and the intron region. at 3' ITR.
[00311] [00311] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the cargo region useful and the intron region.
[00312] [00312] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the cargo region useful and the reinforcement region.
[00313] [00313] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the cargo region useful and the polyadenylation signal sequence region.
[00314] [00314] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the cargo region useful and the MCS region.
[00315] [00315] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the cargo region useful and the exon region.
[00316] [00316] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the cargo region useful and the 3'ITR.
[00317] [00317] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00318] [00318] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00319] [00319] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00320] [00320] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the intron region and the exon region.
[00321] [00321] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the intron region easILTR.
[00322] [00322] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00323] [00323] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the booster region and the MCS region.
[00324] [00324] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the booster region and the exon region.
[00325] [00325] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00326] [00326] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00327] [00327] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00328] [00328] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00329] [00329] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00330] [00330] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the MCS region and at 3'ITR.
[00331] [00331] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the intron region, and the second stuffer sequence can be located between the exon region easILTR.
[00332] [00332] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and the payload region.
[00333] [00333] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and the intron region.
[00334] [00334] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and the reinforcement region.
[00335] [00335] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region.
[00336] [00336] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and the MCS region.
[00337] [00337] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and the exon region.
[00338] [00338] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the promoter region and at 3' ITR.
[00339] [00339] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the cargo region useful and the intron region.
[00340] [00340] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the cargo region useful and the reinforcement region.
[00341] [00341] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the cargo region useful and the polyadenylation signal sequence region.
[00342] [00342] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the cargo region useful and the MCS region.
[00343] [00343] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the cargo region useful and the exon region.
[00344] [00344] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the cargo region useful and the 3'ITR.
[00345] [00345] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00346] [00346] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00347] [00347] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00348] [00348] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the intron region and the exon region.
[00349] [00349] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the intron region easILTR.
[00350] [00350] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00351] [00351] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the booster region and the MCS region.
[00352] [00352] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the booster region and the exon region.
[00353] [00353] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00354] [00354] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00355] [00355] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00356] [00356] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00357] [00357] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00358] [00358] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the MCS region and at 3'ITR.
[00359] [00359] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the booster region, and the second stuffer sequence can be located between the exon region easILTR.
[00360] [00360] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the payload region.
[00361] [00361] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the intron region.
[00362] [00362] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the enhancer region.
[00363] [00363] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region.
[00364] [00364] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the MCS region.
[00365] [00365] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the exon region.
[00366] [00366] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the promoter region and the 3' ITR.
[00367] [00367] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the intron region.
[00368] [00368] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00369] [00369] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00370] [00370] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00371] [00371] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the exon region.
[00372] [00372] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the 3' ITR.
[00373] [00373] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00374] [00374] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00375] [00375] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00376] [00376] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the exon region.
[00377] [00377] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the 3' ITR.
[00378] [00378] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the polyadenylation signal sequence region.
[00379] [00379] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the MCS region.
[00380] [00380] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the exon region.
[00381] [00381] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00382] [00382] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region.
[00383] [00383] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the exon region.
[00384] [00384] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the 3' ITR.
[00385] [00385] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00386] [00386] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00387] [00387] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00388] [00388] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter and MCS region. the payload region.
[00389] [00389] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter and MCS region. the intron region.
[00390] [00390] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter and MCS region. the reinforcement region.
[00391] [00391] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter and MCS region. the polyadenylation signal sequence region.
[00392] [00392] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter and MCS region. the MCS region.
[00393] [00393] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter region and the exon region.
[00394] [00394] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the promoter and MCS region. at 3' ITR.
[00395] [00395] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the cargo region useful and the intron region.
[00396] [00396] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the cargo region useful and the reinforcement region.
[00397] [00397] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the cargo region useful and the polyadenylation signal sequence region.
[00398] [00398] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the cargo region useful and the MCS region.
[00399] [00399] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the cargo region useful and the exon region.
[00400] [00400] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the cargo region useful and the 3'ITR.
[00401] [00401] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00402] [00402] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00403] [00403] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00404] [00404] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the intron region and the exon region.
[00405] [00405] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the intron region easILTR.
[00406] [00406] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00407] [00407] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the booster region and the MCS region.
[00408] [00408] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the booster region and the exon region.
[00409] [00409] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00410] [00410] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00411] [00411] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00412] [00412] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00413] [00413] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. the exon region.
[00414] [00414] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the MCS region and at 3'ITR.
[00415] [00415] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the MCS region, and the second stuffer sequence can be located between the exon region ea3g3ITR.
[00416] [00416] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. the payload region.
[00417] [00417] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. the intron region.
[00418] [00418] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. the reinforcement region.
[00419] [00419] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. the polyadenylation signal sequence region.
[00420] [00420] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. the MCS region.
[00421] [00421] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. the exon region.
[00422] [00422] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the promoter and exon region. at 3' ITR.
[00423] [00423] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the cargo region useful and the intron region.
[00424] [00424] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the cargo region useful and the reinforcement region.
[00425] [00425] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the cargo region useful and the polyadenylation signal sequence region.
[00426] [00426] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the cargo region useful and the MCS region.
[00427] [00427] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the cargo region useful and the exon region.
[00428] [00428] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the cargo region useful and the 3' ITR.
[00429] [00429] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00430] [00430] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00431] [00431] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00432] [00432] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the intron region and the exon region.
[00433] [00433] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the intron region ea3g3ITR.
[00434] [00434] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00435] [00435] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the booster region and the MCS region.
[00436] [00436] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the booster region and the exon region.
[00437] [00437] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00438] [00438] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00439] [00439] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00440] [00440] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00441] [00441] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00442] [00442] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the MCS region and at 3'ITR.
[00443] [00443] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the 5' ITR and the exon region, and the second stuffer sequence can be located between the exon region ea3ITR
[00444] [00444] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the cargo region useful and the intron region.
[00445] [00445] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the cargo region useful and the reinforcement region.
[00446] [00446] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the cargo region useful and the polyadenylation signal sequence region.
[00447] [00447] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the cargo region useful and the MCS region.
[00448] [00448] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the cargo region useful and the exon region.
[00449] [00449] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the cargo region useful and the 3' ITR.
[00450] [00450] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00451] [00451] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00452] [00452] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00453] [00453] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the intron region and the exon region.
[00454] [00454] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the intron region and the 3' ITR.
[00455] [00455] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the enhancer region and the polyadenylation signal sequence region.
[00456] [00456] In one embodiment, a viral genome can comprise two loader sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the enhancer region and the MCS region.
[00457] [00457] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the enhancer region and the exon region.
[00458] [00458] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the enhancer region and the 3' ITR.
[00459] [00459] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00460] [00460] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00461] [00461] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00462] [00462] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00463] [00463] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the MCS region and at 3' ITR.
[00464] [00464] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the payload region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00465] [00465] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the payload region and the intron region.
[00466] [00466] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00467] [00467] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00468] [00468] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00469] [00469] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the payload region and the exon region.
[00470] [00470] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the payload region and the 3' ITR.
[00471] [00471] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the intron region and the intron region. the reinforcement region.
[00472] [00472] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the intron region and the intron region. the polyadenylation signal sequence region.
[00473] [00473] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the intron region and the intron region. the MCS region.
[00474] [00474] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the intron and the intron region. the exon region.
[00475] [00475] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the intron region and the intron region. at 3' ITR.
[00476] [00476] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the enhancer region and the intron region. the polyadenylation signal sequence region.
[00477] [00477] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the enhancer region and the intron region. the MCS region.
[00478] [00478] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the enhancer region and the intron region. the exon region.
[00479] [00479] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the enhancer region and the intron region. at 3' ITR.
[00480] [00480] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the MCS region.
[00481] [00481] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the exon region.
[00482] [00482] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the 3' ITR.
[00483] [00483] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the MCS region and the intron region. exon region.
[00484] [00484] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the MCS region and the intron region. 3' ITR.
[00485] [00485] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the intron region, and the second stuffer sequence can be located between the exon and the intron region. at 3' ITR.
[00486] [00486] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the payload region and the intron region.
[00487] [00487] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00488] [00488] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00489] [00489] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00490] [00490] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the payload region and the exon region.
[00491] [00491] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the payload region and the 3' ITR.
[00492] [00492] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00493] [00493] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00494] [00494] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00495] [00495] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the intron region and the exon region.
[00496] [00496] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the intron region and at 3' ITR.
[00497] [00497] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the booster region, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00498] [00498] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the booster region, and the second stuffer sequence can be located between the booster region and the MCS region.
[00499] [00499] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the booster region, and the second stuffer sequence can be located between the booster region and the exon region.
[00500] [00500] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the booster region, and the second stuffer sequence can be located between the booster region and at 3' ITR.
[00501] [00501] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the MCS region.
[00502] [00502] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the exon region.
[00503] [00503] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the 3' ITR.
[00504] [00504] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00505] [00505] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00506] [00506] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the enhancer region, and the second stuffer sequence can be located between the exon region and at 3' ITR.
[00507] [00507] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and intron region.
[00508] [00508] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and reinforcement region.
[00509] [00509] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00510] [00510] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00511] [00511] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the exon region.
[00512] [00512] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the payload region and the 3' ITR.
[00513] [00513] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00514] [00514] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00515] [00515] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00516] [00516] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and exon region.
[00517] [00517] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the 3' ITR.
[00518] [00518] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the polyadenylation signal sequence region.
[00519] [00519] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the MCS region.
[00520] [00520] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the exon region.
[00521] [00521] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the 3' ITR.
[00522] [00522] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region.
[00523] [00523] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the exon region.
[00524] [00524] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the easILTR polyadenylation signal sequence region.
[00525] [00525] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00526] [00526] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00527] [00527] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00528] [00528] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the payload region and the intron region.
[00529] [00529] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00530] [00530] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00531] [00531] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00532] [00532] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the payload region and the exon region.
[00533] [00533] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the payload region and the 3' ITR.
[00534] [00534] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the intron and exon region. the reinforcement region.
[00535] [00535] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the intron region and the exon region. the polyadenylation signal sequence region.
[00536] [00536] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the intron and exon region. the MCS region.
[00537] [00537] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the intron region and the exon region. the exon region.
[00538] [00538] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the intron region and the exon region. at 3' ITR.
[00539] [00539] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the booster region and the exon region. the polyadenylation signal sequence region.
[00540] [00540] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the booster region and the exon region. the MCS region.
[00541] [00541] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the booster region and the exon region. the exon region.
[00542] [00542] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the booster region and the exon region. at 3' ITR.
[00543] [00543] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the MCS region.
[00544] [00544] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the exon region.
[00545] [00545] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the 3' ITR.
[00546] [00546] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region. exon region.
[00547] [00547] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region. 3' ITR.
[00548] [00548] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the exon region, and the second stuffer sequence can be located between the exon and exon region. at 3' ITR.
[00549] [00549] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the payload region and the intron region.
[00550] [00550] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00551] [00551] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00552] [00552] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the payload region and the MCS region.
[00553] [00553] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the payload region and the exon region.
[00554] [00554] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the payload region and the 3' ITR.
[00555] [00555] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the intron region and the MCS region. the reinforcement region.
[00556] [00556] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the intron region and the MCS region. the polyadenylation signal sequence region.
[00557] [00557] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the intron region and the MCS region. the MCS region.
[00558] [00558] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the intron region and the MCS region. the exon region.
[00559] [00559] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the intron region and the MCS region. at 3' ITR.
[00560] [00560] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the enhancer region and the MCS region. the polyadenylation signal sequence region.
[00561] [00561] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the enhancer region and the MCS region. the MCS region.
[00562] [00562] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the enhancer region and the MCS region. the exon region.
[00563] [00563] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the enhancer region and the MCS region. at 3' ITR.
[00564] [00564] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the MCS region.
[00565] [00565] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the exon region.
[00566] [00566] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the 3' ITR.
[00567] [00567] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. exon region.
[00568] [00568] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. 3' ITR.
[00569] [00569] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and the MCS region, and the second stuffer sequence can be located between the exon and MCS region. at 3' ITR.
[00570] [00570] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the payload region and the intron region.
[00571] [00571] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the payload region and the reinforcement region.
[00572] [00572] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the payload region and the polyadenylation signal sequence region.
[00573] [00573] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the payload region and the MCS region.
[00574] [00574] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the payload region and the exon region.
[00575] [00575] In one embodiment, a viral genome can comprise two payload sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the payload region and at 3'ITR.
[00576] [00576] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the intron region and the 3'ITR. reinforcement region.
[00577] [00577] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the intron region and the 3'ITR. polyadenylation signal sequence region.
[00578] [00578] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the intron region and the 3'ITR. MCS region.
[00579] [00579] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the intron region and the 3'ITR. exon region.
[00580] [00580] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the intron region and the 3g3iITR.
[00581] [00581] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the enhancer region and the 3'ITR. polyadenylation signal sequence region.
[00582] [00582] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the enhancer region and the 3'ITR. MCS region.
[00583] [00583] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the enhancer region and the 3'ITR. exon region.
[00584] [00584] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the booster region and the 3'ITR. 3' ITR.
[00585] [00585] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the signal sequence region of polyadenylation and the MCS region.
[00586] [00586] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the signal sequence region of polyadenylation and the exon region.
[00587] [00587] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the signal sequence region of polyadenylation and the 3' ITR.
[00588] [00588] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the MCS region and the 3'ITR region. of exon.
[00589] [00589] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the MCS region and the 3'ITR. 'ITR.
[00590] [00590] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the promoter region and 3'ITR, and the second stuffer sequence can be located between the easIlTR exon region.
[00591] [00591] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. intron and the reinforcement region.
[00592] [00592] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. intron and the polyadenylation signal sequence region.
[00593] [00593] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region and the intron region. intron and the MCS region.
[00594] [00594] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. intron and exon region.
[00595] [00595] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region and the intron region. intron and the 3' ITR.
[00596] [00596] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region and the intron region. booster and the polyadenylation signal sequence region.
[00597] [00597] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. reinforcement and the MCS region.
[00598] [00598] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. reinforcement and the exon region.
[00599] [00599] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. reinforcement and the 3' ITR.
[00600] [00600] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the MCS region.
[00601] [00601] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the exon region.
[00602] [00602] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the 3' ITR.
[00603] [00603] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00604] [00604] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00605] [00605] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the intron region, and the second stuffer sequence can be located between the payload region and the intron region. exon and the 3' ITR.
[00606] [00606] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. intron and the reinforcement region.
[00607] [00607] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. intron and the polyadenylation signal sequence region.
[00608] [00608] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. intron and the MCS region.
[00609] [00609] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. intron and exon region.
[00610] [00610] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. intron and the 3' ITR.
[00611] [00611] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. booster and the polyadenylation signal sequence region.
[00612] [00612] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. reinforcement and the MCS region.
[00613] [00613] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. reinforcement and the exon region.
[00614] [00614] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. reinforcement and the 3' ITR.
[00615] [00615] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the MCS region.
[00616] [00616] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the exon region.
[00617] [00617] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the 3' ITR.
[00618] [00618] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00619] [00619] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00620] [00620] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the booster region, and the second stuffer sequence can be located between the payload region. exon and the 3' ITR.
[00621] [00621] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the reinforcement region.
[00622] [00622] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the polyadenylation signal sequence region.
[00623] [00623] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the MCS region.
[00624] [00624] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the exon region.
[00625] [00625] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the intron region and the 3' ITR.
[00626] [00626] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the polyadenylation signal sequence region.
[00627] [00627] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the MCS region.
[00628] [00628] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the exon region.
[00629] [00629] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00630] [00630] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region.
[00631] [00631] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the exon region.
[00632] [00632] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the 3ITR.
[00633] [00633] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00634] [00634] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00635] [00635] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00636] [00636] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. intron and the reinforcement region.
[00637] [00637] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. intron and the polyadenylation signal sequence region.
[00638] [00638] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. intron and the MCS region.
[00639] [00639] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. intron and exon region.
[00640] [00640] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. intron and the 3' ITR.
[00641] [00641] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. booster and the polyadenylation signal sequence region.
[00642] [00642] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. reinforcement and the MCS region.
[00643] [00643] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. reinforcement and the exon region.
[00644] [00644] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. reinforcement and the 3' ITR.
[00645] [00645] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the MCS region.
[00646] [00646] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the exon region.
[00647] [00647] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the 3' ITR.
[00648] [00648] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00649] [00649] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00650] [00650] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the MCS region, and the second stuffer sequence can be located between the payload region. exon and the 3' ITR.
[00651] [00651] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. intron and the reinforcement region.
[00652] [00652] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. intron and the polyadenylation signal sequence region.
[00653] [00653] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region and the exon region. intron and the MCS region.
[00654] [00654] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. intron and exon region.
[00655] [00655] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. intron and the 3' ITR.
[00656] [00656] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. booster and the polyadenylation signal sequence region.
[00657] [00657] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. reinforcement and the MCS region.
[00658] [00658] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. reinforcement and the exon region.
[00659] [00659] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. reinforcement and the 3' ITR.
[00660] [00660] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the MCS region.
[00661] [00661] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region and the exon region. polyadenylation signal sequence and the exon region.
[00662] [00662] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the 3' ITR.
[00663] [00663] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00664] [00664] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00665] [00665] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the exon region, and the second stuffer sequence can be located between the payload region. exon and the 3' ITR.
[00666] [00666] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. intron and the reinforcement region.
[00667] [00667] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. intron and the polyadenylation signal sequence region.
[00668] [00668] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. intron and the MCS region.
[00669] [00669] In one embodiment, a viral genome may comprise two payload sequences, the first stuffer sequence being located between the payload region and the 3 ITR region. and the second stuffing sequence can be located between the intron region and the exon region.
[00670] [00670] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. intron and the 3' ITR.
[00671] [00671] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. booster and the polyadenylation signal sequence region.
[00672] [00672] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. reinforcement and the MCS region.
[00673] [00673] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. reinforcement and the exon region.
[00674] [00674] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. reinforcement and the 3' ITR.
[00675] [00675] In one embodiment, a viral genome may comprise two payload sequences, the first stuffer sequence being located between the payload region and the 3 ITR region. and the second stuffer sequence may be located between the polyadenylation signal sequence region and the MCS region.
[00676] [00676] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the exon region.
[00677] [00677] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. polyadenylation signal sequence and the 3' ITR.
[00678] [00678] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00679] [00679] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00680] [00680] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the payload region and the 3 ITR region, and the second stuffer sequence can be located between the payload region. exon and the 3' ITR.
[00681] [00681] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00682] [00682] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the booster region and the MCS region.
[00683] [00683] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the booster region and the exon region.
[00684] [00684] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00685] [00685] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00686] [00686] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00687] [00687] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00688] [00688] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00689] [00689] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the MCS region and at 3' ITR.
[00690] [00690] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the booster region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00691] [00691] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the polyadenylation signal sequence region.
[00692] [00692] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the MCS region.
[00693] [00693] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the reinforcement region and the exon region.
[00694] [00694] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the booster region and the 3' ITR.
[00695] [00695] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region.
[00696] [00696] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the exon region.
[00697] [00697] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the ea3ITR polyadenylation signal sequence region.
[00698] [00698] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00699] [00699] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00700] [00700] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00701] [00701] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the reinforcer region and the polyadenylation signal sequence region.
[00702] [00702] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the reinforcer region and the MCS region.
[00703] [00703] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the reinforcer region and the exon region.
[00704] [00704] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the reinforcer region and the 3' ITR.
[00705] [00705] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00706] [00706] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00707] [00707] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00708] [00708] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. the exon region.
[00709] [00709] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. at 3' ITR.
[00710] [00710] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the MCS region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00711] [00711] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the reinforcer region and the polyadenylation signal sequence region.
[00712] [00712] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the reinforcer region and the MCS region.
[00713] [00713] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the reinforcer region and the exon region.
[00714] [00714] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the reinforcer region and the 3' ITR.
[00715] [00715] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00716] [00716] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00717] [00717] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00718] [00718] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00719] [00719] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the MCS region and at 3' ITR.
[00720] [00720] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and the exon region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00721] [00721] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the booster region and the polyadenylation signal sequence region.
[00722] [00722] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the booster region and the MCS region.
[00723] [00723] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the booster region and the exon region.
[00724] [00724] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the booster region and at 3' ITR.
[00725] [00725] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron and 3'ITR region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the MCS region.
[00726] [00726] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron and 3'ITR region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the exon region.
[00727] [00727] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron and 3'ITR region, and the second stuffer sequence can be located between the filler sequence region. polyadenylation signal and the 3' ITR.
[00728] [00728] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the MCS region and the exon region.
[00729] [00729] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the MCS region and the 3'ITR.
[00730] [00730] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the intron region and 3'ITR, and the second stuffer sequence can be located between the exon region and the 3ITR .
[00731] [00731] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region.
[00732] [00732] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the polyadenylation signal sequence region and the exon region.
[00733] [00733] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the eagsILTR polyadenylation signal sequence region.
[00734] [00734] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00735] [00735] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00736] [00736] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the polyadenylation signal sequence region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00737] [00737] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00738] [00738] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00739] [00739] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the MCS region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00740] [00740] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. the exon region.
[00741] [00741] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the MCS region, and the second stuffer sequence can be located between the MCS region and the MCS region. at 3' ITR.
[00742] [00742] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the MCS region, and the second stuffer sequence can be located between the exogenous region3 'ITR.
[00743] [00743] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00744] [00744] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00745] [00745] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the exon region, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00746] [00746] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00747] [00747] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the exon region, and the second stuffer sequence can be located between the MCS region and at 3' ITR.
[00748] [00748] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the exon region, and the second stuffer sequence can be located between the exon region3 'ITR.
[00749] [00749] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the 3' ITR, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the MCS region.
[00750] [00750] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the 3' ITR, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the exon region.
[00751] [00751] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the 3' ITR, and the second stuffer sequence can be located between the sequence region of polyadenylation signal and the 3' ITR.
[00752] [00752] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the 3' ITR, and the second stuffer sequence can be located between the MCS region and the exon region.
[00753] [00753] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the 3' ITR, and the second stuffer sequence can be located between the MCS region and at 3'ITR.
[00754] [00754] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the booster region and the 3' ITR, and the second stuffer sequence can be located between the exon region ea3ITR
[00755] [00755] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00756] [00756] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00757] [00757] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the MCS region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00758] [00758] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the exon region, and the second stuffer sequence can be located between the MCS region and the exon region.
[00759] [00759] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the exon region, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00760] [00760] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the exon region, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00761] [00761] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and a3'ITR, and the second stuffer sequence can be located between the MCS region and the exon region.
[00762] [00762] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and the 3' ITR, and the second stuffer sequence can be located between the MCS region and the 3' ITR.
[00763] [00763] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the polyadenylation signal sequence region and a3'ITR, and the second stuffer sequence can be located between the exon region and the 3' ITR.
[00764] [00764] In one embodiment, a viral genome can comprise two cargo sequences, the first stuffer sequence can be located between the MCS region and the exon region, and the second stuffer sequence can be located between the exon region and the 3ITR . AAV production
[00765] [00765] The present invention provides methods for generating parvoviral particles, eg, AAV particles, by replicating the viral genome in a replicating viral cell.
[00766] [00766] According to the invention, the viral genome comprising a payload region will be incorporated into the AAV particle produced in the viral replication cell.
[00767] [00767] Methods for making AAV particles are well known in the literature and are described, for example, in US Patents — No. , US6943019, US6953690, US7022519, US7238526, US7291498 and US7491508, USSO64764, US6194191,
[00768] [00768] In one embodiment, the AAV particles are made by the methods described in WO2015191508, the entirety of which is incorporated herein by reference.
[00769] [00769] Viral replication cells commonly used for the production of recombinant AAV particles include, but are not limited to, 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines, as described in US Patent Nos. US6156303, US5387484, US5741683, US5691176, and USS688676; in US Patent Publication No. 2002/0081721, and International Patent Publication No.* WO 00/47757, WO 00/24916, and WO 96/17947, the entirety of each of which is incorporated herein by reference.
[00770] [00770] In some embodiments, the present invention features a method for producing an AAV particle having improved (increased, enhanced) transduction efficiency comprising the steps of: 1) cotransfecting competent bacterial cells with a bacmid vector and a construct vector virus and/or an AAV payload construct vector, 2) isolating the resulting viral construct expression vector and the AAV payload construct expression vector and separately transfecting the viral replicating cells, 3) isolating and purifying the resulting viral and payload construct particles comprising viral construct expression vector or AAV payload construct expression vector, 4) co-infecting a viral replication cell with the particles of either AAV or AAV payload construct of viral construct comprising viral construct expression vector or AAV payload construct expression vector, and 5) collecting and purifying the virus particle AAV comprising a viral genome.
[00771] [00771] In some embodiments, the present invention features a method for producing an AAV particle comprising the steps of 1) simultaneously co-transfecting mammalian cells, such as, but not limited to, HEK293 cell, with a payload region, a construct expressing rep and cap genes and a helper construct, 2) collecting and purifying the AAV particle comprising a viral genome.
[00772] [00772] In some embodiments, the viral genome of the AAV particle of the invention optionally encodes a selectable marker.
[00773] [00773] The selectable marker may comprise a cell surface marker, such as any protein expressed on the cell surface including, but not limited to, receptors, CD markers, lectins, integrins, or truncated versions thereof.
[00774] [00774] In some embodiments, selectable marker reporter genes described in International Application No. WO 96/23810; Heim et al., Current Biology 2:178-182 (1996); Heim et al., Proc. natl. academy Sci. USA (1995); or Heim et al., Science 373:663-664 (1995), WO 96/30540, the entirety of each of which is incorporated herein by reference). Genome Size.
[00775] [00775] In one embodiment, the AAV particle comprising a payload described in this application may be a single-stranded or double-stranded vector genome.
[00776] [00776] The vector genome size can be small, medium, large, or the maximum size.
[00777] [00777] Additionally, the vector genome may comprise a promoter and a polyA tail.
[00778] [00778] In one embodiment, the vector genome comprising a payload described in this application may be a small single stranded vector genome.
[00779] [00779] A small single stranded vector genome can be 2.7 to 3.5 kb in size such as about 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5 kb in size.
[00780] [00780] As a non-limiting example, the small single strand vector genome can be 3.2 kb in size.
[00781] [00781] Additionally, the vector genome may comprise a promoter and a polyA tail.
[00782] [00782] In one embodiment, the vector genome comprising a payload described in this application may be a small double stranded vector genome.
[00783] [00783] A small double stranded vector genome can be 1.3 to 1.7 kb in size such as about 1.3, 1.4, 1.5, 1.6, and 1.7 kb in size.
[00784] [00784] As a non-limiting example, the small double stranded vector genome can be 1.6 kb in size.
[00785] [00785] Additionally, the vector genome may comprise a promoter and a polyA tail.
[00786] [00786] In one embodiment, the vector genome comprising a payload described in this application may be a medium single stranded vector genome.
[00787] [00787] An average single stranded vector genome can be 3.6 to 4.3 kb in size such as about 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 and 4.3 kb in size.
[00788] [00788] As a non-limiting example, average single stranded vector genome can be 4.0 kb in size.
[00789] [00789] Additionally, the vector genome may comprise a promoter and a polyA tail.
[00790] [00790] In one embodiment, the vector genome comprising a payload described in this application may be a medium double stranded vector genome.
[00791] [00791] An average double stranded vector genome can be 1.8 to 2.1 kb in size such as about 1.8, 1.9, 2.0, and 21 kb in size.
[00792] [00792] As a non-limiting example, the average double stranded vector genome can be 2.0 kb in size.
[00793] [00793] Additionally, the vector genome may comprise a promoter and a polyA tail.
[00794] [00794] In one embodiment, the vector genome comprising a payload described in this application may be a large single stranded vector genome.
[00795] [00795] A large single strand vector genome can be 4.4 to 6.0 kb in size such as about 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0 kb in size.
[00796] [00796] As a non-limiting example, the large single strand vector genome can be 4.7 kb in size.
[00797] [00797] As another non-limiting example, the large single-stranded vector genome can be 4.8 kb in size.
[00798] [00798] As yet another non-limiting example, the large single strand vector genome can be 6.0 kb in size.
[00799] [00799] Additionally, the vector genome may comprise a promoter and a polyA tail.
[00800] [00800] In one embodiment, the vector genome comprising a payload described in this application may be a large double stranded vector genome.
[00801] [00801] “A large double stranded vector genome can be 2.2 to 3.0 kb in size such as about 2.2, 2.3, 2.4, 2.5, 2.6, 2.7 , 2.8, 2.9 and 3.0 kb in size.
[00802] [00802] As a non-limiting example, the large double stranded vector genome can be 2.4 kb in size.
[00803] [00803] Additionally, the vector genome may comprise a promoter and a polyA tail. Payloads of the Invention.
[00804] [00804] The AAV particles of the present invention comprise at least one payload region.
[00805] [00805] “As used in this application, "payload" or "payload region" refers to one or more polynucleotides or polynucleotide regions encoded by or in a viral genome or an expression product of such a polynucleotide or polynucleotide region, for example, a transgene, a polynucleotide encoding a polypeptide or multipeptide, or a modular nucleic acid or regulatory nucleic acid.
[00806] [00806] Payloads of the present invention typically encode polypeptides or fragments or variants thereof.
[00807] [00807] The payload region can be constructed to reflect a similar or mirrored region of the natural organization of an MRNA.
[00808] [00808] The payload region may comprise a combination of coding and non-coding nucleic acid sequences.
[00809] [00809] In some embodiments, the AAV payload region may encode a coding or non-coding RNA.
[00810] [00810] In one embodiment, the AAV particle comprises a viral genome with a payload region comprising nucleic acid sequences encoding more than one polypeptide of interest.
[00811] [00811] In such an embodiment, a viral genome encoding more than one polypeptide can be replicated and packaged into a viral particle.
[00812] [00812] A target cell transduced with a viral particle comprising more than one polypeptide can express each of the polypeptides in a single cell.
[00813] [00813] In one embodiment, the payload region may comprise the components shown in Figure 1.
[00814] [00814] Payload region 110 is located in the viral genome
[00815] [00815] At the 5' and/or 3' end of the payload region 110 there may be at least one inverted terminal region (ITR) 120.
[00816] [00816] In the payload region, there is a promoter region 130, an intron region 140, and a coding region 150.
[00817] [00817] When the payload region of the AAV particle encodes a polypeptide, the polypeptide can be either a peptide or a protein.
[00818] [00818] The viral genomes encoding the polypeptides described in this application may be useful in the fields of human disease, viruses, infections, veterinary applications and a variety of in vivo and in vitro scenarios.
[00819] [00819] In some embodiments, AAV particles are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of neurological diseases and/or disorders.
[00820] [00820] In some embodiments, AAV particles are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of Parkinson's mala.
[00821] [00821] In some embodiments, AAV particles are useful in the field of medicine for the treatment, prophylaxis, palliation or amelioration of central nervous system disorders. The nature of polypeptides and variants.
[00822] [00822] The amino acid sequences encoded by payload regions of the viral genomes of the invention can be translated as an entire polypeptide, a plurality of polypeptides or polypeptide fragments, which can be independently encoded by one or more nucleic acids, acid fragments nucleic acids or variants of any of the above.
[00823] [00823] “As used in this application, "polypeptide" means a polymer of amino acid residues (natural or artificial) linked between the immune system most often by peptide bonds.
[00824] [00824] The term, as used in this application, refers to proteins, polypeptides, and peptides of any size, structure, or function.
[00825] [00825] In some cases, the encoded polypeptide is smaller than about 50 amino acids and the polypeptide is then called a peptide.
[00826] [00826] If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues in length.
[00827] [00827] Accordingly, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of those described above.
[00828] [00828] A polypeptide can be a single molecule or it can be a multimolecular complex such as a dimer, a trimer or a tetramer.
[00829] [00829] They may also comprise single-chain or multi-chain polypeptides and may be associated or linked.
[00830] [00830] The term polypeptide may also be applied to polymers of amino acids in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.
[00831] [00831] The term "polypeptide variant" refers to molecules that differ in their amino acid sequence from a native or reference sequence.
[00832] [00832] Amino acid sequence variants may have substitutions, deletions, and/or insertions at certain positions in the amino acid sequence, relative to a native or reference sequence.
[00833] [00833] Generally, the variants will have at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous). ) to a native or reference sequence.
[00834] [00834] In some embodiments, "variant mimetics" are displayed.
[00835] [00835] As used in this application, the term "variant mimetic" means a mimetic that contains one or more amino acids that would mimic an activated sequence.
[00836] [00836] For example, glutamate can serve as a mimetic for phosphorothreonine and/or phosphoserine.
[00837] [00837] Alternatively, variant mimetics may result in deactivation or an inactivated product containing the mimetic, eg phenylalanine may act as an inactivation substitution for tyrosine; or alanine can act as an inactivating substitution for serine.
[00838] [00838] The term "amino acid sequence variant" refers to molecules with some differences in their amino acid sequences from a native or starting sequence.
[00839] [00839] Amino acid sequence variants may have substitutions, deletions, and/or insertions at certain positions in the amino acid sequence.
[00840] [00840] "Native" or "starter" sequence should not be confused with a wild-type sequence.
[00841] [00841] “As used in this application, a native or starting sequence is a relative term referring to an original molecule with which a comparison can be made.
[00842] [00842] “Native” or “starter” sequences or molecules may represent wild-type (the sequence found in nature), but do not have to be the wild-type sequence.
[00843] [00843] Generally, the variants will have at least about 70% homology to a native sequence, and preferably, they will be at least about 80%, more preferably at least about 90% homologous to a native sequence.
[00844] [00844] "Homology", as used for amino acid sequences, is defined as the percentage of residues in the candidate amino acid sequences that are identical to residues in the amino acid sequence of a second sequence after alignment of the sequences and introduction of gaps, if necessary to achieve the maximum percentage of homology.
[00845] [00845] “Methods and computer programs for alignment are well known in the literature.
[00846] [00846] It is understood that homology depends on a percent identity calculation, but may differ in value due to gaps and penalties introduced in the calculation.
[00847] [00847] —By "homologs", as used in connection with amino acid sequences, is meant the corresponding sequence from other species with substantial identity to a second sequence from a second species.
[00848] [00848] "Analogs" is intended to include polypeptide variants that differ by one or more amino acid changes, for example, substitutions, additions, or deletions of amino acid residues that still retain the properties of the parent polypeptide.
[00849] [00849] Sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C-terminal ends).
[00850] [00850] Sequence tags can be used for peptide purification or localization.
[00851] [00851] Lysines can be used to increase peptide solubility or allow biotinylation.
[00852] [00852] —Alternatively, amino acid residues located in the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted resulting in truncated sequences.
[00853] [00853] Certain amino acids (e.g. C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, such as, for example, expression of the sequence as part of a larger sequence that is soluble, or linked to a solid support.
[00854] [00854] "Substitutional variants", when referring to proteins, are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position.
[00855] [00855] Substitutions can be single, where only one amino acid in the molecule has been replaced, or they can be multiple where two or more amino acids have been replaced in the same molecule.
[00856] [00856] As used in this application the term "conservative amino acid substitution" refers to the replacement of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge or polarity.
[00857] [00857] Examples of conservative substitutions include replacing a non-polar (hydrophobic) residue such as isoleucine, valine and leucine with another non-polar residue.
[00858] [00858] Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between serineglycine and serine.
[00859] [00859] - Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of an acid residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions.
[00860] [00860] Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or or a polar residue for a non-polar residue.
[00861] [00861] "Insertion variants" when referring to proteins are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence.
[00862] [00862] "Immediately adjacent" to an amino acid means connected to the alpha-carboxy or alpha-amino functional group of the amino acid.
[00863] [00863] "Deletion variants" when referring to proteins are those with one or more amino acids removed in the native or starting amino acid sequence.
[00864] [00864] Generally, deletion variants will have one or more amino acids deleted in a particular region of the molecule.
[00865] [00865] “As used in this application, the term "derivative" is used synonymously with the term "variant" and refers to a molecule that has been modified or exchanged in any way with respect to a reference molecule or starting molecule.
[00866] [00866] In some embodiments, derivatives include native or starting proteins that have been modified with a proteinaceous or non-proteinaceous organic derivatizing agent, and post-translational modifications.
[00867] [00867] “Covalent modifications are traditionally introduced by reacting the targeted amino acid residues of the protein with an organic derivatization agent that is capable of reacting with selected side chains or terminal residues, or by taking advantage of post-translational modification mechanisms that work in recombinant host cells. selected.
[00868] [00868] The resulting covalent derivatives are useful in programs aimed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of recombinant glycoprotein.
[00869] [00869] Such modifications are known to the person skilled in the art and are carried out without undue experimentation.
[00870] [00870] Certain post-translational modifications are a result of the action of recombinant host cells on the expressed polypeptide.
[00871] [00871] The glutaminyl and asparaginyl residues are often post-translationally deamidated to the corresponding glutamyl and aspartyl residues.
[00872] [00872] Alternatively, these residues are deamidated under moderately acidic conditions.
[00873] [00873] Any form of these residues may be present in the proteins used in accordance with the present invention.
[00874] [00874] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & Co. San Francisco, pp. 79-86 (1983)).
[00875] [00875] "Characteristics" when referring to proteins are defined as components based on distinct amino acid sequences of a molecule.
[00876] [00876] Characteristics of the proteins of the present invention include surface manifestations, local conformational shapes, coils, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.
[00877] [00877] As used in this application when referring to proteins the term "surface manifestation" refers to a polypeptide-based component of a protein that appears on an outermost surface.
[00878] [00878] As used in this application when referring to proteins the term "local conformational format" means a polypeptide-based structural manifestation of a protein that is located in a definable space of the protein.
[00879] [00879] As used in this application when referring to proteins the term "folding" means the resulting conformation of an amino acid sequence after energy minimization.
[00880] [00880] A winding can occur at the secondary or tertiary level of the winding process.
[00881] [00881] Examples of secondary level windings include beta sheets and alpha helices.
[00882] [00882] Examples of tertiary windings include domains and regions formed due to the aggregation or separation of energetic forces.
[00883] [00883] Regions formed in this way include hydrophobic and hydrophilic pockets, among others.
[00884] [00884] As used in this application the term "turn" when referring to the conformation of a protein means a curve that changes the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.
[00885] [00885] “As used in this application when referring to proteins the term "loop" refers to a structural feature of a peptide or polypeptide that reverses the direction of the backbone of a peptide or polypeptide and comprises four more amino acid residues. Oliva et al. identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).
[00886] [00886] As used in this application when referring to proteins the term "half-loop" refers to a portion of an identified loop having at least half the number of amino acid residues as the loop from which it derives.
[00887] [00887] It is known that loops do not always contain an even number of amino acid residues.
[00888] [00888] Therefore, in cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the integer portion or the integer portion of the loop ( loop amino acid number/2+/-0.5 amino acids).
[00889] [00889] For example, the loop identified as a 7 amino acid loop could produce 3 amino acid or 4 amino acid half-loops (7/2=3.5+/-0.5 being equal to 3 or 4).
[00890] [00890] “As used in this application when referring to proteins, the term "domain" refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding ability, serving as a site for protein-protein interactions).
[00891] [00891] “As used in this application when referring to proteins the term "half domain" means a portion of an identified domain having at least half the number of amino acid residues of the domain from which it is derived.
[00892] [00892] “It is known that domains do not always contain an even number of amino acid residues.
[00893] [00893] Therefore, in cases where a domain contains or is identified as comprising an odd number of amino acids, a half-domain of the odd-numbered domain will comprise the integer portion or the integer portion of the domain (number of domain amino acids /2+/-0.5 amino acids).
[00894] [00894] “For example, a domain identified as a 7 amino acid domain could produce 3 amino acid or 4 amino acid half domains (7/2=3.5+/-0.5 being equal to 3 or 4).
[00895] [00895] It is also known that subdomains can be identified in domains or half-domains, these subdomains having less than all the structural or functional properties identified in the domains or half-domains from which they derive. It is also known that amino acids constituting any of the domain types in this application need not be contiguous along the polypeptide backbone (i.e., non-adjacent amino acids can structurally fold together to produce a domain, a half-domain, or a subdomain) .
[00896] [00896] As used in this application when referring to proteins the term "site" when in the context of amino acid based embodiments is used synonymously with "amino acid residue" and "amino acid side chain".
[00897] [00897] A site represents a position in a peptide or polypeptide that can be modified, manipulated, altered, derivatized or varied in the polypeptide molecules of the present invention.
[00898] [00898] As used in this application the terms "terminal or terminal" when referring to proteins refers to an end of a peptide or polypeptide.
[00899] [00899] Such an end is not limited to just the first or last site of the peptide or polypeptide, but may include additional amino acids in the terminal regions.
[00900] [00900] The polypeptide molecules of the present invention can be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated with an amino acid with a free carboxyl group (COOH)). ).
[00901] [00901] The proteins of the invention, in some cases, are made up of multiple polypeptide chains joined by disulfide bonds or by non-covalent forces (multimers, oligomers).
[00902] [00902] These types of proteins have multiple N- and C-termini.
[00903] [00903] Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide moiety such as an organic conjugate.
[00904] [00904] Once any of the features have been identified or defined as a component of a molecule of the invention, any of the various manipulations and/or modifications of those features can be carried out by displacement, permutation, inversion, deletion, randomization or duplication.
[00905] [00905] Furthermore, it is known that manipulation of features can result in the same outcome as a modification of the molecules of the invention.
[00906] [00906] For example, a manipulation involving the deletion of a domain would result in altering the length of a molecule in the same way as modifying a nucleic acid to encode a molecule of less than total length.
[00907] [00907] Modifications and manipulations can be performed by methods known in the prior art such as site-directed mutagenesis.
[00908] [00908] The resulting modified molecules can be tested for activity by in vitro or in vivo assays such as those described in this application or any other suitable screening assay known in the literature. Payload: AADC polynucleotide constructs.
[00909] [00909] In accordance with the present invention, aromatic L-amino acid decarboxylase (AADC; also known as dopa decarboxylase and DDC) polynucleotides are provided that function alone or in combination with additional nucleic acid sequences to encode the AADC protein.
[00910] [00910] As used in this application an "AADC polynucleotide" is any nucleic acid polymer that encodes an AADC protein and, when present in a vector, plasmid or translatable construct, expresses such AADC protein in a cell, tissue, organ or organism .
[00911] [00911] —AADC polynucleotides include precursor molecules that are processed within the cell.
[00912] [00912] AADC polynucleotides or processed forms thereof may be encoded in a plasmid, vector, genome or other nucleic acid expression vector for delivery to a cell.
[00913] [00913] In some embodiments, the AADC polynucleotides are designed as components of AAV viral genomes and packaged into AAV particles that are processed in the cell to produce the wild-type AADC protein.
[00914] [00914] In some embodiments, the AADC polynucleotide may be and the payload of the AAV particle.
[00915] [00915] As used in this application, the wild-type AADC protein can be any of the naturally occurring isoforms or variants of the DDC gene.
[00916] [00916] Multiple alternatively spliced transcript variants encoding different isoforms of AADC have been identified.
[00917] [00917] Specifically, the DDC gene produces seven transcript variants that encode six distinct isoforms.
[00918] [00918] Both transcript variants of DDC 1 and 2 encode AADC isoform 1.
[00919] [00919] In some embodiments, the AADC polynucleotides encode DDC transcript variant 2, thus encoding an isoform 1 of a native AADC (NCBI Reference Sequence: NP 000781.1).
[00920] [00920] This sequence is shown below: MNASEFRRRGKEMVDYVANYMEGIEGRQVYPDVEPGYLRPLIPAAAPQEPD TFEDIINDVEKIIMPGVTHWHSPYFFAYFPTASSYPAMLADMLCGAIGCIGFS WAASPACTELETVMMDWLGKMLEPKAFLNEKAGEGGGVIQGSASEATLV ALLAARTKVIHRLQAASPELTQAAIMEKLVAYSSDQAHSSVERAGLIGGVKLK AIPSDGNFAMRASALQEALERDKAAGLIPFFMVATLGTTTCCSFDNLLEVGPI CNKEDIWLHVDAAYAGSAFICPEFRHLLNGVEFADSFNFNPHKWLLVNFDCS AMWVKKRTDLTGAFRLDPTYLKHSHQDSGLITDYRHWQIPLGRRFRSLKMW FVFRMYGVKGLQAYIRKHVQLSHEFESLVRQDPRFEICVEVILGLVCFRLKG
[00921] [00921] The AADC polynucleotides of the invention can be genetically engineered to contain modular elements and/or sequence motifs assembled to create AADC polynucleotide constructs.
[00922] [00922] In accordance with the present invention, AADC polynucleotides are provided.
[00923] [00923] Such polynucleotides comprise nucleic acid polymers that comprise a region of linked nucleosides encoding one or more isoforms or variants of the AADC protein.
[00924] [00924] In some embodiments, the AADC polynucleotide comprises a codon-optimized transcript encoding an AADC protein.
[00925] [00925] In some embodiments, the AADC polynucleotide comprises a region of sequence encoding one or more isoforms or wild-type variants of an AADC protein.
[00926] [00926] Such polynucleotides may also comprise a region of sequence encoding any one or more of the following: a 5' ITR, a cytomegalovirus (CMV) booster, a CMV promoter, a 1-law exon, a 1-law intron, a hbBglobin intron 2 , a 3' exon of hBglobin, a 5' UTR, a 3' UTR, a hGH poly(A) signal, and/or a 3' ITR.
[00927] [00927] Such sequence regions are taught in this application or may be any of those known in the prior art.
[00928] [00928] In some embodiments, the AADC polynucleotide comprises SEQ |D NO: 979 or a fragment or variant thereof.
[00929] [00929] In one embodiment, the AADC polynucleotide comprises a sequence that has percent identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00930] [00930] The AADC polynucleotide can have 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% , 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity with any of SEQ ID NO: 979 or a fragment or variant thereof.
[00931] [00931] The AADC polynucleotide can have 1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90%, 50-99%, 50 -100%, 60-70%, 60-80%, 60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99%, 70-100%, 80 -85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00932] [00932] As a non-limiting example, the AADC polynucleotide comprises a sequence that has 80% identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00933] [00933] As another non-limiting example, the AADC polynucleotide comprises a sequence that has 85% identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00934] [00934] "As another non-limiting example, the AADC polynucleotide comprises a sequence that has 90% identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00935] [00935] As another non-limiting example, the AADC polynucleotide comprises a sequence that has 95% identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00936] [00936] As another non-limiting example, the AADC polynucleotide comprises a sequence that has 99% identity to any of SEQ ID NO: 979 or a fragment or variant thereof.
[00937] [00937] In some embodiments, the coding region of the AADC polynucleotide is 1440 nucleotides in length.
[00938] [00938] Such an AADC polynucleotide may have codon-optimized all or part of the polynucleotide.
[00939] [00939] In some embodiments, the AADC polynucleotide comprises any one of SEQ ID NO: 979 or a fragment or variant thereof, but lacks the 5' and/or 3' ITRs.
[00940] [00940] Such a polynucleotide can be incorporated into a plasmid or vector and used to express the encoded AADC protein.
[00941] [00941] In one embodiment, the AADC polynucleotides can be produced in insect cells (eg, Sf9 cells).
[00942] [00942] In one embodiment, AADC polynucleotides can be produced using triple transfection.
[00943] [00943] In one embodiment, the AADC polynucleotide may comprise a codon-optimized open reading frame of an AADC mMRNA, at least one 5'I'TR and at least one 3'UTR where the one or more of the 5S' ITRs can be located at the 5' end of the promoter region and one or more of the 3' ITRs can be located at the 3' end of the poly(A) signal.
[00944] [00944] The AADC mRNA may comprise a promoter region, a 5' untranslated region (UTR), a 3'UTR and a poly(A) signal.
[00945] [00945] The promoter region may include, but are not limited to, a booster element, a promoter element, a first exon region, a first intron region, a second intron region and a second exon region.
[00946] [00946] “As a non-limiting example, the booster element and the promoter element are derived from CMV.
[00947] [00947] “As another non-limiting example, the first exon region is exon 1 law or fragments thereof, the first intron region is intron 1 law or fragments thereof, the second intron region is intron 2 of hbBglobin or fragments thereof and the second exon region is hbBglobin exon 3 or fragments thereof.
[00948] [00948] As yet another non-limiting example, the poly(A) signal is derived from human growth hormone.
[00949] [00949] In one embodiment, at least one element can be used with the AADC polynucleotides described in this application to improve transgene target specificity and expression (see, for example, Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the entirety of which is incorporated by reference).
[00950] [00950] “Non-limiting examples of elements to improve transgene target specificity and expression include promoters, endogenous mIRNAs, post-transcriptional regulatory elements (PREs), polyadenylation signal sequences (PolyA), and upstream enhancers (USEs) , "enhancers" and CMV introns.
[00951] [00951] In one embodiment, at least one element can be used with the AADC polynucleotides described in this application to improve transgene target specificity and expression (see, for example, Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the entirety of which is incorporated herein by reference) as well as promoters.
[00952] [00952] In one embodiment, the AADC polynucleotide is encoded in a plasmid or vector, which may be derived from an adeno-associated virus (AAV).
[00953] [00953] AAV may comprise a capsular serotype such as, but not limited to, PHP.
[00954] [00954] B,PHP.
[00955] [00955] A, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAVA, AAVA4-4, AAV5, AAV6, AAV6.
[00956] [00956] 1,AAVE.
[00957] [00957] 2,AAVE.
[00958] [00958] 1.
[00959] [00959] 2, AAV7, AAV7.
[00960] [00960] 2,AAV8B, AAV9, AAVO.
[00961] [00961] 11, AAVO9.
[00962] [00962] 13, AAVO.
[00963] [00963] 16, AAVO9.
[00964] [00964] 24, AAVO9.
[00965] [00965] 45, AAVO.
[00966] [00966] 47, AAVO.
[00967] [00967] 61, AVO9.
[00968] [00968] 68, AAVO.
[00969] [00969] 84, AAVO.
[00970] [00970] 9,AAVI1O, AAVI1, AAV12, AAVIG.
[00971] [00971] 3, AAV24.
[00972] [00972] 1,AAV27.
[00973] [00973] 3, AAVA42.
[00974] [00974] 12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAVA2-A, AAV42-5a, AAV42-5b, AAVA42-6b, AAV42-8, AAV42-10, AAVA42-11, AAV42 -12, AAV42-13, AAVA42-15, AAV42-aa, AAV43-1, AAVA3-12, AAVA43-20, AAV43-21, AAVA43-23, AAVA43-25, AAVA43-5, AAVA44.
[00975] [00975] 1,AAV44.
[00976] [00976] 2,AAV44.
[00977] [00977] 5,AAV223.
[00978] [00978] 1,AAV223.
[00979] [00979] 2,AAV223.
[00980] [00980] 4,AAV223.
[00981] [00981] 5,AAV223.
[00982] [00982] 6,AAV223.
[00983] [00983] 7, AAV1-7/rh.
[00984] [00984] 48, AAV1-8/rh.
[00985] [00985] 49, AAV2-15/rhr.
[00986] [00986] 62, AAV2-3/rh.
[00987] [00987] 61, AAV2-4/rh.
[00988] [00988] 50.AAV2-5/rhr.
[00989] [00989] 51, AAV3.
[00990] [00990] 1/hu.
[00991] [00991] 6,AAV3.
[00992] [00992] 1/hu.
[00993] [00993] 9,AAV3-9/rh.
[00994] [00994] 52, AAV3-11/rh.
[00995] [00995] 53, AAV4-8/r11.
[00996] [00996] 64, AAV4-9/rh.
[00997] [00997] 54, AAV4-19/rh.
[00998] [00998] 55, AAV5-3/rh.
[00999] [00999] 57, AAV5-22/rh.
[001000] [001000] 58, AAV7.
[001001] [001001] 3/hu.
[001002] [001002] 7,AAV6.
[001003] [001003] 8/hu.
[001004] [001004] 10, AAVIG.
[001005] [001005] 12/hu.
[001006] [001006] 11, AAV29.
[001007] [001007] 3/bb.
[001008] [001008] 1,AAV29.
[001009] [001009] 5/bb.
[001010] [001010] 2,AAV106.
[001011] [001011] 1/hu.
[001012] [001012] 37, AAV1I14.
[001013] [001013] 3/hu.
[001014] [001014] 40, AAV127.
[001015] [001015] 2/hu.
[001016] [001016] 41, AAV127.
[001017] [001017] 5/hu.
[001018] [001018] 42, AAV128.
[001019] [001019] 3/hu.
[001020] [001020] 44, AAV130.
[001021] [001021] 4/hu.
[001022] [001022] 48, AAV145.
[001023] [001023] 1/hu.
[001024] [001024] 53, AAV145.
[001025] [001025] 5/hu.
[001026] [001026] 54, AAV145.
[001027] [001027] G/hu.
[001028] [001028] 55, AAVIG61.
[001029] [001029] 10/hu.
[001030] [001030] 60, AAVIG61.
[001031] [001031] 6/hu.
[001032] [001032] 61, AAV33.
[001033] [001033] 12/hu.
[001034] [001034] 17, AAV33.
[001035] [001035] 4/hu.
[001036] [001036] 15, AAV33.
[001037] [001037] 8/hu.
[001038] [001038] 16, AAV52/hu.
[001039] [001039] 19, AAV52.
[001040] [001040] 1/hu.
[001041] [001041] 20, AVS58.
[001042] [001042] 2/hu.
[001043] [001043] 25, AAVA3.
[001044] [001044] 3, AAVA3.
[001045] [001045] 4,AAVA3.
[001046] [001046] 5, AAVA3.
[001047] [001047] 7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVTh.
[001048] [001048] 72, AAVhu.
[001049] [001049] 8, AAVrh.
[001050] [001050] 68, AAVrh.
[001051] [001051] 70, AAVPpi.
[001052] [001052] 1, AAVpi.
[001053] [001053] 3, AAVpi.
[001054] [001054] 2, AAVrh.
[001055] [001055] 60, AAVrh.
[001056] [001056] 44, AAVrh.
[001057] [001057] 65, AAVrh.
[001058] [001058] 55, AAVrh.
[001059] [001059] 47, AAVrh.
[001060] [001060] 69, AAVrh.
[001061] [001061] 45, AAVrh.
[001062] [001062] 59, AAVhu.
[001063] [001063] 12, AAVH6, AAVLKO3, AAVH-1/hu.
[001064] [001064] 1, AAVH-5/hu.
[001065] [001065] 3, AAVLG-10/rh.
[001066] [001066] 40, AAVLG-4/rh.
[001067] [001067] 38, AAVLG-9/hu.
[001068] [001068] 39, AAVN721-8/rh.
[001069] [001069] 43, AAVCh.
[001070] [001070] 5,AAVCh.
[001071] [001071] 5R1, AAVcy.
[001072] [001072] 2,AAVcy.
[001073] [001073] 3, AAVcy.
[001074] [001074] 4,AVCy.
[001075] [001075] 5,AVCy.
[001076] [001076] 5R1,AVCy.
[001077] [001077] 5R2, AAVCYy.
[001078] [001078] 5R3, AAVCYy.
[001079] [001079] 5R4, AAVCy.
[001080] [001080] 6, AAVhu.
[001081] [001081] 1, AAVhu.
[001082] [001082] 2, AAVhu.
[001083] [001083] 3, AAVhu.
[001084] [001084] 4, AAVhu.
[001085] [001085] 5,AAVhu.
[001086] [001086] 6, AAVhu.
[001087] [001087] 7, AAVhu.
[001088] [001088] 9,AAVhu.
[001089] [001089] 10, AAVhu.
[001090] [001090] 11, AAVhu.
[001091] [001091] 13, AAVhu.
[001092] [001092] 15, AAVhu.
[001093] [001093] 16, AAVhu.
[001094] [001094] 17, AAVhu.
[001095] [001095] 18, AAVhu.
[001096] [001096] 20, AAVhu.
[001097] [001097] 21, AAVhu.
[001098] [001098] 22, AAVhu.
[001099] [001099] 23.
[001100] [001100] 2, AAVhu.
[001101] [001101] 24, AAVhu.
[001102] [001102] 25, AAVhu.
[001103] [001103] 27, AAVhu.
[001104] [001104] 28, AAVhu.
[001105] [001105] 29, AAVhu.
[001106] [001106] 29R, AAVhu.
[001107] [001107] 31, AAVhu.
[001108] [001108] 32, AAVhu.
[001109] [001109] 34, AAVhu.
[001110] [001110] 35, AAVhu.
[001111] [001111] 37, AAVhu.
[001112] [001112] 39, AAVhu.
[001113] [001113] 40, AAVhu.
[001114] [001114] 41, AAVhu.
[001115] [001115] 42, AAVhu.
[001116] [001116] 43, AAVhu.
[001117] [001117] 44, AAVhu.
[001118] [001118] 44R1, AAVhu.
[001119] [001119] 44R2, AAVhu.
[001120] [001120] 44R3, AAVhu.
[001121] [001121] 45, AAVhu.
[001122] [001122] 46, AAVhu.
[001123] [001123] 47, AAVhu.
[001124] [001124] 48, AAVhu.
[001125] [001125] 48R1, AAVhu.
[001126] [001126] 48R2, AAVhu.
[001127] [001127] 48R3, AAVhu.
[001128] [001128] 49, AAVhu.
[001129] [001129] 51, AAVhu.
[001130] [001130] 52, AAVhu.
[001131] [001131] 54, AAVhu.
[001132] [001132] 55, AAVhu.
[001133] [001133] 56, AAVhu.
[001134] [001134] 57, AAVhu.
[001135] [001135] 58, AAVhu.
[001136] [001136] 60, AAVhu.
[001137] [001137] 61, AAVhu.
[001138] [001138] 63, AAVhu.
[001139] [001139] 64, AAVhu.
[001140] [001140] 66, AAVhu.
[001141] [001141] 67, AAVhu.
[001142] [001142] 9/14, AAVhu.
[001143] [001143] t19, AAVrh.
[001144] [001144] 2, AAVrh.
[001145] [001145] 2R, AAVrh.
[001146] [001146] 8, AAVrh.
[001147] [001147] 8R, AAVrh.
[001148] [001148] 10, AAVrh.
[001149] [001149] 12, AAVrh.
[001150] [001150] 13, AAVrh.
[001151] [001151] 13R, AAVrIh.
[001152] [001152] 14, AAVrh.
[001153] [001153] 17, AAVrh.
[001154] [001154] 18, AAVrh.
[001155] [001155] 19, AAVrh.
[001156] [001156] 20, AAVrh.
[001157] [001157] 21, AAVrh.
[001158] [001158] 22, AAVrh.
[001159] [001159] 23, AAVrh.
[001160] [001160] 24, AAVrh.
[001161] [001161] 25, AAVrh.
[001162] [001162] 31, AAVrh.
[001163] [001163] 32, AAVrh.
[001164] [001164] 33, AAVrh.
[001165] [001165] 34, AAVrh.
[001166] [001166] 35, AAVrh.
[001167] [001167] 36, AAVrh.
[001168] [001168] 37, AAVrh.
[001169] [001169] 37R2, AAVrh.
[001170] [001170] 38, AAVrh.
[001171] [001171] 39, AAVrh.
[001172] [001172] 40, AAVrh.
[001173] [001173] 46, AAVrh.
[001174] [001174] 48, AAVrh.
[001175] [001175] 48.
[001176] [001176] 1, AAVrh.
[001177] [001177] 48.
[001178] [001178] 1.
[001179] [001179] 2, AAVrh.
[001180] [001180] 48.
[001181] [001181] 2, AAVrh.
[001182] [001182] 49, AAVrh.
[001183] [001183] 51, AAVrh.
[001184] [001184] 52, AAVrh.
[001185] [001185] 53, AAVrh.
[001186] [001186] 54, AAVrh.
[001187] [001187] 56, AAVrh.
[001188] [001188] 57, AAVrh.
[001189] [001189] 58, AAVrh.
[001190] [001190] 61, AAVrh.
[001191] [001191] 64, AAVrh.
[001192] [001192] 64R1, AAVrh.
[001193] [001193] 64R2, AAVrh.
[001194] [001194] 67, AAVrh.
[001195] [001195] 73, AAVrh.
[001196] [001196] 74, AAVTrhaR, AAVrh8R mutant ASSGR, AAVrhaR mutant R533A, AMAV, BAAV, goat AAV, bovine AAV, AAVhE1.
[001197] [001197] 1, AAVRErI.
[001198] [001198] 5, AAVhER1.
[001199] [001199] 14, AAVREr1.
[001200] [001200] 8, AAVRErI.
[001201] [001201] 16, AAVRhEr1.
[001202] [001202] 18, AAVhEr1.
[001203] [001203] 35, AAVRhEr1.
[001204] [001204] 7, AAVRErI.
[001205] [001205] 36, AAVhEr2.
[001206] [001206] 29, AAVRhEr2.
[001207] [001207] 4, AAVREr2.
[001208] [001208] 16, AAVhEr2.
[001209] [001209] 30, AAVhEr2.
[001210] [001210] 31, AAVhEr2.
[001211] [001211] 36, AAVRER1.
[001212] [001212] 23, AAVhEr3.
[001213] [001213] 1,AAV2.
[001214] [001214] 5T AAV-PAEC, AAV-LKO1, AAV-LKO2, AAV-LKO3, AAV-LKO4, AAV-LKOS, AAV-LKO6, AAV-LKO7, AAV-LKO8, AAV-LKO9, AAV-LK10, AAV- LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAECA, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101 AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 AAV Shuffle 100-1 AAV Shuffle 100 -3, AAV Shuffle 100-
[001215] [001215] 50, AAVrh.
[001216] [001216] 43, AAVrh.
[001217] [001217] 62, AAVrh.
[001218] [001218] 48, AAVhu.
[001219] [001219] 19, AAVhu.
[001220] [001220] 11, AAVhu.
[001221] [001221] 53, AAV4-8/rh.
[001222] [001222] 64, AAVLG-9/hu.
[001223] [001223] 39, AAV5A4.
[001224] [001224] 5/hu.
[001225] [001225] 23, AAVB5A4.
[001226] [001226] 2/hu.
[001227] [001227] 22, AAVB54.
[001228] [001228] 7/hu.
[001229] [001229] 24,AAV54.
[001230] [001230] 1/hu.
[001231] [001231] 21, AAVB54.
[001232] [001232] 4R/hu.
[001233] [001233] 27, AAVA46.
[001234] [001234] 2/hu.
[001235] [001235] 28, AAV46.
[001236] [001236] 6/hu.
[001237] [001237] 29, AAV128.
[001238] [001238] 1/hu.
[001239] [001239] 43, True type AAV (ttAAV), UPENN AAV 10, Japanese AAV serotypes 10, AAV CBr-7.
[001240] [001240] 1, AAV CBr-7.
[001241] [001241] 10, AAV CBr-7.
[001242] [001242] 2, AAV CBr-7.
[001243] [001243] 3, AAV CBr-7.
[001244] [001244] 4, AAV CBr-7.
[001245] [001245] 5, AAV CBr-7.
[001246] [001246] 7, AAV CBr-7.
[001247] [001247] 8, AAV CBr-B7.
[001248] [001248] 3, AAV CBr-B7.
[001249] [001249] 4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.
[001250] [001250] 1, AAV CHt-6.
[001251] [001251] 10, AAV CHt-6.
[001252] [001252] 5, AAV CHt-6.
[001253] [001253] 6, AAV CHt-6.
[001254] [001254] 7, AAV CHt-6.
[001255] [001255] 8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV. CHt-P6, AAV CHtP8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd- 8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd- H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg- F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv- 12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clvi-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV
[001256] [001256] 10, AAV CSp-8.
[001257] [001257] 2,AAV CSp-8.
[001258] [001258] 4,AAV CSp-8.
[001259] [001259] 5,AAV CSp-8.
[001260] [001260] 6, AAV CSp-8.
[001261] [001261] 7, AAV CSp-8.
[001262] [001262] 8, AAV CSp-8.
[001263] [001263] 9, AAV CSp-9, AAV.
[001264] [001264] hu.
[001265] [001265] 48R3, AAV.
[001266] [001266] VR-355, AAV3B, AAV4, AAV5, AAVFI/HSCI1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVFI6/HSC16, AAVF1I7/HSC17, AAVF2/HSC 2 , AAVF3/HSC3, — AAVF4/HSC4, — AAVES/HSC5, — AAVF6/HSCG6, AAVEF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, PHP.
[001267] [001267] B(AAV-PHP.
[001268] [001268] B), PHP.
[001269] [001269] A(AAV.
[001270] [001270] PHP.
[001271] [001271] A), G2B-26, G2B-13, TH1.
[001272] [001272] 1-32, TH1.
[001273] [001273] 1-35, AAVHP.
[001274] [001274] B2, AAVHP.
[001275] [001275] B3, AAVHP.
[001276] [001276] N/PHP.
[001277] [001277] B-DGT, AAVHP.
[001278] [001278] B-EST, AAVHP.
[001279] [001279] B-GGT, AAVPHP.
[001280] [001280] B-ATP, AAVPHP.
[001281] [001281] B-ATT-T, AAVPHP.
[001282] [001282] B-DGT-T, AAVPHP.
[001283] [001283] B-GGT-T, AAVPHP.
[001284] [001284] B-SGS, AAVPHP.
[001285] [001285] B-AQP, AAVPHP.
[001286] [001286] B-QQP, AAVPHP.
[001287] [001287] B-SNP(3), AAVHP.
[001288] [001288] B-SNP, AAVHP.
[001289] [001289] B-QGT, AAVHP.
[001290] [001290] B-NQT, AAVPHP.
[001291] [001291] B-EGS, AAVHP.
[001292] [001292] B-SGN, AAVHP.
[001293] [001293] B-EGT, AAVHP.
[001294] [001294] B-DST, AAVHP.
[001295] [001295] B-DST, AAVPHP.
[001296] [001296] B-STP, AAVHP.
[001297] [001297] B-PQP, AAVPHP.
[001298] [001298] B-SQP, AAVHP.
[001299] [001299] B-QLP, AAVHP.
[001300] [001300] B-TMP, AAVPHP.
[001301] [001301] B-TTP, AAVHP.
[001302] [001302] S/G2A12, AAVG2A15/G2A3, AAVG2B4, and/or AAVG2B5, and variants thereof.
[001303] [001303] According to the present invention the AAV particles can be prepared as pharmaceutical compositions.
[001304] [001304] It will be understood that such compositions necessarily comprise one or more active ingredients and, more often, a pharmaceutically acceptable excipient.
[001305] [001305] The relative amounts of the active ingredient (e.g. AAV particle), a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition according to the present invention may vary depending on the identity, size, and/or the condition of the subject being treated and further depending on the route by which the composition is administered.
[001306] [001306] For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.
[001307] [001307] By way of example, the composition may comprise between 0.1% and 100%, for example, between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (p /p) of active ingredient.
[001308] [001308] In some embodiments, the AAV particle compositions described in this application may comprise at least one payload.
[001309] [001309] As a non-limiting example, pharmaceutical compositions may contain an AAV particle with 1,2,3,4 or 5 payloads.
[001310] [001310] While the descriptions of pharmaceutical compositions presented in this application are primarily directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other animal, for example, to non-human animals, for example, non-human mammals.
[001311] [001311] Modification of pharmaceutical compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals is well known, and the experienced veterinary pharmacologist can design and/or effect such modification by simple routine experimentation, if there is.
[001312] [001312] Subjects to whom administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, dogs, cats, dogs, mice, rats, birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.
[001313] [001313] In some embodiments, the compositions are administered to humans, patients, or human subjects. Formulations.
[001314] [001314] The formulations of the present invention may include, but are not limited to, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with AAV particles (e.g., for transfer or transplantation into an individual) and combinations of the same.
[001315] [001315] Formulations of the pharmaceutical compositions described in this application can be prepared by any method known or developed in the future in the field of pharmacology.
[001316] [001316] As used in this application the term "compositions" refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.
[001317] [001317] In general, such methods of preparation include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.
[001318] [001318] As used in this application, the term "active ingredient" generally refers to an AAV particle carrying a payload region encoding the polypeptides of the invention or to the end product encoded by a viral genome of an AAV particle as described in this order.
[001319] [001319] The AAV particle formulations and pharmaceutical compositions described in this application can be prepared by any method known or developed in the future in the field of pharmacology.
[001320] [001320] In general, such preparation methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product in a desired single-dose or multi-dose unit.
[001321] [001321] A pharmaceutical composition according to the present invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of individual unit doses.
[001322] [001322] As used in this application, a "unit dose" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
[001323] [001323] The amount of active ingredient is generally equal to the dosage of active ingredient that should be administered to an individual and/or a convenient fraction of such dosage such as, for example, half or one-third of such dosage.
[001324] [001324] In one embodiment, the AAV particles of the invention may be formulated in PBS with 0.001% pluronic acid (F-68) at a pH of about 7.0.
[001325] [001325] In some embodiments, the AAV formulations described in this application may contain sufficient AAV particles for expression of at least one expressed functional payload.
[001326] [001326] As a non-limiting example, AAV particles may contain viral genomes encoding 1, 2, 3, 4 or 5 functional payloads.
[001327] [001327] In accordance with the present invention AAV particles can be formulated for delivery to the CNS.
[001328] [001328] Agents that cross the blood-brain barrier may be used.
[001329] [001329] For example, some peptides that penetrate cells that can target molecules to the blood-brain barrier endothelium can be used for formulation (e.g. Mathupala, Expert Opin Ther Pat.
[001330] [001330], 2009, 19, 137-140; the entirety of which is incorporated herein by way of reference). Excipients and Diluents.
[001331] [001331] The AAV particles of the invention can be formulated using one or more excipients or diluents to (1) enhance stability; (2) enhance cell transfection or transduction; (3) allow systematic or delayed release of the payload; (4) alter biodistribution (eg, targeting the viral particle to specific tissues or cell types); (5) increase translation of the encoded protein; (6) alter the release profile of the encoded protein and/or (7) allow regulatable expression of the payload of the invention.
[001332] [001332] In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
[001333] [001333] In some embodiments, an excipient is approved for use in humans and for veterinary use.
[001334] [001334] In some embodiments, an excipient must be approved by the United States Food and Drug Administration.
[001335] [001335] In some embodiments, an excipient must be pharmaceutical grade.
[001336] [001336] In some embodiments, an excipient must meet North American Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and/or International Pharmacopeia standards.
[001337] [001337] Excipients, as used in this application, include, but are not limited to, any and all solvents, dispersion mediums, diluents, or other liquid carriers, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives , inter alia, as appropriate for the particular dosage form desired.
[001338] [001338] Various excipients for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the literature (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; whose entirety is incorporated herein by reference).
[001339] [001339] The use of a conventional excipient medium may be contemplated within the scope of the present invention, except to the extent that any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing an undesirable biological effect or otherwise interacting harmfully with any of the other components of the pharmaceutical composition.
[001340] [001340] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol , sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, etc., and/or combinations thereof.
[001341] [001341] In one embodiment, the AAV particles may be formulated into a hydrogel prior to adm.
[001342] [001342] Hydrogels possess a degree of flexibility that is similar to natural tissue as a result of their significant water content.
[001343] [001343] In another embodiment, a hydrogel can be administered to a subject prior to administration of an AAV particle formulation.
[001344] [001344] As a non-limiting example, the hydrogel delivery site may be at 7.62 cm (3 inches) (e.g., at 7.37, 7.11, 6.86, 6.6, 6.35 , 6.1, 5.84, 5.59, 5.33, 5.08, 4.83, 4.57, 4.32, 4.06, 3.81, 3.56, 3.30, 3 .05, 2.79, 2.54, 2.29, 2.03, 1.78, 1.52, 1.27, 1.02, 076, 0.51, 0.25 cm or less than 0, 25 cm [2.9, 2.8, 2.7, 2.6, 2.5, 24, 2.3, 2.2,, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0, 5, 0.4, 0.3, 0.2, 0.1 or less than 0.1 inch]) from the site of administration of the AAV particle formulation. Inactive Ingredients.
[001345] [001345] In some embodiments, the AAV particle formulations may comprise at least one inactive ingredient.
[001346] [001346] As used in this application, the term "inactive ingredient" refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in the formulations.
[001347] [001347] In some embodiments, all, none, or some of the inactive ingredients that can be used in the formulations of the present invention must be approved by the US Food and Drug Administration (FDA).
[001348] [001348] In one embodiment, the AAV particle pharmaceutical compositions comprise at least one inactive ingredient such as, but not limited to, 1,2,6-Hexanetriol; 1,2-Dimyristoyl-Sn-Glycero-3-(Phospho-S-(1-Glycerol)); 1,2-Dimyristoyl-Sn-Glycero-3-Phosphocholine; 1,2-Dioleoyl-Sn-Glycer-3-Phosphocholine; 1,2-Dipalmitoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); 1,2-Distearoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); 1,2-Distearoyl-Sn-Glycero-3-Phosphocholine; 1-O-Tolylbiguanide; 2-Ethyl-1,6-Hexanediol; Acetic Acid; glacial acetic acid; acetic anhydride; acetone; sodium acetone bisulfite; acetylated lanolin alcohols; acetylated monoglycerides; Acetylcysteine; Acetyltryptophan DL-; acrylate copolymer; acrylic acid-iso-octyl acrylate copolymer; acrylic adhesive 788; activated charcoal; Add 72A103; Scotch tape; adipic acid; Aerotex 3730 resin; Alanine; Aggregated albumin; Colloidal Albumin; Human Albumin; alcohol; dehydrated alcohol; denatured alcohol; diluted alcohol; Alfadex; alginic acid; alkyl ammonium sulfonic acid betaine; sodium alkyl aryl sulfonate; allantoin; Ali Alpha.
[001349] [001349] -lonone; almond oil; Alpha-Terpineol; Alpha-Tocopherol; Alpha-Tocopheryl Acetate, DI-; Alpha-Tocopherol, DI-; aluminum acetate; aluminum chlorohydroxy allantoinate; aluminum hydroxide; aluminum hydroxide - hydrated sucrose; aluminum hydroxide gel; aluminum hydroxide gel F 500; aluminum hydroxide gel F 5000; aluminum monostearate; aluminum oxide; aluminum polyester; aluminum silicate; aluminum starch octenylsuccinate; aluminum stearate; aluminum subacetate; anhydrous aluminum sulfate; Amerchol C; Amerchol-Cab; Aminomethylpropanol; ammonia; ammonia solution; strong ammonia solution; Ammonium acetate; Ammonium hydroxide; ammonium lauryl sulfate; nonoxynol-4 ammonium sulfate; C-12-C-15 linear primary alcohol ammonium salt ethoxylated; ammonium sulfate; Ammonix; Amphoteric-2; Amphoteric-9; Anethole; anhydrous citric acid; anhydrous dextrose; anhydrous lactose; anhydrous trisodium citrate; anise oil; Sbn anoxide; defoamer; Antipyrine; Apaflurane; apricot kernel oil Peg-6 esters; Aquaphor; Arginine; Arlacel; Ascorbic acid; ascorbyl palmitate; asparic acid; Balsam Peru; barium sulphate; beeswax; synthetic beeswax; Beheneth-10; bentonite; benzalkonium chloride; benzenesulfonic acid; benzethonium chloride; benzododecinium bromide; benzoic acid; benzilic alcohol; benzyl benzoate; benzyl chloride; Betadex; Bibapcitide; bismuth subgallate; Boric acid; Brocrinat; butane; butyl alcohol; vinyl methyl ether/anhydride-maleic butyl ester copolymer (125,000 Mw); butyl stearate; butylated hydroxyanisole; butylated hydroxytoluene; butylene glycol; butylparaben; butyric acid; C20-40 Pareth-24; caffeine; calcium; calcium carbonate; calcium chloride; calcium gluceptate; calcium hydroxide; calcium lactate; Chalcobutrol; Sodium Chaldiamide; trisodium caloxetate; Calteridol calcium; Canadian balm; caprylic/capric triglyceride; triglyceride — caprylic/capric/stearic; Capture; captisol; Caramel; Carbomer 1342; Carbomer 1382; Carbomer 934; Carbomer 934p; Carbomer 940; Carbomer 941; Carbomer 980; Carbomer 981; Type B Carbomer Homopolymer (Cross-linked Allyl Pentaerythritol); Type C Carbomer Homopolymer (Cross-linked Alyl Pentaerythritol); carbon dioxide; vinyl carboxy copolymer; carboxymethylcellulose; Sodium carboxymethyl cellulose; Carboxypolymethylene; carrageenan; carrageenan salt; Castor oil; cedar leaf oil; cellulose; microcrystalline cellulose; Cerasint-Se; Ceresin; Ceteareth-12; —“ Ceteareth-15; Ceteareth-30; alcohol! cetearyl/Ceteareth-20; cetearia ethyl hexanoate; Ceteth-10; Ceteth-2; Ceteth-20; Ceteth-23; cetostearyl alcohol; cetrimonium chloride; cetyl alcohol; cetyl esters wax; cetyl palmitate; cetylpyridinium chloride; Chlorobutanol; Chlorobutanol hemihydrate;
[001350] [001350] Beta. -Cyclodextrin; sulfur dioxide; sulfuric acid; sulfurous acid; Surfactol Qs; Tagatose, D-; Baby powder; tallow oil; tallow glycerides; Tartaric acid; tartaric acid, DI-; tenox; Tenox-2; tert-butyl alcohol; tert-butyl tert-butylhydroquinone hydroperoxide; copper (I) tetrakis(2-methoxyisobutylisocyanide) tetrafluorobutate; tetrapropyl orthosilicate; Tetrophosmin; Theophylline; Thimerosal; Threonine; Thimol; tin; titanium dioxide; Tocopherol; Tocofersolan; total parenteral nutrition, lipid emulsion; Triacetin; Tricaprylin; Trichloromonofluoromethane; Trideceth-10; Triethanolamine Lauryl Sulfate; trifluoroacetic acid; medium chain triglycerides; trihydroxystearine; Trilaneth-4 phosphate; Trilaureth-4 phosphate; trisodium citrate dihydrate; Trisodium hedta; Triton 720; Triton X-200; Trolamine; Tromantadine; Tromethamine (TRIS); Tryptophan; Tyloxapol; Tyrosine; undecylenic acid; Union 76 Amsco-Res 6038; Urea; valine; vegetable oil; hydrogenated vegetable oil glyceride; hydrogenated vegetable oil; Versetamide; Viscarine; of Viscose/Cotton; Vitamin E;
[001351] [001351] The pharmaceutical composition formulations of AAV particles disclosed in this application may include cations or anions.
[001352] [001352] In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mn2+, Mg+ and combinations thereof.
[001353] [001353] As a non-limiting example, the formulations may include polymers and complexes with a metal cation (see, for example, US Patent Nos. 6,265,389 and 6,555,525, the entirety of each of which is incorporated herein by reference ).
[001354] [001354] The formulations of the invention may also include one or more pharmaceutically acceptable salts.
[001355] [001355] As used in this application, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds where the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reaction of the base group free with a suitable organic acid).
[001356] [001356] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acid residues tacs and carboxylic acids; between others.
[001357] [001357] Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate,
[001358] [001358] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, among others, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium , methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, among others.
[001359] [001359] Pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
[001360] [001360] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods.
[001361] [001361] Generally, such salts can be prepared by reacting the acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
[001362] [001362] Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed, Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19
[001363] [001363] The term "pharmaceutically acceptable solvate", as used in this application, means a compound of the invention in which molecules of a suitable solvent are incorporated into the crystal lattice.
[001364] [001364] A suitable solvent is physiologically tolerable at the dosage administered.
[001365] [001365] Solvates can be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
[001366] [001366] Examples of suitable solvents are ethanol, water (e.g., mono-, di-, and trihydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N-Ni-dimethylformamide (DMF), N, N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone — (DMPU) , — acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, among others.
[001367] [001367] When the solvent is water, the solvate is called a "hydrate". ADMINISTRATION AND DOSAGE. Administration.
[001368] [001368] In one embodiment, the AAV particle may be administered to a subject (e.g., to an individual's CNS) in a therapeutically effective amount to reduce symptoms of central nervous system disease (e.g., Parkinson's disease) of an individual (e.g. determined by a known assessment method).
[001369] [001369] The AAV particles of the present invention may be administered by any route of delivery that results in a therapeutically effective outcome.
[001370] [001370] These include, but are not limited to, enteral (in the gut), gastroenteral, epidural (in the dura mater), oral (by mouth), transdermal, intracerebral (in the brain), intracerebroventricular (in the ventricles — brain), epicutaneous ( application to the skin), intradermal (to the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular ( into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal cord), intraparenchymal (into brain tissue), intraperitoneal (infusion or injection into the peritoneum), intravesical infusion, intravitreal (through the eye) , intracavernous injection (into a pathological cavity), intracavitary (at the base of the penis), intravaginal, intrauterine, additionally extra-amniotic, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through a mem mucosal membrane), transvaginal, insufflation (swallow), sublingual, sublabial, enema, eye drops (over the conjunctiva), or otic drops, auricular (into the ear or through the ear), buccal (directed to the cheek), conjunctival, cutaneous, dental (on a tooth or teeth), electroosmosis, endocervical, endosinusal, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (in a cartilage), intracaudal (in the cauda equina), intracisternal (in the cisterna magna cerebellomedularis), intracorneal (in the cornea), intracoronal dental, intracoronary (in the coronary arteries), intracorporus cavernosum (in the dilatable spaces of the corporus cavernosa of the penis), intradiscal (in a disc), intraductal (in a duct of a gland), intraduodenal (in the duodenum), intradural (in the dura or below), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (in the stomach), in tragingival (in the gums), intrailiac (in the portion of the small intestine), intralesional (in a localized lesion or introduced directly into it), intraluminal (in a lumen of a tube), intralymphatic (in the lymph), intramedullary (in the medullary cavity of a bone), intrameningeal (in the meninges), intramyocardial (in the myocardium), intraocular (in the eye), intraovarian (in the ovary), intrapericardial (in the pericardium), intrapleural (in the pleura), intraprostatic (in the prostate), intrapulmonary (in the lungs or its bronchi), intrasinusal (in the sinuses or periorbital), intraspinal (in the spine), intrasynovial (in the synovial cavity of a joint), intratendinous (in a tendon), intratesticular (in the testis), intrathecal (in the cerebrospinal fluid in any level of the cerebrospinal axis), intrathoracic (in the chest), intratubular (in the tubules of an organ), intratumoral (in a tumor), intratympanic (in the aurus media), intravascular (in a vessel or vessels), intraventricular (in a ventricle ), iontophoresis (by means of electric current ions of soluble salts migrate to the tissues of the body), irrigation (washing or spraying open wounds or body cavities), laryngeal (directly into the larynx), nastrogastric (through the nose and into the stomach), occlusive dressing technique (topical administration which is then covered with a dressing that occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, epidural, perineural, periodontal, rectal , respiratory (in the respiratory tract by oral or nasal inhalation for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (through or through the tympanic cavity), ureteral (into the ureter), urethral (into the urethra), vaginal, caudal block, diagnostics, nerve block, bile perfusion ary, cardiac perfusion, photophoresis and spinal.
[001371] [001371] In some embodiments, compositions may be administered in a manner that allows them to cross the blood brain barrier, vascular barrier, or other epithelial barrier.
[001372] [001372] The AAV particles of the present invention may be administered in any suitable form, whether as a liquid solution or suspension, or as a solid form suitable for liquid solution or suspension in a liquid solution.
[001373] [001373] The AAV particles may be formulated with any suitable and pharmaceutically acceptable excipient.
[001374] [001374] In one embodiment, the AAV particles of the present invention can be delivered to a subject by a single route of administration.
[001375] [001375] In one embodiment, the AAV particles of the present invention can be delivered to a subject via a multi-site route of administration.
[001376] [001376] An individual may receive administration at 2, 3, 4, 5 or more than 5 sites.
[001377] [001377] In one embodiment, a subject may receive administration of the AAV particles of the present invention by bolus infusion.
[001378] [001378] In one embodiment, a subject may receive administration of the AAV particles of the present invention by systematic delivery over a period of minutes, hours, or days.
[001379] [001379] The infusion rate can be changed depending on the individual, distribution, formulation or other distribution parameter.
[001380] [001380] In one embodiment, the AAV particles of the present invention may be delivered via intramuscular delivery.
[001381] [001381] (See, for example, US Patent No. 6,506,379; the entirety of which is incorporated herein by reference).
[001382] [001382] Non-limiting examples of intramuscular administration include an intravenous injection or a subcutaneous injection.
[001383] [001383] In one embodiment, the AAV particles of the present invention may be delivered by oral administration.
[001384] [001384] Non-limiting examples of oral administration include an administration in the digestive tract and a buccal administration.
[001385] [001385] In one embodiment, the AAV particles of the present invention may be delivered via the intraocular delivery route.
[001386] [001386] A non-limiting example of intraocular administration includes an intravitreal administration.
[001387] [001387] In one embodiment, the AAV particles of the present invention may be delivered via the intranasal delivery route.
[001388] [001388] Non-limiting examples of intranasal delivery include the administration of nasal drops or nasal sprays.
[001389] [001389] In some embodiments, the AAV particles that can be administered to a subject by peripheral injections.
[001390] [001390] Non-limiting examples of peripheral injections include intraperitoneal, intramuscular, intravenous, conjunctival or articular injection.
[001391] [001391] It has already been described in the art that the peripheral administration of AAV particles can be transported to the central nervous system, e.g., to motor neurons (e.g., US Patent Publication Nos. 20100240739; and 20100130594; the entirety of each one of which is incorporated herein by reference).
[001392] [001392] In one embodiment, the AAV particles may be delivered by injection into the CSF pathway.
[001393] [001393] Non-limiting examples of delivery to the CSF pathway include intrathecal and intracerebroventricular administration.
[001394] [001394] In one embodiment, the AAV particles may be distributed by systemic distribution.
[001395] [001395] As a non-limiting example, systemic distribution may be by intravascular administration.
[001396] [001396] In one embodiment, the AAV particles of the present invention may be administered to a subject by intracranial delivery (See, for example, US Patent No. 8,119,611; the entirety of which is incorporated herein by reference).
[001397] [001397] In some embodiments, the AAV particles of the present invention may be administered by injection.
[001398] [001398] As a non-limiting example, the AAV particles of the present invention can be administered to a subject by injection.
[001399] [001399] In some embodiments, the AAV particles of the present invention may be administered by muscle injection.
[001400] [001400] As a non-limiting example, the AAV particles of the present invention can be administered to a subject by muscular administration.
[001401] [001401] In some embodiments, the AAV particles of the present invention may be administered by intramuscular administration.
[001402] [001402] As a non-limiting example, the AAV particles of the present invention can be administered to a subject by intramuscular administration.
[001403] [001403] In one embodiment, the AAV particles of the present invention are administered to a subject and transduce the muscle of a subject.
[001404] [001404] As a non-limiting example, AAV particles are administered by intramuscular administration.
[001405] [001405] In some embodiments, the AAV particles of the present invention may be administered via intraparenchymal injection.
[001406] [001406] As a non-limiting example, the AAV particles of the present invention can be administered to a subject by intraparenchymal administration.
[001407] [001407] In some embodiments, the AAV particles of the present invention may be administered by intravenous administration.
[001408] [001408] As a non-limiting example, the AAV particles of the present invention can be administered to a subject by intravenous administration.
[001409] [001409] In one embodiment, the AAV particles of the present invention may be administered via intravenous delivery.
[001410] [001410] In one embodiment, the AAV particles of the present invention may be administered via a single-dose intravenous delivery.
[001411] [001411] As a non-limiting example, single-dose intravenous delivery may be a one-time treatment.
[001412] [001412] In the context of central nervous system disorders (eg, Parkinson's disease), single-dose intravenous delivery may produce lasting relief for individuals with central nervous system disorders (eg, Parkinson's disease) and/or symptoms related.
[001413] [001413] Relief may last for minutes such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 minutes or more than 59 minutes; hours such as, but not limited to, 1, 2, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
[001414] [001414] In one embodiment, the AAV particles of the present invention may be administered via intravenous delivery to the DRG nociceptive neurons.
[001415] [001415] In one embodiment, the AAV particles of the present invention may be administered via a single-dose intravenous delivery to the DRG nociceptive neurons.
[001416] [001416] As a non-limiting example, single-dose intravenous delivery may be a one-time treatment.
[001417] [001417] In the context of central nervous system disorders (eg, Parkinson's disease), single-dose intravenous delivery may produce lasting relief for individuals with central nervous system disorders (eg, Parkinson's disease) and/or symptoms related.
[001418] [001418] Relief may last for minutes such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 minutes or more than 59 minutes; hours such as, but not limited to, 1, 2, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more than 48 hours; days such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or more than 31 days; weeks such as, but not limited to, 1, 2,3,4,5, 6,7,8,9,10, 11,12, 13, 14, 15, 16, or more than 16 weeks; months such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 months; years such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 years.
[001419] [001419] In some embodiments, the AAV particles of the present invention may be administered by intrathecal injection.
[001420] [001420] As a non-limiting example, the AAV particles of the present invention can be administered by intrathecal injection.
[001421] [001421] In one embodiment, the AAV particle may be administered to the cisterna magna in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
[001422] [001422] As a non-limiting example, the AAV particle can be administered intrathecally.
[001423] [001423] In one embodiment, the AAV particle may be administered using intrathecal infusion and in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
[001424] [001424] In some embodiments, the AAV particles of the present invention may be administered via a single-dose intrathecal injection.
[001425] [001425] As a non-limiting example, single-dose intrathecal injection may be a one-time treatment.
[001426] [001426] In the context of central nervous system disorders (eg, Parkinson's disease), single-dose intrathecal injection may produce lasting relief for individuals with central nervous system disorders (eg, Parkinson's disease) and/or symptoms related.
[001427] [001427] Relief may last for minutes such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 minutes or more than 59 minutes; hours such as, but not limited to, 1, 2,1,2,3,4,5,6,7,8,9, 10,11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, or more than 48 hours; days such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or more than 31 days; weeks such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, or more than 16 weeks; months such as, but not limited to, 1, 2, 3.4, 5, 6.7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 months; years such as, but not limited to, 1, 2,3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 years.
[001428] [001428] In some embodiments, the AAV particles of the present invention may be administered via intrathecal injection to the DRG nociceptive neurons.
[001429] [001429] In some embodiments, the AAV particles of the present invention may be administered via a single-dose intrathecal injection to the DRG nociceptive neurons.
[001430] [001430] As a non-limiting example, single-dose intrathecal injection may be a one-time treatment.
[001431] [001431] In the context of central nervous system disorders (eg, Parkinson's disease), single-dose intrathecal injection may produce lasting relief for individuals with central nervous system disorders (eg, Parkinson's disease) and/or symptoms related.
[001432] [001432] Relief may last for minutes such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 minutes or more than 59 minutes; hours such as, but not limited to, 1, 2, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more than 48 hours; days such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or more than 31 days; weeks such as, but not limited to, 1, 2,3,4,5, 6,7,8,9,10, 11,12, 13, 14, 15, 16, or more than 16 weeks; months such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more than 24 months; years such as, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more than 15 years.
[001433] [001433] In one embodiment, the AAV particle described in this application is administered via intrathecal (IT) infusion at C1.
[001434] [001434] The infusion can be for 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more than 15 hours.
[001435] [001435] In some embodiments, the AAV particles of the present invention may be administered by intraparenchymal injection.
[001436] [001436] As a non-limiting example, the AAV particles of the present invention can be administered to a subject by intraparenchymal injection.
[001437] [001437] In one embodiment, the AAV particle can be administered to the cisterna magna in a therapeutically effective amount to transduce spinal cord motor neurons and/or astrocytes.
[001438] [001438] As a non-limiting example, the AAV particle can be administered by intraparenchymal injection.
[001439] [001439] In some embodiments, the AAV particles of the present invention can be administered by intraparenchymal injection and intrathecal injection.
[001440] [001440] As a non-limiting example, the AAV particles of the present invention can be administered via intraparenchymal injection and intrathecal injection.
[001441] [001441] In some embodiments, the AAV particles of the present invention may be administered by subcutaneous injection.
[001442] [001442] As a non-limiting example, the AAV particles In one embodiment, the AAV particles of the present invention can be administered to a subject by subcutaneous injection.
[001443] [001443] In some embodiments, the AAV particles of the present invention may be administered topically.
[001444] [001444] As a non-limiting example, the AAV particles of the present invention can be administered to a subject topically.
[001445] [001445] In one embodiment, the AAV particles may be delivered by direct injection into the brain.
[001446] [001446] As a non-limiting example, delivery to the brain may be by intrastriatal administration.
[001447] [001447] In one embodiment, the AAV particles of the present invention may be administered via intrastriatal injection.
[001448] [001448] In one embodiment, the AAV particles of the present invention can be administered via intrastriatal injection and another via administration described in this application.
[001449] [001449] In one embodiment, the AAV particles may be delivered by more than one route of administration.
[001450] [001450] As non-limiting examples of combined administrations, the AAV particles can be delivered by intrathecal and intracerebroventricular administration, or by intravenous and intraparenchymal administration.
[001451] [001451] In one embodiment, the AAV particle may be administered to the CNS in a therapeutically effective amount to improve the function and/or survival of an individual with central nervous system disorders (eg, Parkinson's disease).
[001452] [001452] As a non-limiting example, the vector may be administered by direct infusion into the striatum.
[001453] [001453] The AAV particle can be administered in a "therapeutically effective" amount, i.e., an amount that is sufficient to alleviate and/or prevent at least one symptom associated with the disease, or provide improvement in the subject's condition.
[001454] [001454] In one embodiment, the catheter may be located at more than one site in the spine for multi-site delivery.
[001455] [001455] The AAV particle can be delivered as a continuous infusion and/or bolus.
[001456] [001456] Each delivery site can have a different dosing schedule or the same dosing schedule can be used for each delivery site.
[001457] [001457] As a non-limiting example, the distribution sites can be in the cervical region and in the lumbar region.
[001458] [001458] As another non-limiting example, the distribution sites can be in the cervical region.
[001459] [001459] As another non-limiting example, the distribution sites can be in the lumbar region.
[001460] [001460] In one embodiment, an individual is assessed for spinal anatomy and pathology prior to delivery of the AAV particle described in this application.
[001461] [001461] As a non-limiting example, an individual with scoliosis may have a different dosing schedule and/or catheter location compared to an individual without scoliosis.
[001462] [001462] In one embodiment, the orientation of the individual's spine during AAV particle delivery may be vertical to the ground.
[001463] [001463] In another embodiment, the orientation of the individual's spine during AAV particle distribution may be horizontal to the ground.
[001464] [001464] In one embodiment, the subject's spine may be at a certain angle to the ground during the delivery of the AAV particle.
[001465] [001465] The angle of the individual's spine in relation to the earth can be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 180 degrees.
[001466] [001466] In one embodiment, the method and duration of delivery are chosen to provide broad spinal cord transduction.
[001467] [001467] As a non-limiting example, intrathecal delivery is used to provide broad transduction along the rostral-caudal length of the spinal cord.
[001468] [001468] As another non-limiting example, multisite infusions provide more uniform transduction along the rostral-caudal length of the spinal cord.
[001469] [001469] As yet another non-limiting example, prolonged infusions provide more uniform transduction along the rostral-caudal length of the spinal cord.
[001470] [001470] In one embodiment, administration occurs by a posterior surgical delivery approach (eg, behind the head) to the putamen.
[001471] [001471] As a non-limiting example, mean putaminal coverage is 50% with posterior distribution and surgery time is less than 10 hours.
[001472] [001472] In one embodiment, administration occurs by a transfrontal (eg, top of the head) surgical delivery approach to the putamen.
[001473] [001473] As a non-limiting example, the mean putaminal coverage is less than 50% with posterior distribution and the operative time is greater than 10 hours. Parenteral and injectable administration.
[001474] [001474] In some embodiments, the pharmaceutical compositions of AAV particles of the present invention may be administered parenterally.
[001475] [001475] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
[001476] [001476] In addition to the active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, peanut, corn, germ, olive, castor, and sesame oils) glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols fatty acid esters of sorbitan, and mixtures thereof.
[001477] [001477] In addition to inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
[001478] [001478] In certain embodiments for parenteral administration, the compositions are mixed with solubilizing agents such as CREMOPHOR, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
[001479] [001479] In other embodiments, surfactants such as hydroxypropylcellulose are included.
[001480] [001480] Injectable formulations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known prior art using suitable dispersing agents, wetting agents, and/or suspending agents.
[001481] [001481] Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in parenterally acceptable non-toxic diluents and/or solvents, for example, as a solution in 1,3-butanediol.
[001482] [001482] Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
[001483] [001483] Sterile fixed oils are conventionally employed as a solvent or suspending medium.
[001484] [001484] For this purpose, any sweet fixed oil can be used, including synthetic mono- or diglycerides.
[001485] [001485] Fatty acids such as oleic acid can be used in the preparation of injectables.
[001486] [001486] Injectable formulations must be sterilized, for example, by filtration through a bacteria-retaining filter, and/or by incorporation of sterilizing agents in the form of sterile solid compositions that can be dissolved or dissolved in sterile water or other sterile injectable medium. before use.
[001487] [001487] To prolong the effect of the active ingredients, it is often desirable to delay the absorption of the active ingredients from subcutaneous or intramuscular injections.
[001488] [001488] This can be done using liquid suspensions of crystalline or amorphous material with poor water solubility.
[001489] [001489] The rate of absorption of active ingredients depends on the rate of dissolution which, in turn, may depend on the size of the crystals and the crystalline form.
[001490] [001490] Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle.
[001491] [001491] Injectable depot forms are made by forming matrices for microencapsulation of the drug in biodegradable polymers such as polylactide-polyglycolide.
[001492] [001492] Depending on the drug to polymer ratio and the nature of the particular polymer employed, the rate of drug release can be controlled.
[001493] [001493] Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).
[001494] [001494] Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue. Deposit Administration.
[001495] [001495] As described in this application, in some embodiments, the pharmaceutical compositions of AAV particles of the present invention are formulated in depots for sustained release.
[001496] [001496] Generally, specific organs or tissues ("target tissues") are targeted for administration.
[001497] [001497] In some aspects of the invention, the pharmaceutical compositions of AAV particles of the present invention are spatially retained in or near target tissues.
[001498] [001498] Methods are provided for delivering pharmaceutical compositions of AAV particles to target tissues of mammalian subjects by contacting the target tissues (which comprise one or more target cells) with pharmaceutical compositions of AAV particles, under conditions such that they are substantially retained in the target tissues, meaning at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99, 9, 99. 99 or more than 99.99% of the composition is retained in the target tissues.
[001499] [001499] Advantageously, retention is determined by measuring the amount of the pharmaceutical compositions of AAV particles that penetrate one or more target cells.
[001500] [001500] For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or greater than 99.99% of the pharmaceutical compositions of AAV particles administered to subjects are present intracellularly within a period of time after administration.
[001501] [001501] For example, intramuscular injection to mammalian subjects can be accomplished by the use of aqueous compositions comprising pharmaceutical compositions of AAV particles of the present invention and one or more transfection agents, and retention is determined by measuring the amount of the pharmaceutical compositions. of AAV particles present in muscle cells.
[001502] [001502] Certain aspects of the invention pertain to methods for delivering the AAV particle pharmaceutical compositions of the present invention to target tissues of mammalian subjects by contacting the target tissues (comprising one or more target cells) with the target tissues. pharmaceutical compositions of AAV particles under conditions such that they are substantially retained in such target tissues.
[001503] [001503] Pharmaceutical compositions of AAV particles comprise sufficient active ingredient so that the effect of interest is produced in at least one target cell.
[001504] [001504] In some embodiments, pharmaceutical compositions of AAV particles generally comprise one or more cell-penetrating agents, although "naked" formulations (such as without cell-penetrating agents or other agents) are also contemplated, with or without carriers. pharmaceutically acceptable. Distribution to the Central Nervous System.
[001505] [001505] In one embodiment, delivery of pharmaceutical compositions comprising AAV particles to cells of the central nervous system (eg, parenchyma) comprises infusion of up to 1 ml.
[001506] [001506] In one embodiment, delivery of pharmaceutical compositions comprising AAV particles to cells of the central nervous system (e.g., parenchyma) may comprise infusion of 0.001, 0.002, 0.003, 0.004, 0.005, 0.010, 0.015, 0.020, 0.025 , 0.030, 0.040, 0.050, 0.060, 0.070, 0.080, 0.090, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0, 9 ml.
[001507] [001507] In one embodiment, delivery of the pharmaceutical composition comprising AAV particles to cells of the central nervous system (e.g., parenchyma) comprises infusion of between about 1 mL to about 120 mL.
[001508] [001508] In one embodiment, delivery of the pharmaceutical composition comprising AAV particles to the cells of the central nervous system (e.g., parenchyma) may comprise infusion of 0.1, 1, 1.1, 1.2, 1.3 , 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3.4, 5.6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42,43,44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 ,
[001509] [001509] In one embodiment, delivery of AAV particles to central nervous system cells (eg, parenchyma) comprises infusion of at least 3 mL.
[001510] [001510] In one embodiment, delivery of AAV particles to central nervous system cells (eg, parenchyma) consists of a 3 mL infusion.
[001511] [001511] In one embodiment, delivery of AAV particles to central nervous system cells (eg, parenchyma) comprises infusion of at least 10 mL.
[001512] [001512] In one embodiment, delivery of AAV particles to central nervous system cells (eg, parenchyma) consists of 10 mL infusion.
[001513] [001513] In one embodiment the volume of the pharmaceutical composition comprising AAV particles delivered to central nervous system cells (e.g. parenchyma) of a subject is 50 µl, 100 µl, 200 µl, 300 µl, 400 µl, 500 µl , 600ul, 700ul, 800ul, 900ul, 1000ul, 1100ul, 1200ul, 1300ul, 1400ul, 1500ul, 1600ul, 1700ul, 1800ul, 1900ul, 2000ul or more than 2000ul .
[001514] [001514] In one embodiment the volume of the pharmaceutical composition comprising AAV particles distributed to a region in both hemispheres of a subject's brain is 50 ul, 100 ul, 200 ul, 300 ul, 400 ul, 500 ul, 600 ul, 700ul, 800ul, 900ul, 1000ul, 1100ul, 1200ul, 1300ul, 1400ul, 1500ul, 1600ul, 1700ul, 1800ul, 1900ul, 2000ul or more than 2000ul.
[001515] [001515] As a non-limiting example, the volume distributed for a region in both hemispheres is 200 ul.
[001516] [001516] As another non-limiting example, the volume distributed for a region in both hemispheres is 900 ul.
[001517] [001517] As yet another non-limiting example, the volume distributed for a region in both hemispheres is 1800 ul.
[001518] [001518] In one embodiment, the volume of the pharmaceutical composition comprising AAV particles delivered to the putamen in both hemispheres of a subject's brain is 50ul, 100ul, 200ul, 300ul, 400ul, 450ul, 500ul , 600ul, 700ul, 800ul, 900ul, 1000ul, 1100ul, 1200ul, 1300ul, 1400ul, 1500ul, 1600ul, 1700ul, 1800ul, 1900ul, 2000ul or more than 2000ul .
[001519] [001519] As a non-limiting example, the volume distributed to the putamen in both hemispheres is 100 ul.
[001520] [001520] As another non-limiting example, the volume distributed to the putamen in both hemispheres is 200 ul.
[001521] [001521] As a non-limiting example, the volume distributed to the putamen in both hemispheres is 300 ul.
[001522] [001522] As another non-limiting example, the volume distributed to the putamen in both hemispheres is 450 ul.
[001523] [001523] As another non-limiting example, the volume distributed to the putamen in both hemispheres is 900 ul.
[001524] [001524] As yet another non-limiting example, the volume distributed to both hemispheres of the putamen is 1800 ul.
[001525] [001525] In one embodiment the volume of the pharmaceutical composition comprising AAV particles delivered to an individual is 900 µl to each putamen.
[001526] [001526] In one embodiment, the volume of the pharmaceutical composition comprising AAV particles delivered to an individual is 450 µl for each putamen.
[001527] [001527] In one embodiment, the total volume dispensed to an individual may be divided among one or more sites of administration, eg 1, 2, 3, 4, 5 or more than 5 sites.
[001528] [001528] As a non-limiting example, the total volume is divided between administration to the right and left putamen.
[001529] [001529] As another non-limiting example, the total volume is divided between two delivery sites each of the left and right putamen.
[0015380] [0015380] In one embodiment, the pharmaceutical composition comprising AAV particles is administered via a fenestrated needle.
[001531] [001531] Non-limiting examples of fenestrated needles are disclosed in patent US No. * 8,333,734, 7,135,010, 7,575,572, 7,699,852, 4,411,657, 6,890,319, 6,613,026, 6,726,659, 6,565,572, 6,520,949, 6,382,212, 5,848,996, 5,759,179, 5 674.267, 5,588,960, 5,484,401, 5,199,441 , 5,012,818, 4,474,569, 3,766,907, 3,552,394, the entirety of each of which is incorporated herein by reference.
[001532] [001532] In one embodiment, a composition comprises at least one payload described in this application and the payloads are components of a viral genome packaged in an AAV particle.
[001533] [001533] The percentage ratio (%) of AAV particles comprising the payload to the AAV particles without the payload (in this application also called empty capsids) in the composition may be 0:100, 1:99, 0:90 , 15:85, 25:75, 30:70, 50:50, 70:30, 85:15, 90:10, 99:1 or 100:0.
[001534] [001534] As a non-limiting example, the percentage ratio of AAV particles comprising payload to empty capsids is 50:50.
[001535] [001535] As another non-limiting example, the percentage ratio of AANV particles comprising payload to empty capsids is 70:30.
[001536] [001536] As another non-limiting example, the percentage ratio of AAV particles comprising payload to empty capsids is 85:15.
[001537] [001537] As another non-limiting example, the percentage ratio of AAV particles comprising payload to empty capsids is 100:0.
[001538] [001538] In one embodiment, the composition described in this application comprises at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 60, 65 , 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more than 99% of AAV particles comprising the payload.
[001539] [001539] As a non-limiting example, the composition comprises at least 50% AAV particles comprising the payload.
[001540] [001540] As another non-limiting example, the composition comprises at least 52% AAV particles comprising the payload.
[001541] [001541] As another non-limiting example, the composition comprises at least 58% AAV particles comprising the payload.
[001542] [001542] As another non-limiting example, the composition comprises at least 70% AAV particles comprising the payload.
[001543] [001543] As another non-limiting example, the composition comprises at least 83% AAV particles comprising the payload.
[001544] [001544] As another non-limiting example, the composition comprises at least 85% AAV particles comprising the payload.
[001545] [001545] As another non-limiting example, the composition comprises at least 99% AAV particles comprising the payload.
[001546] [001546] As another non-limiting example, the composition comprises 100% AAV particles comprising the payload.
[001547] [001547] In one embodiment, the composition described in this application comprises 1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90%, 50- 99%, 50-100%, 60-70%, 60-80%, 60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99%, 70- 100%, 80-85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% of AAV particles comprising the filler useful.
[001548] [001548] As a non-limiting example, the composition described in this application comprises 950-100% AAV particles comprising the payload.
[001549] [001549] As another non-limiting example, the composition described in this application comprises 50-60% AAV particles comprising the payload.
[001550] [001550] As another non-limiting example, the composition described in this application comprises 80-99% AAV particles comprising the payload.
[001551] [001551] As another non-limiting example, the composition described in this application comprises 80-90% AAV particles comprising the payload.
[001552] [001552] As a non-limiting example, the composition described in this application comprises 80-95% AAV particles comprising the payload.
[001553] [001553] As a non-limiting example, the composition described in this application comprises 80-85% AAV particles comprising the payload.
[001554] [001554] In one embodiment, the composition described in this application comprises less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 60, 65 , 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% empty particles .
[001555] [001555] As a non-limiting example, the composition comprises less than 50% empty particles.
[001556] [001556] As a non-limiting example, the composition comprises less than 45% empty particles.
[001557] [001557] As a non-limiting example, the composition comprises less than 40% empty particles.
[001558] [001558] As a non-limiting example, the composition comprises less than 35% empty particles.
[001559] [001559] As a non-limiting example, the composition comprises less than 30% empty particles.
[001560] [001560] As a non-limiting example, the composition comprises less than 25% empty particles.
[001561] [001561] As a non-limiting example, the composition comprises less than 20% empty particles.
[001562] [001562] As a non-limiting example, the composition comprises less than 15% empty particles.
[001563] [001563] As a non-limiting example, the composition comprises less than 10% empty particles.
[001564] [001564] As a non-limiting example, the composition comprises less than 5% empty particles.
[001565] [001565] As a non-limiting example, the composition comprises less than 1% empty particles.
[001566] [001566] The composition described in this application comprises 1-10%, 10-20%, 30-40%, 50-60%, 50-70%, 50-80%, 50-90%, 50-99%, 50 - 100%, 60-70%, 60-80%, 60-90%, 60-99%, 60-100%, 70-80%, 70-90%, 70-99%, 70-100%, 80 -85%, 80-90%, 80-95%, 80-99%, 80-100%, 90-95%, 90-99%, or 90-100% of empty particles.
[001567] [001567] As a non-limiting example, the composition described in this application comprises 30-40% empty particles.
[001568] [001568] As another non-limiting example, the composition described in this application comprises 30-50% empty particles.
[001569] [001569] As another non-limiting example, the composition described in this application comprises 30-60% empty particles.
[001570] [001570] As another non-limiting example, the composition described in this application comprises 30-70% empty particles.
[001571] [001571] As a non-limiting example, the composition described in this application comprises 30-80% empty particles.
[001572] [001572] As a non-limiting example, the composition described in this application comprises 30-90% empty particles.
[001573] [001573] In one embodiment, the ratio of the volume of distribution in the parenchyma of an area of an individual to the infusion volume of an area of an individual may be 0.1, 0.2, 0.3, 0.4 , 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1 .7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9 , 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4 .2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 , 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or more than 6.0.
[001574] [001574] As a non-limiting example, the ratio of parenchymal volume of distribution to infusion volume was 1.6 in the caudate nucleus.
[001575] [001575] As a non-limiting example, the ratio of parenchymal volume of distribution to infusion volume was 3.1 in the putamen.
[001576] [001576] As a non-limiting example, the distribution of AAV particles in the putamen can be 2-3 times the infused volume.
[001577] [001577] In one embodiment, the effectiveness of the dose, route of administration and/or volume of administration can be evaluated by various methods described in this application such as, but not limited to, PET imaging, L-DOPA challenge testing (for for example, see Forsayeth et al., 2006, Mol. Ther. 14(4): 571-577), UPDRS scores, and patient diaries (eg, Hauser diary).
[001578] [001578] As a non-limiting example, a subject may have reduced dyskinesia or periods of reduced dyskinesia after administration of the pharmaceutical composition comprising AAV particles.
[001579] [001579] As another non-limiting example, an individual may have a reduction in Parkinson's related symptoms including limited mobility and dyskinesia.
[001580] [001580] As yet another non-limiting example, an individual may show improvement in resting time and motor fluctuations.
[001581] [001581] The improvement can be at least 10%, 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 more than 90%.
[001582] [001582] Improvement can last for minutes (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 15, 20, 25, 30, 35, 40, 45, 50, 55 or more than 55), hours (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more than 24), days (e.g. 1, 2, 3, 4.5, 6 or more than 7), weeks (1, 2, 3, 4, 5, 6, 7 or more than 7), months (1,2, 3, 4, 5,6,7, 8.9, 10, 11 or more than 11) or years (e.g. 1,2,3,4, 5, 6, 7, 8, 9 or more than 9).
[001583] [001583] In one embodiment, the selection of subjects for administration of the AAV particles described in this application and/or the effectiveness of dose, route of administration, and/or volume of administration can be assessed by imaging the perivascular spaces (PVS) which are also known as Virchow-Robin spaces.
[001584] [001584] PVS surround arterioles and venules as they perforate the brain parenchyma and are filled with cerebrospinal fluid (CSF)/interstitial fluid.
[001585] [001585] PVS are common in the midbrain, in the BG, and in the semi-oval center.
[001586] [001586] While not wishing to be bound by theory, PVS play a role in the normal clearance of metabolites and have been associated with impaired cognition and various disease states including Parkinson's disease.
[001587] [001587] PVS are usually of normal size, but can increase in size in a number of disease states. Potter et al. (Cerebrovasc Dis. 2015 Jan; 39(4): 224-231; the entirety of which is incorporated herein by way of reference) developed a classification method where they studied a complete range of PVS and classified the PVS of the basal ganglia, the central semi- oval and midbrain.
[001588] [001588] They used the frequency and range of PVS used by Mac and Lullich et al. (J Neurol Neurosurg Psychiatry. 2004 Nov;75(11):1519-23; the entirety of which is incorporated herein by reference) and Potter et al. showed 5 degrees for the PVS of the basal ganglia and semi-oval center: O (none), 1 (1-10), 2 (11-20), 3 (21-40) and 4 (>40) and 2 degrees for midbrain PVS: O (not visible) or 1 (visible).
[001589] [001589] The user's guide to the classification system by Potter et al. can be found at: www.sbirc.ed.ac.uk/documents/epvs-rating-scale-user-quide pdf.
[001591] [001591] In one embodiment, the selection of subjects for administration of the AAV particles described in this application and/or the effectiveness of the dose, route of administration, and/or volume of administration can be evaluated by measurements made by emission tomography. positrons (PET) from neuroimaging biomarkers such as, but not limited to, FDOPA.
[001592] [001592] Neuroimaging biomarkers such as FDOPA can be used to identify affected individuals and/or can be used to detect a nigrostriatal defect before the onset of clinical manifestations.
[001593] [001593] In addition, PET-based criteria can be used to classify individuals based on their nigrostriatal neuronal integrity (eg, normal, normal, or doubtful nigrostriatal neuronal integrity) (Rachette et al. Am J Med Genet B Neuropsychiatr Genet. 2006 Apr 5; 141B(3): 245-249; the entirety of which is incorporated herein by reference).
[001594] [001594] In one embodiment, an individual who can receive a dose of the AAV particles described in this application may have advanced PD and still respond to levodopa therapy, but the individual also experiences clinically refractory motor complications (e.g., severe motor fluctuations and/or dyskinesias that occur during therapy with levodopa and other dopaminergics despite medication adjustments and optimization).
[001595] [001595] The individual may be healthy enough to undergo a neurosurgical procedure which can be determined by methods known in the literature.
[001596] [001596] As a non-limiting example, the individual may meet the selection criteria for deep brain stimulation (DBS).
[001597] [001597] The individual may have idiopathic PD, be younger than 69 years of age, have marked responses to levodopa, have medication-refractory symptoms (eg, motor fluctuation and/or dyskinesia), and/or have little or no cognitive dysfunction.
[001598] [001598] In one embodiment, an individual who may receive a dose of the AAV particles described in this application may also suffer from dementia or cognitive impairment.
[001599] [001599] In one embodiment, an individual who may receive a dose of the AAV particles described in this application may have been previously treated with the same or a similar drug.
[001600] [001600] In another embodiment, an individual may have been treated with a drug that reduced the symptoms of Parkinson's disease.
[001601] [001601] In one embodiment, an individual who may receive a dose of the AAV particles described in this application may have failed to obtain adequate benefit from traditional medical therapy.
[001602] [001602] As a non-limiting example, the individual may not have responded to treatment.
[001603] [001603] As another non-limiting example, an individual may have residual disability despite treatment.
[001604] [001604] In one embodiment, an individual who may receive a dose of the AAV particles described in this application may undergo examinations to assess levels of neurotransmitter analytes to determine the effectiveness of the dose.
[001605] [001605] As a non-limiting example, CSF neurotransmitters, plasma AADC activity and/or VLA in urine can be analyzed.
[001606] [001606] In one embodiment, an individual who may receive a dose of the AAV particles described in this application may be filmed or recorded to monitor the individual's progress during the course of treatment. Ddistribution to the putamen.
[001607] [001607] In one embodiment, AAV particles may be delivered to the right putamen and/or the left putamen.
[001608] [001608] Administration may occur at one or more sites in the putamen such as, but not limited to, 2 sites, 3 sites, 4 sites or more than 4 sites.
[001609] [001609] As a non-limiting example, AAV particles are distributed to 2 sites in the left putamen and 2 sites in the right putamen.
[001610] [001610] In one embodiment, administration of the AAV particle formulation to an individual provides coverage of an individual's putamen (eg, the left and/or right putamen).
[001611] [001611] In one aspect, the administration of the AAV particles can provide at least 8%, 9%, 10%, 13%, 14%, 15%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% , 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95% or more than 95% for the left and/or right putamen of an individual.
[001612] [001612] As a non-limiting example, coverage is at least 20%.
[001613] [001613] As a non-limiting example, coverage is at least 40%.
[001614] [001614] In another aspect, the administration of AAV particles can provide at least 8%, 9%, 10%, 13%, 14%, 15%, 19%, 20%, 21%, 22%, 23% , 24%.
[001615] [001615] 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% , 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95% or more than 95% surface area coverage of an individual's left and/or right putamen.
[001616] [001616] As a non-limiting example, total coverage is at least 20%.
[001617] [001617] As a non-limiting example, total coverage is at least 40%.
[001618] [001618] In yet another aspect, the administration of the AAV particles can provide 10-40%, 20-40%, 20-30%, 20-35%, 20-50%, 30-40%, 35-40 %, 30-60%, 40-70%, 50-80% or 60-90% coverage for an individual's left and/or right putamen or for the total surface area of an individual's left and/or right putamen .
[001619] [001619] In one embodiment, administration of the AAV particle formulation to an individual provides coverage of an individual's posterior putamen (eg, the left and/or right posterior putamen).
[001620] [001620] In one aspect, the administration of the AAV particles can provide at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% , 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, or greater than 95% for an individual's left and/or right posterior putamen.
[001621] [001621] As a non-limiting example, coverage is at least 20%.
[001622] [001622] As a non-limiting example, coverage is at least 40%.
[001623] [001623] In another aspect, the administration of AAV particles can provide at least 10%, 15%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28% , 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45 %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95% coverage of the surface area of an individual's left and/or right posterior putamen.
[001624] [001624] As a non-limiting example, total coverage is at least 20%.
[001625] [001625] As a non-limiting example, total coverage is at least 40%.
[001626] [001626] In yet another aspect, the administration of the AAV particles can provide 10-40%, 20-50%, 30-60%, 40-70%, 50-80% or 60-90% coverage for the an individual's left and/or right posterior putamen or to the total surface area of an individual's left and/or right putamen.
[001627] [001627] In one embodiment, the AAV particles described in this application may be administered by acute bilateral placement of catheters in each putamen.
[001628] [001628] Placement may use magnetic resonance imaging (MRI) guided neurosurgical techniques known in the literature or described in this application.
[001629] [001629] Additionally, a contrast agent such as, but not limited to, a gadolinium-based contrast agent (eg PROHANCE®) can be used in the formulation to monitor and confirm the distribution of the formulation.
[001630] [001630] In one embodiment, a subject may receive administration of the AAV particles in a stereotaxic CED-assisted staggered bilateral infusion into the putamen (eg, the post-commissural putamen).
[001631] [001631] In one embodiment, a subject may be administered the AAV particles of the present invention at a dose of 4.5 x 10 12 vector genomes in a volume of 900 µl per putamen.
[001632] [001632] In one embodiment, a subject may be administered the AAV particles of the present invention at a dose of 1.5 x 10 12 vector genomes in a volume of 900 µl per putamen.
[001633] [001633] In one embodiment, a subject may receive administration of the AAV particles of the present invention at a dose of 7.5 x 10 11 vector genomes in a volume of 450 µl per putamen.
[001634] [001634] In one embodiment, a subject may receive administration of the AAV particles with a bilateral surgical infusion into at least one putamen via a posterior surgical delivery approach (i.e., behind the head).
[001635] [001635] The number of subsequent bilateral surgical infusions may be one or more such as, but not limited to, 1 infusion, 2 infusions, 3 infusions, 4 infusions, or more than 4 infusions.
[001636] [001636] As a non-limiting example, AAV particles are delivered into the left putamen with a posterior bilateral surgical infusion.
[001637] [001637] As a non-limiting example, AAV particles are delivered into the right putamen with a posterior bilateral surgical infusion.
[001638] [001638] As a non-limiting example, AAV particles are delivered into the left putamen with two posterior bilateral surgical infusions.
[001639] [001639] As a non-limiting example, AAV particles are delivered into the right putamen with two posterior bilateral surgical infusions.
[001640] [001640] As a non-limiting example, AAV particles are delivered into the right and left putamen with two posterior bilateral surgical infusions.
[001641] [001641] As a non-limiting example, AAV particles are delivered into the left putamen with three subsequent bilateral surgical infusions.
[001642] [001642] As a non-limiting example, AAV particles are delivered into the right putamen with three posterior bilateral surgical infusions.
[001643] [001643] As a non-limiting example, AAV particles are delivered into the right and left putamen with three posterior bilateral surgical infusions.
[001644] [001644] As a non-limiting example, AAV particles are delivered into the left putamen with four posterior bilateral surgical infusions.
[001645] [001645] As a non-limiting example, AAV particles are delivered into the right putamen with four posterior bilateral surgical infusions.
[001646] [001646] As a non-limiting example, AAV particles are delivered to the right and left putamen with four posterior bilateral surgical infusions.
[001647] [001647] In one embodiment, an individual may receive the administration of the AAV particles with a bilateral surgical infusion into at least one putamen by means of a transfrontal (i.e., on top of the head) surgical delivery technique.
[001648] [001648] The number of bilateral surgical infusions may be two or more such as, but not limited to, 2 infusions, 3 infusions, 4 infusions or more than 4 infusions.
[001649] [001649] As a non-limiting example, AAV particles are delivered into the left putamen with 2 posterior bilateral surgical infusions.
[001650] [001650] As a non-limiting example, AAV particles are delivered into the right putamen with 2 posterior bilateral surgical infusions.
[001651] [001651] As a non-limiting example, AAV particles are delivered into the left and right putamen with 2 posterior bilateral surgical infusions.
[001652] [001652] In one embodiment, a subject may receive administration of the AAV particles of the present invention delivered safely to the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) via bilateral infusions, or alternatively, by intrastriatal route (in the caudate nucleus and putamen), or in the subthalamic nucleus (STN). Distribution, Dose and Regimen.
[001653] [001653] The present invention provides methods of administering the AAV particles according to the invention to a subject in need thereof.
[001654] [001654] The AAV particles and pharmaceutical, diagnostic, or prophylactic compositions of the present invention can be administered to a subject using any amount and any route of administration effective for the prevention, treatment, control, or diagnosis of diseases, disorders and/or conditions.
[001655] [001655] The exact amount needed will vary from individual to individual, depending on the species, age, and general condition of the individual, the severity of the disease, the particular composition, its mode of administration, its mode of activity, among others. others.
[001656] [001656] The subject may be a human, a mammal, or an animal.
[001657] [001657] Compositions according to the invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage.
[001658] [001658] It will be understood, however, that the total daily dose of the compositions of the present invention can be decided by the attending practitioner within the scope of sound medical judgment.
[001659] [001659] The appropriate specific therapeutically effective, prophylactically effective, or diagnostic dose level for any particular individual will depend on a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health status, sex and diet of the patient; the time of administration, the route of administration, and the rate of excretion of the specific AAV particle employed; the duration of treatment; drugs used in combination with or coincident with the specific AAV particle employed; and similar factors well known in the medical field.
[001660] [001660] In one embodiment, the distribution of the AAV particles of the present invention results in minimal serious adverse events (SAEs) as a result of the distribution of the AAV particles.
[001661] [001661] In one embodiment, the AAV particle may be delivered in a multiple dose regimen.
[001662] [001662] The multiple dose regimen may be 2, 3, 4, 5, 6, 7, 8, 9, or more than 10 doses.
[001663] [001663] In one embodiment, the AAV particle may be delivered to an individual by a multiple-site route of administration.
[001664] [001664] An individual may be administered the AAV particle at 2, 3, 4, 5 or more than 5 sites. Dosage Levels.
[001665] [001665] In certain embodiments, the AAV particle pharmaceutical compositions of the present invention can be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.0001 mg/kg to about 100 mg/kg 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0 .05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg of the subject's body weight per day, one or more times daily, to obtain the desired therapeutic, diagnostic, or prophylactic effect.
[001666] [001666] It will be understood that the above dosage concentrations may be converted to vg or viral genomes per kg or to total genomes administered by one skilled in the art.
[001667] [001667] In certain embodiments, the AAV particle pharmaceutical compositions of the present invention can be administered at about 10 to about 600 ul/site, 50 to about 500 ul/site, 100 to about 400 ul /site, 120 to about 300 ul/site, 140 to about 200 ul/site, about 160 ul/site.
[001668] [001668] As non-limiting examples, AAV particles can be administered at 50 ul/site and/or 150 ul/site.
[001669] [001669] In one embodiment, the delivery of compositions according to the present invention to cells comprises a delivery rate defined by [VG/hour = mL/hour * VG/mL] where VG is viral genomes, VG/mL is the concentration of the composition, and mL/hour is the sustained delivery rate.
[001670] [001670] In one embodiment, the delivery of compositions according to the present invention to cells may comprise a total concentration per subject of between about 1x10 6 VG and about 1x10 18 vc.
[001671] [001671] In some embodiments, the distribution may comprise a concentration of the composition of about 1x108, 2x108, 3x108, 4x106, 5x106, 6x108, 7x1086, 8x108, 9x108, 1x107, 2x107, 3x107, 4x107, 5x107, 7" , 8x10/, 9x10”, 1x108, 2x108, 3x108,
[001672] [001672] In one embodiment, the concentration of the AAV particle in the composition is 1x1018 vG/subject.
[001673] [001673] In one embodiment, the concentration of the AAV particle in the composition is 3x1012 VG/subject.
[001674] [001674] As a non-limiting example, the composition administered to the subject has a concentration of about 3x10 11 VG/subject.
[001675] [001675] As a non-limiting example, the composition administered to the subject has a concentration of about g9x1011 VG/subject.
[001676] [001676] In one embodiment, the concentration of the AAV particle in the composition is 2.3x10] 1 VG/subject.
[001677] [001677] In one embodiment, the concentration of the AAV particle in the composition is 7.2x1011 VG/subject.
[001678] [001678] In one embodiment, the concentration of the AAV particle in the composition is 7.5x1011 VG/subject.
[001679] [001679] In one embodiment, the concentration of the AAV particle in the composition is 1.4x10] 2 VG/subject.
[001680] [001680] In one embodiment, the concentration of the AAV particle in the composition is 4.8x1012 VG/subject.
[001681] [001681] In one embodiment, the concentration of the AAV particle in the composition is 8.8x10] 2 VG/subject.
[001682] [001682] In one embodiment, the concentration of the AAV particle in the composition is 2.3x1012 VG/subject.
[001683] [001683] In one embodiment, the delivery of compositions according to the present invention to cells may comprise a total concentration per subject of between about 1x10 6 VG/kg and about 1x10 16 VG/Kkg.
[001684] [001684] In some embodiments, the distribution may comprise a concentration of the composition of about 1x108, 2x108, 3x108, 4x1086, 5x108, 6x108, 7x106, 8x108, 9x108, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107. 8x107, 9x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x108, 2x10%, 3x108, 2x10%, 3x108, 4x10, 5x10%, 6x109, 7x10, 8x109, 9x10%, 1x109, 9x10%, 1x1010, 2x1010, 3x101º 4x1010, 5x1010, 7x1010, 8x1010, 9x1010, 1x1011, 2x1011, 2x1011, 2,11011, 2x1011, 23x1011, 23x1011, 24x1011, 25x1071, 26x1011, 2,7x1011, 2,8x1011, 2,9x1011, 2x1011, 3x1011, 4x1011, 5x1011, 6x1011, 7x1011, 7.1x1011, 7.2x1011, 7.3x101], 7.4x1011, 7.5x1011, 7.6x10], 7.1x1011, 7.8x1011, 7.9x1071, 8x1011, 9x1011, 1x1012, 1. ,
[001685] [001685] In one embodiment, the delivery of AAV particles to cells of the central nervous system (e.g., parenchyma) may comprise a total dose between about 1x106 VG and about 1x1016 vc.
[001686] [001686] In some embodiments, the delivery may comprise a total dose of about 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107, 7x107 , 9x107, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x10%, 3x108, 2x10%, 3x108, 4x10, 5x109, 6x10%, 7x10, 8x10, 9x10, 1x1010, 1.9x1010, 2x1010, 3x1019, 3. 73x1010, 4x1010, 6x1010, 7x1010, 8x1010, 1x1010, 1x1011, 2x1011, 2x1011, 2x1011, 3x1011, 4x1011, 4x1011, 4x1011, 5x1011, 6x1011, 7x1071, 8x1071, GX1011, 1x1012, 2x1012, 3x1012, 2x1012, 3x1012, 4x1012, 4x1012, 5x1012, 7x1012 , 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x10138, 6x1013, 7x1013, 8x1013, 9x1013, 1x1014, 2x1014, 3x1014, 4x1014, 5x1014, 6x1014, 7x1014, 8x1014, 9x1014, 1x1015, 2x1015, 3x1015, 4x1015, 5x1015 , 6x1015, 7x1015, 8x1015, 9x1015, or 1x1018 vc.
[001687] [001687] As a non-limiting example, the total dose is 1x1018 vc.
[001688] [001688] As another non-limiting example, the total dose is 21x1012 vG..
[001689] [001689] In one embodiment, about 10th to 106th viral genomes (unit) may be administered per dose.
[001690] [001690] In one embodiment, the delivery of the compositions according to the present invention to cells may comprise total concentration between about 1x10 6 VG/ml and about 1x10 16 VGM/ml.
[001691] [001691] In some embodiments, the distribution may comprise a concentration of the composition of about 1x108, 2x108, 3x1086, 4x106, 5x108, 6x108, 7x108, 8x106, 9x106, 1x107, 2x107, 3x107, 4x107, 5x107, 6x107 8x10 /, 9x10 /, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x108, 2x10%, 3x10, 4x108, 5x10, 4x10, 5x10, 6x109, 7x109, 8x10%, 9x10, 1x1010, 2x1010, 3x1010, 3x1010 4x1010, 5x1010, 7x1010, 7x1010, 9x1010, 1x1011, 2x1011, 2x1011, 3x1011, 4x1011, 5x1011, 5x1011, 6x1011, 7x1011, 8x1011, 8x1011, 8x1011, 9x1011, 1x10012, 11x1012, 1,2x1012, 1.3x1012, 1,4x1012, 1,5x1012, 1,5x1012 1.6x10012, 1.7x1012, 1.8x1012, 1.9x1012, 2x1012, 2.1x1012, 2.2x1012, 2.3x1012, 2.4x1012, 2.5x1012, 2.6x1012, 2.7x1012, 2.8x10 2.9x1012, 3x1012, 3.1x1012, 32x1012, 3.3x1012, 34x1012, 3.5x1012, 3.6x1012, 3.7x1012, 3.8x1012, 3.9x1012, 4x1012, 4.1x1012, 2. 3x101 2, 4.4x1012, 4.6x1012, 4.7x1012, 4,8x1012, 4.9x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x10138, 3x1013, 3x1013, 4x10138, 5x1013, 6x1013, 6.7x1013, 7x1 013, 8x1013, g9x10138, 1x1014, 2x1014, 3x1014, 4x1014, 5x1014, 6x1014, 7x1014, 8x1014, 9x1014,
[001692] [001692] In one embodiment, the concentration of the AAV particle in the composition is 1x1018 VG/mL.
[001693] [001693] In one embodiment, the concentration of the AAV particle in the composition is 3x1012 VG/mL.
[001694] [001694] In one embodiment, the concentration of the AAV particle in the composition is 1.1x1012 VG/mL.
[001695] [001695] In one embodiment, the concentration of the AAV particle in the composition is 3.7x1012 VG/mL.
[001696] [001696] In one embodiment, the concentration of the AAV particle in the composition is 8x101 1 VG/mL.
[001697] [001697] In one embodiment, the concentration of the AAV particle in the composition is 2.6x1012 VG/mL.
[001698] [001698] In one embodiment, the concentration of the AAV particle in the composition is 4.9x1012 VG/mL.
[001699] [001699] In one embodiment, the concentration of the AAV particle in the composition is 0.8x1012 VG/mL.
[001700] [001700] In one embodiment, the concentration of the AAV particle in the composition is 0.83x1012 VG/mL.
[001701] [001701] In one embodiment, the concentration of the AAV particle in the composition is the maximum final dose that can be contained in a vial particle.
[001702] [001702] In one embodiment, the delivery of AAV particles to the cells of the central nervous system (e.g., parenchyma) may comprise a composition concentration between about 1x1086 VG/mL and about 1x1016 VG/mL.
[001703] [001703] In some embodiments, the distribution may comprise a concentration of the composition of about 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x10%, 2x107, 3x107, 4x107, 5x107, 6x107 , 8x107, 9x108, 1x108, 2x108, 3x108, 4x108, 5x108, 6x108, 7x108, 8x108, 9x108, 1x109, 2x108, 3x108, 4x10%, 5x10%, 6x108, 7x108, 8x10, 7x10, 8x10, 9x10%, 1x1010, 2x1010, 3x1010 4x1010, s5x1010, 6x101º, 7x101º, 8x1019, 9x1010, 1x1011, 2x1011, axt1o11, 4x1071, 5x1011, ex1o11, 7x1011, 8x1011, 9x1011, 1x1012, 2x1012, 3x1012, 4x1012, 5x1012, 6x1012, 7x1012, 8x1012, 9x1012, 1x1013, 2x1013, 3x1013, 4x1013, 5x1013, 6x1013, 7x10138, 8x1013, 9x1018, 1x1014, 2x1019, 3x1014, 4x1019, 5x1014, 6x1014, 7x1014, 8x1019, 9x1014, 1x1015, 2x1015, 3x1015, 4x1015, 5x1015, 6x101S, 7x1015, 8x1015, 9x1015, or 1x1018 VG/mL.
[001704] [001704] In one embodiment, the distribution comprises a composition concentration of 1x1018 VvG/mL.
[001705] [001705] In one embodiment, the distribution comprises a concentration of the composition of 2.1x1012 VG/mL. Regime.
[001706] [001706] The desired dosage of the AAV particles of the present invention can be delivered once a day, three times a day, twice a day, once a day, every other day, once every three days, every week, every two weeks, every three weeks, or every four weeks.
[001707] [001707] In certain embodiments, the desired dosage may be distributed over multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations) .
[001708] [001708] When multiple administrations are employed, fractional dosing regimens such as those described in this application should be used.
[001709] [001709] As used in this application, a "fractionated dose" is the division of a "single unit dose" or total daily dose into two or more doses, for example two or more administrations of the "single unit dose".
[001710] [001710] As used in this application, a "single unit dose" is a dose of any drug administered in one dose/once/single route/single point of contact, ie, single administration event.
[001711] [001711] The desired dosage of the AAV particles of the present invention can be administered as a "pulsed dose" or as a "continuous stream".
[001712] [001712] As used in this application, a "pulsed dose" is a series of single unit doses of any drug administered at an established frequency over a period of time.
[001713] [001713] As used in this application, a "continuous flow" is a dose of a drug administered continuously for a period of time at a single route/single point of contact, i.e., a continuous administration event.
[001714] [001714] A total daily dose, an amount given or prescribed in a 24 hour period, may be administered by any of these methods, or as a combination of these methods, or by any other methods suitable for pharmaceutical administration.
[001715] [001715] In one embodiment, delivery of the AAV particles of the present invention to a subject provides AADC regulatory activity in a subject.
[001716] [001716] The regulatory activity may be an increase in the production of AADC in an individual.
[001717] [001717] The regulatory activity can be for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months,
[001718] [001718] In some embodiments, the AAV particle of the present invention can be administered to a subject using a single dose, with a single application.
[001719] [001719] The single application dose can be administered by any method known in the literature and/or described in this application.
[001720] [001720] As used in this application, a "single application" refers to a composition that is administered only once.
[001721] [001721] If necessary, a booster dose may be administered to the individual to ensure that appropriate efficacy is achieved.
[001722] [001722] A booster can be given 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years , 8 years, 9 years, 10 years, or more than 10 years of single application. Distribution Methods.
[001723] [001723] In one embodiment, the AAV particles or pharmaceutical compositions of the present invention may be administered or delivered by the methods of treating diseases described in US Patent No. 8,999,948, or International Publication No. WO 2014178863, the entirety of which is incorporated herein by title. of reference.
[001724] [001724] In one embodiment, the AAV particles or pharmaceutical compositions of the present invention may be administered or delivered by the methods of delivering gene therapy for Alzheimer's disease or other neurodegenerative conditions described in
[001725] [001725] In one embodiment, the AAV particles or pharmaceutical compositions of the present invention may be administered or delivered by the methods of delivering gene therapy to the CNS described in US Patent Nos. 6,436,/708, and 8,946,152, and International Publication No. WO2015168666 , the entirety of which is incorporated herein by reference.
[001726] [001726] In one embodiment, the AAV particle comprising a polynucleotide AADC may be administered or delivered by the methods for delivering AAV virions described in European Patent Application No. EP1857552, the entirety of which is incorporated herein by reference.
[001727] [001727] In one embodiment, the AAV particle or pharmaceutical compositions of the present invention can be administered or delivered by the methods for delivering proteins using the AAV particles described in European Patent Application No. EP2678433, the entirety of which is incorporated herein by title. of reference.
[001728] [001728] In one embodiment, the AAV particle comprising an AADC polynucleotide may be administered or delivered by the methods for delivering DNA molecules using the AAV particles described in US Patent No. US 5,858,351, the entirety of which is incorporated herein by title. of reference.
[001729] [001729] In one embodiment, the AAV particle or pharmaceutical compositions of the present invention may be administered or delivered by the methods for delivering DNA to the bloodstream described in US Patent No. 6,211,163, the entirety of which is incorporated herein by reference. .
[001730] [001730] In one embodiment, the AAV particle or pharmaceutical compositions of the present invention may be administered or delivered by the methods for delivering a payload to the central nervous system described in US Patent No. US 7,588,757, the entirety of which is incorporated herein by reference title.
[001731] [001731] In one embodiment, the AAV particle may be administered or delivered by the methods for delivering AAV virions described in US Patent No. 6,325,998, the entirety of which is incorporated herein by reference.
[001732] [001732] In one embodiment, the AAV particle or pharmaceutical compositions of the present invention may be administered or delivered by the methods for delivering a payload described in US Patent No. US 8,283,151, the entirety of which is incorporated herein by reference.
[001733] [001733] In one embodiment, the AAV particle or pharmaceutical compositions of the present invention may be administered or delivered by methods for delivering a payload using a glutamic acid decarboxylase (GAD) delivery vector described in International Patent Publication No. WO2001089583, the entirety of which is incorporated herein by reference.
[001734] [001734] In one embodiment, the AAV particle or pharmaceutical compositions of the present invention may be administered or delivered by the methods for delivering a payload to neural cells described in International Patent Publication No. WO2012057363, the entirety of which is incorporated herein by title. of reference. Distribution to Cells.
[001735] [001735] The present invention provides a method for delivering to a cell or tissue any of the AAV particles described above, comprising contacting the cell or tissue with said AAV particle or contacting the cell or tissue with a formulation comprising said AAV particle, or contacting the cell or tissue with any of the described compositions, including pharmaceutical compositions.
[001736] [001736] The method of delivering the AAV particle to a cell or tissue can be performed in vitro, ex vivo, or in vivo. Distribution to Individuals.
[001737] [001737] The present invention further provides a method for delivering to a subject, including a mammalian subject, any of the above-described AAV particles comprising administering to the subject said AAV particle, or administering to the subject a formulation comprising said AAV particle. AAV, or administering to the subject any of the described compositions, including pharmaceutical compositions. combinations.
[001738] [001738] AAV particles may be used in combination with one or more other therapeutic, prophylactic, research or diagnostic agents.
[001739] [001739] By "in combination with" it is not intended to imply that the agents need to be administered at the same time and/or formulated for joint delivery, although such delivery methods are within the scope of the present invention.
[001740] [001740] The compositions may be administered concomitantly, before, or subsequent to one or more other desired therapeutics or medical procedures.
[001741] [001741] In general, each agent will be administered at a dose and/or on a schedule determined for that agent.
[001742] [001742] In some embodiments, the present invention encompasses the delivery of pharmaceutical, prophylactic, research, or diagnostic compositions in combination with agents that can increase their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify its distribution in the body.
[001743] [001743] In one embodiment, the AAV particles described in this application can be administered to an individual who is also undergoing levodopa therapy.
[001744] [001744] As a non-limiting example, the individual may have a positive response to levodopa therapy and at least one PD symptom is reduced.
[001745] [001745] As another non-limiting example, the subject may have a response to levodopa therapy where the PD symptoms experienced by the subject are stable.
[001746] [001746] As yet another non-limiting example, the subject may have a negative response to levodopa therapy where the PD symptoms experienced by the subject increase.
[001747] [001747] In one embodiment, the dose of levodopa administered to the subject prior to the AAV particles is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more than 25 mg/kg.
[001748] [001748] As a non-limiting example, the dose is 3 mg/kg.
[001749] [001749] As another non-limiting example, the dose is 10 mg/kg.
[001750] [001750] As yet another non-limiting example, the dose is 20 mg/kg.
[001751] [001751] The subject's response (eg, behavioral response) to levodopa should be assessed prior to administration of AAV particles.
[001752] [001752] Additionally, the subject may receive levodopa once more after administration of the AADC polynucleotides (e.g., 1 week, 2, weeks, 3 weeks, | month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more than 1 year after administration of AAV particles).
[001753] [001753] The behavioral response can be re-evaluated and compared with the initial response to determine the effects of AAV particles.
[001754] [001754] Individual can have 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35 %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% improvement in behavior.
[001755] [001755] In one embodiment, Levodopa may be administered several times after administration of the AAV particles.
[001756] [001756] Levodopa can be given on a repeat schedule (eg, every 5 days, once a week, every 10 days, every 15 days, every 30 days, month, every two months, every every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or once a year) or when symptoms arise.
[001757] [001757] As a non-limiting example, 3 years after administration of AADC polynucleotides an individual may have 1-10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50 %, 30-50%, 40-50%, 40-60%, 50-70%, 50-80%, 60-70%, 60-75%, 60-80%, 60-90%, 70-80 %, 70-90%, 75-90%, 80-90%, 90-100% of striatal neurons in the infused region of the putamen immunoreactive to AADC.
[001758] [001758] As a non-limiting example, 6 years after administration of AAV particles an individual may have 1-10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50 %, 30-50%, 40-50%, 40-60%, 50-70%, 50-80%, 60-70%, 60-75%, 60-80%, 60-90%, 70-80 %, 70-90%, 75-90%, 80-90%, 90-100% of striatal neurons in the infused region of the putamen immunoreactive to AADC.
[001759] [001759] As a non-limiting example, 9 years after administration of AADC polynucleotides an individual may have 1-10%, 5-15%, 10-20%, 15-30%, 20-40%, 25-50 %, 30-50%, 40-50%, 40-
[001760] [001760] In one embodiment, an individual who can receive the AAV particles described in this application has a documented response to levodopa therapy but has clinically refractory fluctuations and is considered a good candidate for surgery.
[001761] [001761] The decision as to whether an individual is a good candidate for surgery can be made by the physician treating the individual for PD or by the physician administering the AAV particles taking into account the overall risk-benefit ratio to the patient for the intervention surgical procedure required for distribution of AAV particles. Expression Measurement.
[001762] [001762] The expression of payloads from viral genomes can be determined by various methods known in the literature, such as, but not limited to, immunochemistry (eg, IHC), in situ hybridization (ISH), enzyme-linked immunosorbent assay (ELISA), affinity ELISA, ELISPOT, flow cytometry, immunocytology, surface plasmon resonance analysis, kinetic exclusion assay, liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), BCA assay , immunoelectrophoresis, western blot analysis, SDS-PAGE, protein immunoprecipitation, and/or PCR.
[001763] [001763] The pharmaceutical compositions of AAV particles described in this application may be characterized by one or more of - bioavailability, therapeutic window and/or volume of distribution.
[001764] [001764] Bioavailability.
[001765] [001765] AAV particles, when formulated into a composition with a delivery agent described in this application, may exhibit an increase in bioavailability compared to a composition without a delivery agent described in this application.
[001766] [001766] As used in this application, the term "bioavailability" refers to the systemic availability of a given amount of AAV particles or expressed payload administered to a mammal.
[001767] [001767] Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (Cmax) of the composition.
[001768] [001768] AUC is an area under curve determination that plots the serum or plasma concentration of a compound (e.g., AAV particles or expressed payloads) along the ordinates (Y-axis) against time along the abscissa ( X axis).
[001769] [001769] Generally, the AUC for a particular compound can be calculated by methods known to those skilled in the art and described by G.S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, the entirety of which is incorporated herein by reference.
[001770] [001770] The Cmax value is the maximum concentration of the AAV particle or expressed payload achieved in the serum or plasma of a mammal after administration of the AAV particle to the mammal.
[001771] [001771] The Cmax value can be measured by methods known to those skilled in the art.
[001772] [001772] The phrases "increased bioavailability" or "improved pharmacokinetics: as used in this application means that the systemic bioavailability of a first AAV particle or expressed payload, measured as AUC, Cmax, or Cmin in a mammal is higher , when co-administered with a delivery agent described in this application, than when no such co-administration occurs.
[001773] [001773] In some embodiments, bioavailability may increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 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 55% 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%. Therapeutic Window.
[001774] [001774] As used in this application "therapeutic window" refers to the range of plasma concentrations, or the range of levels of the therapeutically active substance at the site of action, with a high probability of eliciting a therapeutic effect.
[001775] [001775] In some embodiments, the therapeutic window of the AAV particle described in this application may increase by at least about 2%, 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 50%, at least about 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 85% 90%, at least about 95%, or about 100%. Distribution Volume.
[001776] [001776] As used in this application, the term "volume of distribution" refers to the volume of fluid that would be required to contain the total amount of drug in the body at the same concentration as in blood or plasma: Vdist is equal to the amount of drug in the body/concentration of drug in blood or plasma.
[001777] [001777] For example, for a dose of 10 mg and a plasma concentration of 10 mg/L, the volume of distribution would be equal to 1 litre.
[001778] [001778] The volume of distribution reflects the extent to which the drug is present in extravascular tissue.
[001779] [001779] A large volume of distribution reflects the tendency of a compound to bind to tissue components compared to binding to plasma proteins.
[001780] [001780] In a clinical setting, Vdist can be used to determine a loading dose to achieve a steady-state concentration.
[001781] [001781] In some embodiments, the volume of distribution of the AAV particles described in this application may decrease by at least about 2%, 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 50%, at least about of 55%, at least about 60%, at least about 65%, at least about 70%. Biological Effect.
[001782] [001782] In one embodiment, the biological effect of AAV particles delivered to the animals can be classified by analyzing the expression of payload in the animals.
[001783] [001783] Payload expression can be determined from the analysis of a biological sample collected from a mammal that received the AAV particles of the present invention.
[001784] [001784] For example, a protein expression of 50-200 pg/ml for the protein encoded by the AAV particles delivered to the mammal — can be regarded as a therapeutically effective amount of protein in the mammal. METHODS AND USES OF THE COMPOSITIONS OF THE INVENTION. CNS diseases.
[001785] [001785] The polynucleotides of the present invention can be used in the treatment, prophylaxis or amelioration of any disease or disorder characterized by aberrant or unwanted target expression.
[001786] [001786] In one embodiment, the invention relates to AAV particles for use in treating Parkinson's disease.
[001787] [001787] In some embodiments, AAV particles may be used in the treatment, prophylaxis or amelioration of any disease or disorder characterized by aberrant or unwanted target expression in which the payload, ie, AADC, is exchanged for a payload alternative.
[001788] [001788] The present invention provides a method for the treatment of a disease, disorder and/or condition in a mammalian subject, including a human subject, comprising administering to the subject the AAV particles described in this application.
[001789] [001789] In one embodiment, the disease, disorder and/or condition is a neurological disease, disorder and/or condition.
[001790] [001790] CNS diseases can be diseases that affect any component of the brain (including the cerebral hemispheres, diencephalon, brainstem, and cerebellum) or the spinal cord.
[001791] [001791] In some embodiments, the viral particles of the present invention, through the delivery of a functional payload that can be a therapeutic product that can modulate the level or function of a gene product in the CNS, can be used to treat diseases neurodegenerative disorders and/or diseases or disorders that are characteristic of neurodegeneration, neuromuscular diseases,
[001792] [001792] A functional payload can alleviate or reduce symptoms that result from the abnormal level and/or function of a gene product (e.g., an absence or a defect in a protein) in an individual in need of it or who otherwise confers a benefit to a CNS disorder in an individual in need of it.
[001793] [001793] As non-limiting examples, the therapeutic products delivered by the AAV particles of the present invention may include, but are not limited to, growth factors and trophic factors, cytokines, hormones, neutrotransmitters, enzymes, anti-apoptotic factors, angiogenic factors, and any protein that is known to be mutated in pathological disorders such as "motor neuron survival" (SMN) RNA or antisense RNAi vectoring messenger RNAs encoding proteins of therapeutic interest in any of the CNS diseases discussed in this application; or microRNAs that function in gene silencing and post-transcriptional regulation of gene expression in the CNS (eg, brain-specific Mir-128a, see Adlakha and Saini, Molecular cancer, 2014, 13:33).
[001794] [001794] For example, an RNAi vectoring the enzyme superoxide dismutase can be packaged by the viral particles of the present invention, for the treatment of ALS.
[001795] [001795] Growth and trophic factors may include, but are not limited to, brain-derived growth factor (BDNF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF) , corticotropin releasing factor (CRF), glial cell lineage-derived growth factor (GDNF), insulin-like growth factor 1 (IGF-1), nerve growth factor (NGF), neurotrophin-3 (NT- 3), neurotrophin-4 (NTH4), and vascular endothelial growth factor (VEGF).
[001796] [001796] Cytokines may include interleukin-10 (IL-10), interleukin-6, interleukin-8, chemokine CXCL12 (SDF-1), TGF-beta, and growth and differentiation factor (GDF-1/10).
[001797] [001797] In some modalities, the neurological disorders may be neurodegenerative disorders including, but not limited to, Alzheimer's disease (AD); amyotrophic lateral sclerosis (ALS); Creutzfeldt-Jakob disease (CJD); Huntingtin disease (HD); Friedreich's ataxia (FA); Parkinson's disease (PD); multiple systemic atrophy (MSA); spinal muscular atrophy (SMA), multiple sclerosis (MS); primary progressive aphasia; progressive supranuclear palsy (PSP); insanity; brain cancer, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders that cause neurodegeneration, retinitis pigmentosa (RP), head and brain malformations, hydrocephalus, stroke, prion disease, infantile neuronal ceroid lipofuscinosis (INCL) (a neurodegenerative disease of children caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1)), and others.
[001798] [001798] In some embodiments, the viral particles of the present invention can be used to treat diseases that are associated with impaired growth and development of the CNS, i.e., neurodevelopmental disorders.
[001799] [001799] In some respects, such neurodevelopmental disorders can be caused by genetic mutations including, but not limited to, fragile X syndrome (caused by mutations in the FMR1 gene), Down syndrome (caused by trisomy 21), Down syndrome (caused by trisomy 21), Rett syndrome, Williams syndrome, Angelman syndrome, Smith-Magenis syndrome, ATR-X syndrome, Barth syndrome, immune dysfunction and/or childhood infectious diseases such as Sydenham chorea, schizophrenia, congenital toxoplasmosis, rubella syndrome congenital, metabolic disorders such as diabetes mellitus and phenylketonuria; nutritional defects and/or head trauma, autism and the autism spectrum.
[001800] [001800] In some embodiments, the viral particles of the present invention can be used to treat a tumor in the CNS, including, but not limited to, acoustic neuroma, astrocytoma (Grades I, II, III and IV), chordoma, CNS lymphoma, craniopharyngioma, gliomas (eg, brainstem glioma, ependymoma, optic nerve glioma, subependymoma), medulloblastoma, meningioma, metastatic brain tumors, oligodendroglioma, pituitary tumors, primitive neuroectoderm (PNET), and schwannoma.
[001801] [001801] In some modalities, the neurologic disorders may be functional neurologic disorders with motor and/or sensory symptoms that have a neurologic origin in the CNS.
[001802] [001802] As non-limiting examples, functional neurological disorders can be chronic pain, seizures, speech problems, involuntary movements, and sleep disorders.
[001803] [001803] In some modalities, neurological disorders can be white matter disorders (a group of disorders that affect nerve fibers in the CNS) including, binding partners, Pelizaeus-Merzbacher disease, hypermyelination with atrophy of the basal ganglia and cerebellum, Aicardi-Goutieres syndrome, megaloencephalic leuencephalopathy with subcortical cytos, congenital muscular dystrophies, myotonic dystrophy, Wilson's disease,
[001804] [001804] In some embodiments, neurological disorders can be lysosomal storage disorders (LSDs) caused by the inability of CNS cells to break down metabolic end products including, but not limited to, Niemann-Pick disease (an LSD resulting from an inherited deficiency in acid sphingomyelinase (ASM); metachromatic leukodystrophy (MLD) (an LSD characterized by the accumulation of sulfatides in glial cells and neurons, the result of an inherited deficiency in arylsulfatase A (ARSA)); globoid cell leukodystrophy (GLD) (an LSD caused by mutations in galactosylceramidase); Fabry disease (an LSD caused by mutations in the alpha-galactosidase A (GLA) gene); Gaucher disease (caused by mutations in the beta-glucocerebrosidase (GBA) gene); gangliosidosis GM1/GM2 ; mucopolysaccharidosis disorder; Pompe disease; and neuronal ceroid lipofuscinosis.
[001805] [001805] In another embodiment, the disease, disorder and/or neurological condition is Friedreich's ataxia.
[001806] [001806] In one embodiment, the AAV particle used to treat Friedreich's ataxia comprises a nucleic acid sequence such as, but not limited to, SEQ ID NO: 979 or a fragment or variant thereof, wherein the payload is replaced by frataxin or any other payload known in the art to treat Friedreich's ataxia.
[001807] [001807] In another embodiment, the disease, disorder and/or neurological condition is amyotrophic lateral sclerosis (ALS).
[001808] [001808] In one embodiment the AAV particle used to treat ALS comprises a nucleic acid sequence such as, but not limited to, SEQ ID NO: 979 or a fragment or variant thereof, wherein the payload is replaced by an sShRNA, miRNA, siRNA, RNAi for SOD1 or any other payload known in the prior art to treat ALS.
[001809] [001809] In another embodiment, the neurological disease, disorder and/or condition is Huntington's disease.
[001810] [001810] In one embodiment the AAV particle used to treat Huntington's disease comprises a nucleic acid sequence such as, but not limited to, SEQ ID NO: 9/79 or a fragment or variant thereof, wherein the payload is substituted by a shRNA, miRNA, siRNA, RNAi to Htt or any other payload known in the art to treat Huntington's disease.
[001811] [001811] In another embodiment, the disease, disorder or neurological condition is spinal muscular atrophy (SMA).
[001812] [001812] In another embodiment, the disease, disorder and/or neurological condition is Friedreich's ataxia.
[001813] [001813] In one embodiment the AAV particle used to treat NMS comprises a nucleic acid sequence such as, but not limited to, SEQ ID NO: 979 or a fragment or variant thereof, wherein the payload is replaced by frataxin or any another payload known in the art to handle SMA. Parkinson's disease.
[001814] [001814] In one embodiment, the disease, disorder and/or neurological condition is Parkinson's disease.
[001815] [001815] In one embodiment the AAV particle used to treat Parkinson's disease comprises a payload such as, but not limited to, SEQ ID NO: 979 or a fragment or variant thereof.
[001816] [001816] In one embodiment, the subject is a human patient who has a minimum motor score of about 30 to a maximum score of about 100, about 10 to a maximum score of about 100, about 20 to a maximum score of about 100. maximum of about 100 on the Unified Parkinson's Disease Rating Scale.
[001817] [001817] In one embodiment, the subject has been diagnosed with Parkinson's disease within the last 5 years prior to treatment with the compositions described in this application.
[001818] [001818] As a non-limiting example, the individual may have been diagnosed with Parkinson's in the last week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 1 year , 2 years, 3 years, 4 years or less than 5 years prior to treatment with the compositions described in this application.
[001819] [001819] In one embodiment, the subject has been diagnosed with Parkinson's disease between 5 and 10 years prior to treatment with the compositions described in this application.
[001820] [001820] As a non-limiting example, the individual may have been diagnosed with Parkinson's disease 5, 5.5,, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 years prior to treatment with the compositions described in this application.
[001821] [001821] In one embodiment, the subject has been diagnosed with Parkinson's disease more than 10 years prior to treatment with the compositions described in this application.
[001822] [001822] As a non-limiting example, the individual may have been diagnosed with Parkinson's 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15 .5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,
[001823] [001823] In one embodiment, an individual is 50-65 years of age.
[001824] [001824] As a non-limiting example, the individual is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 years of age.
[001825] [001825] As a non-limiting example, the individual is 50 years old.
[001826] [001826] As a non-limiting example, the individual is 51 years old.
[001827] [001827] As a non-limiting example, the individual is 52 years old.
[001828] [001828] As a non-limiting example, the individual is 53 years old.
[001829] [001829] As a non-limiting example, the individual is 54 years of age.
[001830] [001830] As a non-limiting example, the individual is 55 years of age.
[001831] [001831] As a non-limiting example, the individual is 56 years of age.
[001832] [001832] As a non-limiting example, the individual is 57 years old.
[001833] [001833] As a non-limiting example, the individual is 58 years of age.
[001834] [001834] As a non-limiting example, the individual is 59 years old.
[001835] [001835] As a non-limiting example, the individual is 60 years of age.
[001836] [001836] As a non-limiting example, the individual is 61 years of age.
[001837] [001837] As a non-limiting example, the individual is 62 years of age.
[001838] [001838] As a non-limiting example, the individual is 63 years of age.
[001839] [001839] As a non-limiting example, the individual is 64 years old.
[001840] [001840] As a non-limiting example, the individual is 65 years of age.
[001841] [001841] In one embodiment, an individual is 30 to 50 years of age.
[001842] [001842] As a non-limiting example, the individual is 30 years old.
[001843] [001843] As a non-limiting example, the individual is 31 years old.
[001844] [001844] As a non-limiting example, the individual is 32 years old.
[001845] [001845] As a non-limiting example, the individual is 33 years old.
[001846] [001846] As a non-limiting example, the individual is 34 years old.
[001847] [001847] As a non-limiting example, the individual is 35 years old.
[001848] [001848] As a non-limiting example, the individual is 36 years old.
[001849] [001849] As a non-limiting example, the individual is 37 years old.
[001850] [001850] As a non-limiting example, the individual is 38 years old.
[001851] [001851] As a non-limiting example, the individual is 39 years old.
[001852] [001852] As a non-limiting example, the individual is 40 years old.
[001853] [001853] As a non-limiting example, the individual is 41 years old.
[001854] [001854] As a non-limiting example, the individual is 42 years old.
[001855] [001855] As a non-limiting example, the individual is 43 years old.
[001856] [001856] As a non-limiting example, the individual is 44 years old.
[001857] [001857] As a non-limiting example, the individual is 45 years old.
[001858] [001858] As a non-limiting example, the individual is 46 years old.
[001859] [001859] As a non-limiting example, the individual is 47 years old.
[001860] [001860] As a non-limiting example, the individual is 48 years of age.
[001861] [001861] As a non-limiting example, the individual is 49 years old.
[001862] [001862] As a non-limiting example, the individual is 50 years old.
[001863] [001863] In one embodiment, an individual is 65 to 85 years of age.
[001864] [001864] As a non-limiting example, the individual is 65 years of age.
[001865] [001865] As a non-limiting example, the individual is 66 years old.
[001866] [001866] As a non-limiting example, the individual is 67 years old.
[001867] [001867] As a non-limiting example, the individual is 68 years of age.
[001868] [001868] As a non-limiting example, the individual is 69 years old.
[001869] [001869] As a non-limiting example, the individual is 70 years old.
[001870] [001870] As a non-limiting example, the individual is 71 years old.
[001871] [001871] As a non-limiting example, the individual is 72 years old.
[001872] [001872] As a non-limiting example, the individual is 73 years old.
[001873] [001873] As a non-limiting example, the individual is 74 years old.
[001874] [001874] As a non-limiting example, the individual is 75 years of age.
[001875] [001875] As a non-limiting example, the individual is 76 years old.
[001876] [001876] As a non-limiting example, the individual is 77 years old.
[001877] [001877] As a non-limiting example, the individual is 78 years of age.
[001878] [001878] As a non-limiting example, the individual is 79 years old.
[001879] [001879] As a non-limiting example, the individual is 80 years of age.
[001880] [001880] As a non-limiting example, the individual is 81 years of age.
[001881] [001881] As a non-limiting example, the individual is 82 years old.
[001882] [001882] As a non-limiting example, the individual is 83 years old.
[001883] [001883] As a non-limiting example, the individual is 84 years old.
[001884] [001884] As a non-limiting example, the individual is 85 years of age.
[001885] [001885] In one embodiment, a subject exhibits a change in motor symptoms such as tremors and movements prior to administration of the composition described in this application.
[001886] [001886] Non-limiting examples of tremors include mild unilateral or bilateral tremors, moderate bilateral or midline tremors or untreatable tremors.
[001887] [001887] Non-limiting examples of movements include mild bradykinesia, moderate bradykinesia, severe bradykinesia and morning akinesia.
[001888] [001888] In one modality, an individual may have alterations in balance such as, but not limited to, impaired balance, impaired postural reflexes, significant balance disturbance, or a fall.
[001889] [001889] In one embodiment, an individual may have a reduced quality of life.
[001890] [001890] As a non-limiting example, the individual may have a moderate impact on quality of life such as having some limitations in daily activities.
[001891] [001891] As another non-limiting example, the individual may have a quality of life that has been diminished by the disease.
[001892] [001892] In one embodiment, a subject will observe a change in non-motor symptoms prior to administration of the composition described in this application.
[001893] [001893] As a non-limiting example, the subject may experience mild to moderate cognitive impairment prior to administration of the composition described in this application.
[001894] [001894] As another non-limiting example, the individual may have significant cognitive impairment such as dementia which may also include behavioral disorders such as hallucinations.
[001895] [001895] In one embodiment, a subject may have a satisfactory response with limited fluctuations in one or more dopaminergic drugs prior to administration of the composition described in this application.
[001896] [001896] In one embodiment, a subject may have motor fluctuations causing mild to moderate disability on one or more dopaminergic drugs prior to administration of the composition described in this application.
[001897] [001897] In one embodiment, a subject may have clinically refractory motor fluctuations consisting of "wearing off" and/or levodopa-induced dyskinesias causing significant disability prior to administration of the composition described in this application.
[001898] [001898] In one embodiment, an individual may have mild symptoms associated with Parkinson's disease such as, but not limited to, no cognitive impairment, diagnosed within the last 5 years, satisfactory response with limited fluctuations in one or more dopaminergic drugs, mild unilateral tremors or bilateral, little to no impact on quality of life, and/or no impairment of balance.
[001899] [001899] In one modality, an individual may have moderate symptoms associated with Parkinson's disease such as, but not limited to, mild to moderate cognitive impairment, early signs of compromised balance and postural reflexes, motor fluctuations causing mild to moderate disability in a or more dopaminergic medications, diagnosed within the last 5 to 10 years, moderate bilateral or midline tremors, moderate bradykinesia, and/or an individual experiencing some limitations in daily activities.
[001900] [001900] In one modality, an individual may have advanced symptoms associated with Parkinson's disease such as, but not limited to, having been diagnosed with Parkinson's more than 10 years ago, average refractory motor fluctuations, wasting and/or levodopa-induced dyskinesia that cause significant disability, untreatable tremors, significant balance disturbance and/or fall, significant cognitive impairment (such as dementia with or without behavioral disorders), severe bradykinesia, markedly impaired quality of life from the disease, and/or morning akinesia.
[001901] [001901] In one embodiment, an individual was referred to a movement disorder specialist (MDS) but did not undergo deep brain stimulation.
[001902] [001902] In one embodiment, a subject is making use of DUOPATM in combination with the compositions described in this application.
[001903] [001903] As a non-limiting example, the individual may be successful with the use of DUOPAT TM only.
[001904] [001904] As a non-limiting example, the individual may have no or limited success using DUOPA TM alone.
[001905] [001905] In one embodiment, an individual is an individual who was a candidate for surgical intervention including, but not limited to, deep brain stimulation.
[001906] [001906] As a non-limiting example, deep brain stimulation has been suggested because of disabling motor complications despite treatment with optimal anti-Parkinsonian medication.
[001907] [001907] In one embodiment, an individual has an average wake time of 7.5-14 hours based on the individual's day.
[001908] [001908] As a non-limiting example, the average wake time is 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8 .4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9 .7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11 , 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3 , 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6 , 13.7, 13.8, 13.9, or 14 hours.
[001909] [001909] In one embodiment, an individual has an average wake time of 10.5 hours based on the individual's day.
[001910] [001910] In one embodiment, an individual experiences more than about 1.5 hours of daily waking time (no bothersome dyskinesia), compared to baseline, 6 months after administration of the present invention.
[001911] [001911] As a non-limiting example, an individual experiences more than about 1.5 hours of daily waking time 6 months after administration of the present invention at a dose volume of up to 900 µL per putamen and a total dose of 4 .5 x 1012 vector genome.
[001912] [001912] In one embodiment, an individual experiences more than about 2.
[001913] [001913] Two hours of daily waking time (no bothersome dyskinesia), compared to baseline, 6 months after administration of the present invention.
[001914] [001914] As a non-limiting example, an individual experiences more than about 2.
[001915] [001915] Two hours of daily waking time 6 months after administration of the present invention in a dose volume of up to 900 µl per putamen and a total dose of 1.5 x 10 12 vector genomes.
[001916] [001916] In one embodiment, an individual experiences more than about 4 hours of daily waking time (without bothersome dyskinesia), compared to baseline, 6 months after administration of the present invention.
[001917] [001917] As a non-limiting example, an individual experiences more than about 4 hours of daily waking time 6 months after administration of the present invention at a dose volume of up to 900 µL per putamen and a total dose of 1.5 x 1012 vector genomes.
[001918] [001918] In one embodiment, an individual experiences more than about 1.6 hours of daily waking time (without bothersome dyskinesia), compared to baseline, 12 months after administration of the present invention.
[001919] [001919] As a non-limiting example, an individual experiences more than about 1.6 hours of daily wake time 12 months after administration of the present invention at a dose volume of up to 450 µL per putamen and a total dose of 7 .5 x 1011 vector genomes.
[001920] [001920] In one embodiment, an individual experiences more than about 3.3 hours of daily waking time (no bothersome dyskinesia), compared to baseline, 12 months after administration of the present invention.
[001921] [001921] As a non-limiting example, an individual experiences more than about 3.3 hours of daily waking time 12 months after administration of the present invention at a dose volume of up to 900 ul per putamen and a total dose of 1 .5 x 1012 vector genomes.
[001922] [001922] In one embodiment, an individual experiences more than about 4 hours of daily waking time (no bothersome dyskinesia), compared to baseline, 12 months after administration of the present invention.
[001923] [001923] As a non-limiting example, an individual experiences more than about 4 hours of daily waking time 12 months after administration of the present invention at a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes.
[001924] [001924] In one embodiment, an individual experiences more than about 2.3 hours of daily waking time (without bothersome dyskinesia), compared to baseline, 24 months after administration of the present invention.
[001925] [001925] As a non-limiting example, an individual experiences more than about 2.3 hours of daily waking time 24 months after administration of the present invention at a dose volume of up to 450 µL per putamen and a total dose of 7 .5 x 1011 vector genomes.
[001926] [001926] In one embodiment, an individual has an average rest time of 2-7 hours based on the individual's day.
[001927] [001927] As a non-limiting example, the average resting time is 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2 .9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2 , 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5 , 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 , 6.9, or 7.
[001928] [001928] In one embodiment, an individual has an average rest time of 4.6 hours based on the individual's day.
[001929] [001929] In one embodiment, a subject spends less than about 1.3 hours of daily rest time, compared to baseline, 6 months after administration of the present invention.
[001930] [001930] As a non-limiting example, a subject experiences less than about 1.3 hours of daily rest time 6 months after administration of the present invention at a dose volume of up to 900 uL per putamen and a total dose of 4 5 x 1012 vector genomes.
[001931] [001931] In one embodiment, a subject experiences less than about 1.1 hours of daily rest time, compared to baseline, 6 months after administration of the present invention.
[001932] [001932] As a non-limiting example, a subject experiences less than about 1.1 hours of daily rest time 6 months after administration of the present invention at a dose volume of up to 900 µL per putamen and a total dose of 1 .5 x 1012 vector genomes.
[001933] [001933] In one embodiment, a subject experiences less than about 0.8 hours of daily rest time, compared to baseline, 6 months after administration of the present invention.
[001934] [001934] As a non-limiting example, a subject experiences less than about 0.8 hours of daily rest time 6 months after administration of the present invention at a dose volume of up to 450 uL per putamen and a total dose of 7 .5 x 1011 vector genomes.
[001935] [001935] In one embodiment, a subject experiences less than about 2.3 hours of daily rest time, compared to baseline, 12 months after administration of the present invention.
[001936] [001936] As a non-limiting example, a subject experiences less than about 2.3 hours of daily rest time 12 months after administration of the present invention at a dose volume of up to 900 µL per putamen and a total dose of 1 .5 x 1012 vector genomes.
[001937] [001937] In one embodiment, a subject experiences less than about 1.4 hours of daily rest time, compared to baseline, 12 months after administration of the present invention.
[001938] [001938] As a non-limiting example, a subject experiences less than about 1.4 hours of daily rest time 12 months after administration of the present invention at a dose volume of up to 450 uL per putamen and a total dose of 7 .5 x 1011 vector genomes.
[001939] [001939] In one embodiment, a subject experiences less than about 1.8 hours of daily rest time, compared to baseline, 24 months after administration of the present invention.
[001940] [001940] As a non-limiting example, a subject experiences less than about 1.8 hours of daily rest time 24 months after administration of the present invention at a dose volume of up to 450 uL per putamen and a total dose of 7 .5 x 1011 vector genomes.
[001941] [001941] In one embodiment, a subject experiences 10% less daily rest time 6 months after administration of the present invention.
[001942] [001942] In one embodiment, a subject experiences 10% less daily rest time 12 months after administration of the present invention.
[001943] [001943] In one embodiment, a subject experiences 20% less daily rest time 6 months after administration of the present invention.
[001944] [001944] In one embodiment, a subject experiences 20% less daily rest time 12 months after administration of the present invention.
[001945] [001945] In one embodiment, a subject experiences 30% less daily rest time 6 months after administration of the present invention.
[001946] [001946] In one embodiment, a subject experiences 30% less daily rest time 12 months after administration of the present invention.
[001947] [001947] In one embodiment, a subject experiences 40% less daily rest time 6 months after administration of the present invention.
[001948] [001948] In one embodiment, a subject experiences 40% less daily rest time 12 months after administration of the present invention.
[001949] [001949] In one embodiment, a subject experiences 50% less daily rest time 6 months after administration of the present invention.
[001950] [001950] In one embodiment, a subject experiences 50% less daily rest time 12 months after administration of the present invention.
[001951] [001951] In one embodiment, a subject experiences 60% less daily rest time 6 months after administration of the present invention.
[001952] [001952] In one embodiment, a subject experiences 60% less daily rest time 12 months after administration of the present invention.
[001953] [001953] In one embodiment, a subject experiences 70% less daily rest time 6 months after administration of the present invention.
[001954] [001954] In one embodiment, a subject experiences 70% less daily rest time 12 months after administration of the present invention.
[001955] [001955] In one embodiment, a subject experiences 80% less daily rest time 6 months after administration of the present invention.
[001956] [001956] In one embodiment, a subject experiences 80% less daily rest time 12 months after administration of the present invention.
[001957] [001957] In one embodiment, a subject experiences 90% less daily rest time 6 months after administration of the present invention.
[001958] [001958] In one embodiment, a subject experiences 90% less daily rest time 12 months after administration of the present invention.
[001959] [001959] In one embodiment, a subject experiences 95% less daily rest time 6 months after administration of the present invention.
[001960] [001960] In one embodiment, a subject experiences 95% less daily rest time 12 months after administration of the present invention.
[001961] [001961] In one embodiment, an individual's UPDRS-3 (or UPDRS-III) medication score is assessed prior to administration of the present invention.
[001962] [001962] As a non-limiting example, the subject's UPDRS-3 (or UPDRS-III) medication score prior to administration of the present invention is between 1-40, 1-10, 1-15, 1-20, 1 -25, 1-30, 1-35, 1-40, 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 10-15, 10-20 , 10-25, 10-30, 10-35, 10-40, 15-20, 15-25, 15-30, 15-35, 15-40, 20-25, 20-30, 20-35, 20 -40, 25-30, 25-35, 25-40, 30-35, 30-40, or 35-40.
[001963] [001963] As a non-limiting example, the subject's UPDRS-3 (or UPDRS-III) medication score prior to administration of the present invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.
[001964] [001964] As a non-limiting example, the subject's UPDRS-3 (or UPDRS-III) medication score prior to administration of the present invention is 10.1, 10.2, 10.3, 10.4, 10, 5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11, 8, 11.9, 12, 12.1, 12.2,
[001965] [001965] As a non-limiting example, the subject's UPDRS-3 medication score prior to administration of the present invention is 13.5.
[001966] [001966] In one embodiment, a subject's UPDRS-3 (or UPDRS-IlI) medication score is reduced after administration of the present invention.
[001967] [001967] An individual's UPDRS-3 (or UPDRS-III) medication score may be reduced by a percentage such as, but not limited to, 10%, 15%, 20%, 25%, 30%, 35% , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 95%.
[001968] [001968] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 10% after administration of the present invention.
[001969] [001969] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 20% after administration of the present invention.
[001970] [001970] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 30% after administration of the present invention.
[001971] [001971] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 40% after administration of the present invention.
[001972] [001972] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 50% after administration of the present invention.
[001973] [001973] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 60% after administration of the present invention.
[001974] [001974] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 70% after administration of the present invention.
[001975] [001975] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 80% after administration of the present invention.
[001976] [001976] As a non-limiting example, a subject's UPDRS-3 medication score is reduced by 90% after administration of the present invention.
[001977] [001977] An individual's UPDRS-3 (or UPDRS-III) medication score can be changed by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0, 7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 24, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3, 4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4, 7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 71.3, RT, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8, 7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15 points.
[001978] [001978] As a non-limiting example, an individual's UPDRS-3 medication score is changed by 0.4 points.
[001979] [001979] As a non-limiting example, an individual's UPDRS-3 medication score is changed by 1.6 points.
[001980] [001980] As a non-limiting example, an individual's UPDRS-3 medication score is changed by 1.8 points.
[001981] [001981] As a non-limiting example, an individual's UPDRS-3 medication score is changed by 8.6 points.
[001982] [001982] As a non-limiting example, an individual's UPDRS-3 medication score is changed by 9.6 points.
[001983] [001983] An individual's UPDRS-3 (or UPDRS-III) medication score may increase by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2 .1, 2.2, 2.3, 24, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 , 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6 .1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 74 , 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7 , 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10 .1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11 .4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12 .7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14 , 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15 points.
[001984] [001984] As a non-limiting example, an individual's UPDRS-3 medication score is increased by 0.4 points.
[001985] [001985] As a non-limiting example, an individual's UPDRS-3 medication score is increased by 1.6 points.
[001986] [001986] As a non-limiting example, an individual's UPDRS-3 medication score is increased by 1.8 points.
[001987] [001987] As a non-limiting example, an individual's UPDRS-3 medication score is increased by 8.6 points.
[001988] [001988] As a non-limiting example, an individual's UPDRS-3 medication score is increased by 9.6 points.
[001989] [001989] An individual's UPDRS-3 (or UPDRS-III) medication score may decrease by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 , 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2 .1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3 .4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4 .7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 ,
[001990] [001990] As a non-limiting example, an individual's UPDRS-3 medication score is reduced by 0.4 points.
[001991] [001991] As a non-limiting example, an individual's UPDRS-3 medication score is reduced by 1.6 points.
[001992] [001992] As a non-limiting example, an individual's UPDRS-3 medication score is reduced by 1.8 points.
[001993] [001993] As a non-limiting example, an individual's UPDRS-3 medication score is reduced by 8.6 points.
[001994] [001994] As a non-limiting example, an individual's UPDRS-3 medication score is reduced by 9.6 points.
[001995] [001995] In one embodiment, a subject's UPDRS-3 (or UPDRS-II!) medication score is reduced by 8.6 6 months after administration of the present invention at a dose volume of up to 900 ul per putamen and a total dose of 4.5 x 1012 vector genomes.
[001996] [001996] In one embodiment, a subject's UPDRS-3 (or UPDRS-II!) medication score is reduced by 9.6 6 months after administration of the present invention at a dose volume of up to 900 ul per putamen and a total dose of 1.5 x 1012 vector genomes.
[001997] [001997] In one embodiment, a subject's UPDRS-3 (or UPDRS-III) medication score is reduced by 9.6 12 months after administration of the present invention at a dose volume of up to
[001998] [001998] In one embodiment, the average amount of medication for Parkinson's disease in a subject was about 1500 mg per day prior to administration of the present invention.
[001999] [001999] As a non-limiting example, the medication for Parkinson's is levodopa.
[002000] [002000] In one embodiment, an individual's UPDRS-I score is assessed prior to administration of the present invention.
[002001] [002001] A subject's UPDRS-I score prior to administration of the present invention is between 20 and 50, such as, but not limited to, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.
[002002] [002002] An individual's UPDRS-2 (or UPDRS-II) score may change by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 .8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 , 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6 .1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7 .4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8 .7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 , 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3 , 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6 , 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 , 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15 points.
[002003] [002003] An individual's UPDRS-2 (or UPDRS-II) score may increase by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 .8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 24, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3 .5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4 .8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1 , 6.2, 6.3,
[002004] [002004] An individual's UPDRS-2 (or UPDRS-II) score may decrease by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0 .8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 , 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6 .1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7 .4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8 .7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 , 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3 , 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6 , 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9 , 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or 15 points.
[002005] [002005] As a non-limiting example, a reduction of 3.6 points is observed 12 months after administration of the present invention.
[002006] [002006] As a non-limiting example, a reduction of 3.6 points is observed 6 months after administration of the present invention.
[002007] [002007] In one embodiment, a subject's UPDRS-II score is decreased by 2 to 4 points after administration of the present invention relative to the UPDRS-II score before administration.
[002008] [002008] In one embodiment, an individual's UPDRS-I score is decreased by 2 to 3 points 6 months after administration of the present invention at a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes, in relation to UPDRS-ll medication score before administration.
[002009] [002009] In one embodiment, a subject's UPDRS-I score is decreased by 2 to 3 points 12 months after administration of the present invention at a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes, in relation to UPDRS-ll medication score before administration.
[002010] [002010] In one embodiment, a subject's UPDRS-I score is decreased by 3 to 4 points 6 months after administration of the present invention at a dose volume of up to 900 uL per putamen and a total dose of 4.5 x 1012 vector genomes, in relation to UPDRS-ll medication score before administration.
[002011] [002011] In one embodiment, the present invention is used to improve the motor function of an individual.
[002012] [002012] In one embodiment, the present invention is used to control an individual's motor function and improve their quality of life.
[002013] [002013] In one embodiment, the present invention is used to reduce the dosage of Parkinson's medication in a subject who needs it to improve the subject's motor function.
[002014] [002014] In one embodiment, a single administration of the present invention into a subject's putamen provides improved motor function compared to motor function prior to treatment.
[002015] [002015] In one embodiment, a single administration of the present invention to a subject's putamen provides improved motor function and a reduction in the amount of levodopa the subject requires to control symptoms.
[002016] [002016] In one embodiment, a single administration of the present invention to a subject's putamen provides improved motor function and a reduction in the amount of dopaminergic medication the subject requires to control symptoms.
[002017] [002017] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 10-50% after administration of the present invention.
[002018] [002018] As a non-limiting example, the reduction is observed 6 months after administration of the present invention.
[002019] [002019] As a non-limiting example, the reduction is observed 12 months after administration of the present invention.
[002020] [002020] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 10-20% after administration of the present invention.
[002021] [002021] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 10%.
[002022] [002022] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 11%.
[002023] [002023] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 12%.
[002024] [002024] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 13%.
[002025] [002025] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 14%.
[002026] [002026] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 15%.
[002027] [002027] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 16%.
[002028] [002028] As a non-limiting example, the reduction in Parkinson's medication (eg Levodopa) is 17%.
[002029] [002029] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 18%.
[002030] [002030] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 19%.
[002031] [002031] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 20%.
[002032] [002032] In one embodiment, a 14% reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 6 months after administration of the present invention in a dose volume of up to 450 ul per putamen and a total dose of 7.5 x 1011 vector genomes.
[002033] [002033] In one embodiment, a 10% reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 12 months after administration of the present invention in a dose volume of up to 450 uL per putamen and a total dose of 7.5 x 1011 vector genomes.
[002034] [002034] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 20-30% after administration of the present invention.
[002035] [002035] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 20%.
[002036] [002036] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 21%.
[002037] [002037] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 22%.
[002038] [002038] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 23%.
[002039] [002039] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 24%.
[002040] [002040] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 25%.
[002041] [002041] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 26%.
[002042] [002042] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 27%.
[002043] [002043] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 28%.
[002044] [002044] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 29%.
[002045] [002045] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 30%.
[002046] [002046] In one embodiment, a 27% reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 12 months after administration of the present invention in a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes.
[002047] [002047] In one embodiment, a 28% reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 12 months after administration of the present invention in a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes.
[002048] [002048] In one embodiment, a 29% reduction in the daily dose amount of Parkinson's medication (eg Levodopa)
[002049] [002049] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 30-40% after administration of the present invention.
[002050] [002050] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 30%.
[002051] [002051] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 31%.
[002052] [002052] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 32%.
[002053] [002053] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 33%.
[002054] [002054] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 34%.
[002055] [002055] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 35%.
[002056] [002056] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 36%.
[002057] [002057] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 37%.
[002058] [002058] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 38%.
[002059] [002059] “As a non-limiting example, the reduction in Parkinson's medication (eg Levodopa) is 39%.
[002060] [002060] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 40%.
[002061] [002061] In one embodiment, a 34% reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 6 months after administration of the present invention in a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes.
[002062] [002062] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 40-50% after administration of the present invention.
[002063] [002063] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 40%.
[002064] [002064] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 41%.
[002065] [002065] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 42%.
[002066] [002066] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 43%.
[002067] [002067] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 44%.
[002068] [002068] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 45%.
[002069] [002069] “As a non-limiting example, the reduction in Parkinson's medication (eg Levodopa) is 46%.
[002070] [002070] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 47%.
[002071] [002071] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 48%.
[002072] [002072] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 49%.
[002073] [002073] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 50%.
[002074] [002074] In one embodiment, a 34% reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 6 months after administration of the present invention in a dose volume of up to 900 ul per putamen and a total dose of 4.
[002075] [002075] 5x 1012 vector genomes.
[002076] [002076] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 108-641 mg after administration of the present invention.
[002077] [002077] As a non-limiting example, the reduction is observed 6 months after administration of the present invention.
[002078] [002078] As a non-limiting example, the reduction is observed 12 months after administration of the present invention.
[002079] [002079] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 108-339 mg after administration of the present invention.
[002080] [002080] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 108 mg.
[002081] [002081] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 134 mg.
[002082] [002082] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 159 mg.
[002083] [002083] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 154 mg.
[002084] [002084] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 208 mg.
[002085] [002085] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 231 mg.
[002086] [002086] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 254 mg.
[002087] [002087] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 276 mg.
[002088] [002088] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 298 mg.
[002089] [002089] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 319 mg.
[002090] [002090] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 339 mg.
[002091] [002091] In one embodiment, a 208 mg reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 6 months after administration of the present invention in a dose volume of up to 450 uL per putamen and a total dose of 7.5 x 1011 vector genomes.
[002092] [002092] In one embodiment, a 108 mg reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 12 months after administration of the present invention in a dose volume of up to 450 ul per putamen and a total dose of 7.5Xx 1011 vector genomes.
[002093] [002093] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 339-505 mg after administration of the present invention.
[002094] [002094] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 339 mg.
[002095] [002095] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 358 mg.
[002096] [002096] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 377 mg.
[002097] [002097] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 396 mg.
[002098] [002098] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 413 mg.
[002099] [002099] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 430 mg.
[002100] [002100] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 446 mg.
[002101] [002101] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 462 mg.
[002102] [002102] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 477 mg.
[002103] [002103] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 491 mg.
[002104] [002104] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 505 mg.
[002105] [002105] In one embodiment, a 462 mg reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 12 months after administration of the present invention in a dose volume of up to 900 ul per putamen and a total dose of 1.5x 1012 vector genomes.
[002106] [002106] In one embodiment, a 477 mg reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 12 months after administration of the present invention in a dose volume of up to 900 ul per putamen and a total dose of 1.5Xx 1012 vector genomes.
[002107] [002107] In one embodiment, a 491 mg reduction in the daily dose amount of Parkinson's medication (eg Levodopa)
[002108] [002108] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 505-606 mg after administration of the present invention.
[002109] [002109] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 505 mg.
[002110] [002110] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 518 mg.
[002111] [002111] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 530 mg.
[002112] [002112] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 542 mg.
[002113] [002113] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 553 mg.
[002114] [002114] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 563 mg.
[002115] [002115] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 573 mg.
[002116] [002116] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 582 mg.
[002117] [002117] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 591 mg.
[002118] [002118] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 599 mg.
[002119] [002119] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 606 mg.
[002120] [002120] In one embodiment, a 553 mg reduction in the amount of daily dose of Parkinson's medication (e.g. Levodopa) required by a subject to control symptoms occurs 6 months after administration of the present invention in a dose volume of up to 900 uL per putamen and a total dose of 1.5 x 1012 vector genomes.
[002121] [002121] In one embodiment, the daily dose amount of Parkinson's medication (e.g. Levodopa) is reduced by 606-641 mg after administration of the present invention.
[002122] [002122] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 606 mg.
[002123] [002123] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 612 mg.
[002124] [002124] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 618 mg.
[002125] [002125] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 623 mg.
[002126] [002126] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 628 mg.
[002127] [002127] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 632 mg.
[002128] [002128] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 635 mg.
[002129] [002129] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 637 mg.
[002130] [002130] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 639 mg.
[002131] [002131] As a non-limiting example, the reduction of Parkinson's medication (eg Levodopa) is 641 mg.
[002132] [002132] In one embodiment, a 553 mg reduction in the daily dose amount of Parkinson's medication (eg,
[002133] [002133] 5x1012 vector genomes.
[002134] [002134] In one embodiment, putaminal AADC enzyme activity is increased in a subject following administration of the present invention.
[002135] [002135] As a non-limiting example, the increase being observed for at least 6 months from baseline.
[002136] [002136] In one embodiment, the activity of the putaminal AADC enzyme is increased by 10-20% in a subject after administration of the present invention.
[002137] [002137] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 10%.
[002138] [002138] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 11%.
[002139] [002139] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 12%.
[002140] [002140] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 13%.
[002141] [002141] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 14%.
[002142] [002142] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 15%.
[002143] [002143] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 16%.
[002144] [002144] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 17%.
[002145] [002145] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 18%.
[002146] [002146] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 19%.
[002147] [002147] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 20%.
[002148] [002148] In one embodiment, putaminal AADC enzyme activity is increased by about 13% in a subject after administration of the present invention at a dose volume of up to 450 ul per putamen and a total dose of 7.5 x 1011 vector genomes.
[002149] [002149] In one embodiment, the activity of the putaminal AADC enzyme is increased by 50-60% in a subject after administration of the present invention.
[002150] [002150] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 50%.
[002151] [002151] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 51%.
[002152] [002152] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 52%.
[002153] [002153] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 53%.
[002154] [002154] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 54%.
[002155] [002155] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 55%.
[002156] [002156] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 56%.
[002157] [002157] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 57%.
[002158] [002158] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 58%.
[002159] [002159] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 59%.
[002160] [002160] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 60%.
[002161] [002161] In one embodiment, putaminal AADC enzyme activity is increased by about 56% in a subject after administration of the present invention at a dose volume of up to 900 µl per putamen and a total dose of 1.5 x 10 12 vector genomes.
[002162] [002162] In one embodiment, the activity of the putaminal AADC enzyme is increased by 70-85% in a subject after administration of the present invention.
[002163] [002163] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 70%.
[002164] [002164] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 71%.
[002165] [002165] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 72%.
[002166] [002166] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 73%.
[002167] [002167] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 74%.
[002168] [002168] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 75%.
[002169] [002169] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 76%.
[002170] [002170] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 77%.
[002171] [002171] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 78%.
[002172] [002172] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 79%.
[002173] [002173] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 80%.
[002174] [002174] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 81%.
[002175] [002175] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 82%.
[002176] [002176] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 83%.
[002177] [002177] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 84%.
[002178] [002178] As a non-limiting example, the increase in putaminal AADC enzyme activity is about 85%.
[002179] [002179] In one embodiment, putaminal AADC enzyme activity is increased by about 79% in a subject after administration of the present invention at a dose volume of up to 900 uL per putamen and a total dose of 4.5 x 1012 vector genomes.
[002180] [002180] In one embodiment, a subject's dopamine level increases after administration of the present invention.
[002181] [002181] As a non-limiting example, the amount of dopamine is increased by 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50% , 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100% , or 95-100%. Circadian Rhythm and Sleep-Wake Cycles.
[002182] [002182] Circadian rhythms are physical, mental and behavioral changes that tend to follow a 24-hour cycle.
[002183] [002183] Circadian rhythms can influence sleep-wake cycles, hormone release, body temperature and other bodily functions.
[002184] [002184] Changes in the circadian cycle can cause conditions and/or disorders such as, but not limited to, sleep disorders (eg, insomnia), depression, bipolar disorder, seasonal affective disorder, obesity, and diabetes.
[002185] [002185] In one embodiment, the AAV particles described in this application can be used to treat insomnia.
[002186] [002186] The sleep-wake cycle comprises periods of sleep and periods of wakefulness.
[002187] [002187] Generally, in a 24-hour period the number of total hours of sleep is less than the number of total waking hours.
[002188] [002188] As a non-limiting example, the sleep-wake cycle comprises 7-9 hours of sleep and 15-17 hours of wakefulness.
[002189] [002189] As a non-limiting example, the sleep-wake cycle comprises 8 hours of sleep and 16 hours of wakefulness.
[002190] [002190] As a non-limiting example, the sleep-wake cycle comprises 8-10 hours of sleep and 14-16 hours of wakefulness.
[002191] [002191] In one embodiment, an individual's sleep-wake cycle is improved by administering to the individual the AAV particles described in this application.
[002192] [002192] In one embodiment, an individual's sleep-wake cycle is regulated through the administration to the individual of the AAV particles described in this application.
[002193] [002193] As a non-limiting example, regulation could be correction of more sleep periods occurring at night and fewer sleep periods occurring.
[002194] [002194] In one embodiment, the sleep-wake cycle of a subject who has received the AAV particles described in this application improves compared to the sleep-wake cycle of the subject prior to administration of the AAV particles.
[002195] [002195] As a non-limiting example, the individual has an increased sleep period and a reduced wake period.
[002196] [002196] As another non-limiting example, the individual has a reduced sleep period and an increased wake period.
[002197] [002197] In one embodiment, the sleep-wake cycle of a subject who has received the AAV particles described in this application is regulated in comparison to the sleep-wake cycle of the subject prior to administration of the AAV particles.
[002198] [002198] As a non-limiting example, the duration of sleep periods and waking periods can be approximately the same (eg +/- 1 hour) for at least 2 days.
[002199] [002199] As another non-limiting example, the duration of sleep periods and waking periods if a 24 hour period can be within 10 minutes, 15 minutes, 20 minutes, 25 minutes, minutes, 35 minutes, 40 minutes , 45 minutes, 50 minutes, 55 minutes, one hour, 1.5 hours, or two hours from the previous 24-hour period.
[002200] [002200] In one embodiment, the amount of rapid eye movement (REM) sleep that an individual experiences in a 24 hour period is altered after the individual receives the AAV particles described in this application.
[002201] [002201] REM sleep is generally considered a period of active sleep marked by intense brain activity where brain waves are fast and out of sync.
[002202] [002202] An adult spends, on average, about 20-25% of their total daily sleep period in REM sleep.
[002203] [002203] As a non-limiting example, the amount of REM sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, T%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002204] [002204] As a non-limiting example, the amount of REM sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%, 20-25 %, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002205] [002205] As a non-limiting example, the amount of REM sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002206] [002206] As a non-limiting example, the amount of REM sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25 %, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002207] [002207] In one embodiment, the amount of non-REM (NREM) sleep that an individual experiences in a 24 hour period is altered after the individual receives the AAV particles described in this application.
[002208] [002208] NREM sleep is generally characterized by a reduction in physiological activity as brain waves, as measured by EEG, slow down and have greater amplitude.
[002209] [002209] NREM has four stages: Stage 1 is the time of drowsiness or transition from being awake to falling asleep where brain waves and muscle activity start to slow down; Stage 2 is a period of light sleep during which eye movements stop and brain waves slow down with occasional bursts of fast waves (sometimes called sleep spindles); Stage 3 and Stage 4 (together called slow wave sleep) are characterized by the presence of slow brain waves (delta waves) interspersed with smaller, faster waves where there is no eye movement.
[002210] [002210] An adult spends, on average, about 75-80% of their total daily sleep period in NREM sleep with about half of their total daily sleep period in stage 2 NREM sleep.
[002211] [002211] In one embodiment, the amount of NREM sleep that an individual experiences is altered after the individual receives the AAV particles described in this application.
[002212] [002212] As a non-limiting example, the amount of NREM sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002213] [002213] As a non-limiting example, the amount of NREM sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%, 20-25 %, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002214] [002214] As a non-limiting example, the amount of NREM sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002215] [002215] As a non-limiting example, the amount of NREM sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25 %, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002216] [002216] In one embodiment, the amount of Stage 1 NREM sleep that an individual experiences is altered after the individual receives the AAV particles described in this application.
[002217] [002217] As a non-limiting example, the amount of NREM Stage 1 sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, T%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002218] [002218] As a non-limiting example, the amount of NREM Stage 1 sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%, 15-
[002219] [002219] As a non-limiting example, the amount of NREM Stage 1 sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002220] [002220] As a non-limiting example, the amount of NREM Stage 1 sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15 -25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002221] [002221] In one embodiment, the amount of NREM Stage 2 sleep an individual experiences is altered after the individual receives the AAV particles described in this application.
[002222] [002222] As a non-limiting example, the amount of NREM Stage 2 sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, T%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002223] [002223] As a non-limiting example, the amount of NREM Stage 2 sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25%, 20 -25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002224] [002224] As a non-limiting example, the amount of NREM Stage 2 sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, T%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65%.
[002225] [002225] As a non-limiting example, the amount of NREM Stage 2 sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15 -25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002226] [002226] In one embodiment, the amount of NREM Stage sleep
[002227] [002227] As a non-limiting example, the amount of NREM Stage 3 and 4 sleep is decreased by 1%, 2%, 3%, 4%, 5%, 6%, T%, 8%, 9%, 10% , 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65% .
[002228] [002228] As a non-limiting example, the amount of NREM Stage 3 and 4 sleep is decreased by 1-10%, 5-10%, 5-15%, 10-15%, 15-20%, 15-25% , 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60%.
[002229] [002229] As a non-limiting example, the amount of NREM Stage 3 and 4 sleep is increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% , 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% or more than 65% .
[002230] [002230] As a non-limiting example, the amount of NREM Stage 3 and 4 sleep is increased by 1-5%, 1-10%, 5-10%, 5-15%, 10-15%, 15-20% , 15-25%, 20-25%, 20-30%, 25-30%, 25-35%, 30-35%, 30-40%, 35-40%, 40-50% or 40-60% .
[002231] [002231] In one embodiment, the periods of the NREM and REM cycles are more consistent in a subject after the subject has received the AAV particles described in this application.
[002232] [002232] Generally the NREM and REM cycles alternate at 90 to 110 minute intervals four to six times a night. KITS AND DEVICES. kits.
[002233] [002233] In one embodiment, the invention features a variety of kits for conveniently and/or efficiently carrying out the methods of the present invention.
[002234] [002234] Kits will typically comprise amounts and/or numbers of components sufficient to allow the user to perform multiple treatments of an individual and/or perform multiple experiments.
[002235] [002235] Any of the AAV particles of the present invention may be comprised in a kit.
[002236] [002236] In some embodiments, the kits may further include reagents and/or instructions for creating and/or synthesizing the compounds and/or compositions of the present invention.
[002237] [002237] In some embodiments, kits may also include one or more buffers.
[002238] [002238] In some embodiments, kits of the invention may include components for making arrays or libraries of proteins or nucleic acids, and therefore may include, for example, solid supports.
[002239] [002239] In some embodiments, kit components may be packaged in aqueous media or in lyophilized form.
[002240] [002240] Kit containers will generally include at least one vial, test tube, flask, bottle, syringe, or other container, into which a component can be placed, and preferably appropriately aliquoted.
[002241] [002241] oted.
[002242] [002242] When there is more than one component in the kit, (marking reagent and marker can be packaged together), Kits can usually also contain a third or third container or other additional containers in which the additional components can be placed separately.
[002243] [002243] In some embodiments, the kits may also comprise a second container to contain sterile pharmaceutically acceptable buffers and/or other diluents.
[002244] [002244] In some embodiments, multiple combinations of components may be contained in one or more vials.
[002245] [002245] Kits of the present invention may also include temperature means for containing the compounds and/or compositions of the present invention, for example, proteins, nucleic acids, and any other reagent containers in one commercial package.
[002246] [002246] Such containers may include injection molded or blow molded plastic containers in which the desired vials are contained.
[002247] [002247] In some embodiments, kit components are presented in one and/or more liquid solutions.
[002248] [002248] In some embodiments, liquid solutions are “aqueous” solutions, with sterile aqueous solutions being particularly preferred.
[002249] [002249] In some embodiments, kit components may be presented as dry powders.
[002250] [002250] When the reagents and/or components are presented as dry powders, such powders can be reconstituted by the addition of adequate volumes of solvent.
[002251] [002251] In some embodiments, it is provided that the solvents can also be presented in another container.
[002252] [002252] In some embodiments, the marking dyes are presented as dry powders.
[002253] [002253] In some embodiments, it is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200 , 300, 400, 500, 600, 700, 800, 900, 1000 micrograms or at least or at most such amounts of dyes are shown in the kits of the invention.
[002254] [002254] In such embodiments, the dye may then be resuspended in any suitable solvent, such as DMSO.
[002255] [002255] In some embodiments, kits may include instructions for use of kit components as well as instructions for use of any other reagent not included in the kit.
[002256] [002256] Instructions may include variations that can be implemented. devices
[002257] [002257] In some embodiments, the AAV particles of the present invention may be combined, applied as a coating, or embedded in a device.
[002258] [002258] Devices may include, but are not limited to, stents, pumps, and/or other implantable therapeutic devices.
[002259] [002259] Additionally AAV particles can be delivered to an individual while the individual is wearing a compression device such as, but not limited to, a compression device to reduce the chances of deep vein thrombosis (DVT) in an individual.
[002260] [002260] The present invention features devices that can incorporate AAV particles.
[002261] [002261] These devices contain AAV particles in a suitable formulation that can be immediately delivered to an individual in need thereof, such as a human patient.
[002262] [002262] Delivery devices may be employed to deliver the AAV particles of the present invention in accordance with the single-dose, multiple-dose, or fractionated-dose regimens taught in this application.
[002263] [002263] Methods and devices known in the literature for multiple administrations to cells, organs and tissues are contemplated for use in conjunction with the methods and compositions disclosed in this application as embodiments of the present invention.
[002264] [002264] These include, for example, methods and devices that have multiple needles, hybrid devices that employ, for example, lumens or catheters as well as devices that use mechanisms driven by heat, electrical current or radiation.
[002265] [002265] In some embodiments, the AAV particles of the present invention may be delivered by a device such as, but not limited to, a stent, a tube, a catheter, a tubing, a straw, a needle, and/or a duct.
[002266] [002266] Methods of using these devices are described in this application and are known in the state of the art.
[002267] [002267] In one embodiment, the AAV particles of the present invention may be delivered to a subject by delivery systems that integrate image-guided therapy and image-integration such as, but not limited to, laser surgery devices, MRgFUS, endoscopic and robotics.
[002268] [002268] In one embodiment, the AAV particles of the present invention may be administered to a subject via the CLEARPOINT® neurointervention system from MRI Interventions, Inc.
[002269] [002269] The CLEARPOINT® Neurointervention System can be used alone or in combination with any other delivery methods and devices described in this application.
[002270] [002270] The CLEARPOINTº Neurointervention System helps provide stereotaxic guidance in the placement and operation of instruments or devices during the planning and operation of neurological procedures.
[002271] [002271] In one embodiment, the AAV particles of the present invention may be delivered to a subject via Renishaw PLC's NEUROMATES stereotaxic robot system.
[002272] [002272] The NEUROMATES system may be used alone or in combination with any other delivery methods and devices described in this application.
[002273] [002273] As a non-limiting example, the NEUROMATE can be used with headrests, CT image finders, structural accessories, remote controls and software.
[002274] [002274] In one embodiment, the AAV particles of the present invention may be administered to a subject via Elekta AB's MICRODRIVE TM device.
[002275] [002275] The MICRODRIVE TM device may be used alone or in combination with any other administration methods and devices described in this order.
[002276] [002276] As a non-limiting example, the MICRODRIVETM device can be used to position electrodes (e.g. for microelectrode recording (MER), implantation of macrostimulation and deep brain stimulation (DBS) electrodes), implantation of catheters, tubes, or DBS electrodes using crossed wire holders and AP for position verification, biopsies, injections and aspirations, brain damage, endoscope guidance and GAMMA KNIFEº radiosurgery.
[002277] [002277] In one embodiment, the AAV particles of the present invention may be delivered to a subject via the AXIIISº stereotaxic ministructure from MONTERISº Medical, Inc.
[002278] [002278] The AXIIISº stereotaxic ministructure can be used alone or in combination with any other delivery methods and devices described in this application.
[002279] [002279] The AXIIISº stereotaxic ministructure is a path alignment device that can be used for laser coagulation, biopsies, catheter staining, electrode implantation, endoscopy, and clot elimination.
[002280] [002280] The miniframe provides a 360 degree interface and provides access to multiple intracranial targets with a simple adjustment.
[002281] [002281] Also, the ministructure is MRI compatible.
[002282] [002282] In one embodiment, the AAV particles of the present invention may be administered to a subject via the INTEGRA TM CRWº system from Integra LifeSciences Corporation.
[002283] [002283] System INTEGRA! M CRWº can be used alone or in combination with any other delivery methods and devices described in this application.
[002284] [002284] The CRWº system can be used for various applications such as, but not limited to, stereotaxic surgery, microsurgery, catheterization and biopsy.
[002285] [002285] The CRWº system is designed to provide precision to those who use the system (eg thumb lock screws, Vernier scale, double screw attachment and a solid frame).
[002286] [002286] In one embodiment, the AAV particles of the present invention may be administered to an individual via the EPOCH® solutions system from Stereotaxis, Inc. which may include the NIOBE® ES magnetic navigation system, the VDRIVES robotic navigation system, and /or the ODYSSEY information solution (all from Stereotaxis, Inc.).
[002287] [002287] The EPOCHº Solutions System may be used alone or in combination with any other delivery methods and devices described in this application.
[002288] [002288] As a non-limiting example, the NIOBEº ES magnetic navigation system can be used to precisely contact an individual.
[002289] [002289] As another non-limiting example, the NIOBES ES magnetic navigation system can be used with the VDRIVES robotic navigation system to provide precise movement and stability.
[002290] [002290] In one embodiment, the AAV particles of the present invention may be delivered to a subject via a NeuroStation workstation that uses frameless stereotaxic methods to provide image guidance for applications such as, but not limited to, surgical planning , biopsies, craniotomies, endoscopy, intraoperative ultrasound and radiotherapy.
[002291] [002291] In one embodiment, the AAV particles of the present invention may be administered to a subject by means of a robotic stereotaxic system such as, but not limited to, the device described in US Patent No. 5,078,140, the entirety of which is incorporated herein by reference title.
[002292] [002292] The robotic arm of the device can be used to precisely guide surgical tools or other implements used to conduct a procedure.
[002293] [002293] In one embodiment, the AAV particles of the present invention may be administered to a subject via an automatic delivery system such as, but not limited to, the device described in US Patent No. 5,865,744, the entirety of which is incorporated herein by reference title.
[002294] [002294] Based on the images gathered by the delivery system, the computer adjusts the needle delivery to be at the appropriate depth for the particular individual.
[002295] [002295] In one embodiment, the AAV particles of the present invention may be administered to a subject who is simultaneously using during administration, and/or uses for a period of time before and/or after administration, a compression device such as, but without limitation, a compression device that reduces the chances of deep vein thrombosis (DVT) in an individual.
[002296] [002296] The compression device can be used for at least 5 minutes, 15 minutes, 30 minutes, 45 minutes, one hour, two hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, or more than 8 hours before an individual receives AAV particles.
[002297] [002297] The compression device can be used for at least 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, two weeks, 3 weeks or one month after the AAV particles are administered.
[002298] [002298] As a non-limiting example, the compression device is used simultaneously during the process of distributing the AAV particles.
[002299] [002299] As another non-limiting example, the compression device is used prior to administration of the AAV particles.
[002300] [002300] As another non-limiting example, the compression device is used after the administration of AAV particles.
[002301] [002301] As another non-limiting example, the compression device is used before, during and after the administration of the AAV particles.
[002302] [002302] Non-limiting examples of compression devices include ActiveCare +SFT intermittent compression device, ActiveCare +SFT pneumatic compression device, Venowave from DVTIite, compression pump from KCI system, pneumatic compression therapy equipment from Aircast VenaFlow system, SCD Express Compression System or Bio Compression Systems, Inc. (for example, the pump can be selected from Model SC-2004, Model SC-2004-FC, Model SC-3004, Model SC-3004-FC, Model SC-2008, Model SC-2008-DL, Model SC-3008-T, by BioCryo system,
[002303] [002303] In one embodiment, the AAV particles may be delivered to an individual via an AAV particle delivery device and a head attachment frame.
[002304] [002304] The head fixation frame can be, but not limited to, any of the head fixation frames marketed by MRI! interventions.
[002305] [002305] As a non-limiting example, the head attachment frame may be any of the frames described in US Patent Nos.* 8,099,150, 8,548,569 and 9,031,636 and in International Patent Publication Nos. WO201108495 and WO2014014585, the entirety of each being incorporated herein by reference.
[002306] [002306] A head fixation frame can be used in combination with an MRI compatible drill such as, but not limited to, the MRI compatible drills described in International Patent Publication No. WO2013181008 and in US Patent Publication No. US20130325012, whose in its entirety is incorporated herein by reference.
[002307] [002307] In one embodiment, the AAV particles may be delivered by a method, system and/or computer program to position the apparatus to a target point on an individual to deliver the AAV particles.
[002308] [002308] As a non-limiting example, method, system and/or computer program may be the methods, systems and/or computer programs described in US Patent No. 8,340,743, the entirety of which is incorporated herein by reference.
[002309] [002309] The method may include: determining a target point on the body and a reference point, where the target point and the reference point define a planned trajectory line (PTL) that extend through each other; determine a view plane, on which the PTL intersects the view plane at a crosshair point; adjusting the guide device relative to the body to move relative to the PTL, where the guide device does not intersect the viewing plane; determine an intersection point (GPP) between the guide axis and the viewing plane; and align the GPP with the crosshair point on the viewing plane.
[002310] [002310] In one embodiment, AAV particles may be delivered to an individual via a convection-enhanced delivery device.
[002311] [002311] Non-limiting examples of vectored drug delivery using convection are described in US Patent Publication Nos.* US20100217228, US20130035574 and US20130035660 and International Patent Publication Nos. WOZ2Z013019830 and WOZ2008144585, the entirety of each of which is incorporated herein by reference. .
[002312] [002312] In one embodiment, an individual may have images taken before, during and/or after delivery of the AAV particles.
[002313] [002313] The imaging method may be a method known in the literature and/or described in this application, such as, but not limited to, magnetic resonance imaging (MRI).
[002314] [002314] As a non-limiting example, images can be used to assess the therapeutic effect.
[002315] [002315] As another non-limiting example, images can be used for assisted distribution of AAV particles.
[002316] [002316] In one embodiment, the AAV particles may be delivered by an MRI guided device.
[002317] [002317] Non-limiting examples of MRI guided devices are described in US Patent Nos. 9,055,884, 9,042,958, 8,886,288,
[002318] [002318] As a non-limiting example, the MRI guided device must also be capable of providing real-time data such as those described in US Patent Nos.* 8,886,288 and 8,768,433, the entirety of which is incorporated herein. by way of reference.
[002319] [002319] As another non-limiting example, the MRI guided device or system may be used with a guiding cannula such as the systems described in 8.175.6/7 and
[002320] [002320] As yet another non-limiting example, the MRI guided device includes a trajectory-guiding frame for guiding an interventional device as described, for example, in US Patent No. 9,055,884 and US Patent Application No. US20140024927, the entirety of each of which is incorporated herein by reference.
[002321] [002321] In one embodiment the AAV particles may be delivered through an MRI compatible nozzle assembly.
[002322] [002322] Non-limiting examples of MRI compatible nozzle assemblies are described in US Patent Publication No. US20140275980, the entirety of which is incorporated herein by reference.
[002323] [002323] In one embodiment, the AAV particles may be delivered through a cannula that is MRI compatible.
[002324] [002324] Non-limiting examples of MRI compatible cannulas include those taught in International Patent Publication No.
[002325] [002325] In one embodiment, the AAV particles may be delivered through a catheter that is MRI compatible.
[002326] [002326] Non-limiting examples of MRI compatible catheters include those taught in International Patent Publication No. WO2012116265, US Patent Publication No.
[002327] [002327] In one embodiment, the AAV particles may be delivered through a device having an elongated tubular body and a diaphragm as described in US Patent Publication Nos. US20140276582 and US20140276614, the entirety of each being incorporated herein by title. of reference.
[002328] [002328] In one embodiment, the AAV particles may be delivered through an MRI-compatible tracking and/or guidance system such as, but not limited to, those described in US Patent Publications Nos. US20150223905 and US20150230871, the entirety of each of which is incorporated herein by reference.
[002329] [002329] As a non-limiting example, MRI compatible locating and/or guiding systems may comprise a frame adapted for attachment to a patient, a guiding cannula with a lumen configured to be weighted to the frame so that it can be controllable in at least three dimensions, and an elongated probe configured to smoothly advance by sliding and retracting into the lumen of the guiding cannula, the elongated probe comprising at least one of a stimulation or recording electrode.
[002330] [002330] In one embodiment, AAV particles may be delivered to an individual via a trajectory chart as described US Patent Publication Nos. US20150031982 and US20140066750 and International Patent Publication Nos. WO2015057807 and WOZ2014039481, the entirety of each being incorporated herein by reference.
[002331] [002331] In one embodiment, the AAV particles may be delivered to an individual via a gene gun. DEFINITIONS
[002332] [002332] In various parts of this specification, the substituents of the compounds of the present invention are shown in groups or bands.
[002333] [002333] This report is specifically intended to include any and all individual sub-combinations of the members of such groups and bands.
[002334] [002334] About: As used in this application, the term "about" means +/- 10% of the mentioned value.
[002335] [002335] Activity: As used in this application, the term "activity" refers to the condition in which things are happening or being done.
[002336] [002336] The compositions described in this application may have activity and this activity may involve one or more biological events.
[002337] [002337] Adeno-associated virus: The term "adeno-associated virus" or "AAV" as used in this application refers to members of the dependovirus genus comprising any particle, sequence, gene, protein, or component derived therefrom.
[002338] [002338] The term "AAV particle", as used in this application, comprises a capsid and a polynucleotide.
[002339] [002339] The AAV particle can be derived from any serotype, described in this application or known in the literature, including combinations of serotypes (ie, "pseudotified" AAV) or from multiple —“genomes (e.g. single stranded or self-complementary) .
[002340] [002340] In addition, the AAV particle may be replication defective and/or directed.
[002341] [002341] Administered in combination: As used in this application, the term "administered in combination" or "combined administration" means that two or more agents (e.g., AAV) are administered to an individual at the same time or within a range of so that there may be an overlap of an effect of each agent on the patient and/or on the individual at some time simultaneously exposed to both.
[002342] [002342] In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of each other or within about 24 hours, 12 hours, 6 hours, 3 hours of at least one dose of one or more other agents.
[002343] [002343] In some embodiments, administration occurs in overlapping dosage regimens.
[002344] [002344] As used in this application, the term "dosing regimen" refers to a plurality of doses spaced apart in time.
[002345] [002345] Such doses may occur at regular intervals or may include one or more gaps in administration.
[002346] [002346] In some embodiments, administrations of agents are spaced close enough together to achieve a combinatorial (eg, synergistic) effect.
[002347] [002347] Amelioration: As used in this application, the term "improvement" or "improvement" refers to a reduction in the severity of at least one indicator of a condition or disease.
[002348] [002348] For example, in the context of neurodegeneration disorders, improvement includes reduced loss of neurons.
[002349] [002349] Animal: As used in this application, the term "animal" refers to any member of the animal kingdom.
[002350] [002350] In some embodiments, "animal" refers to human beings at any stage of development.
[002351] [002351] In some embodiments, "animal" refers to non-human animals at any stage of development.
[002352] [002352] In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig).
[002353] [002353] In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms.
[002354] [002354] In some embodiments, the animal is a transgenic animal, genetically modified animal, or a clone.
[002355] [002355] Antisense strand: As used in this application, the term "the antisense strand" or "the first strand" or "the guide strand" of a siRNA molecule refers to a strand that is substantially complementary to a section of about 10 -50 nucleotides, eg about 15-30, 16-25, 18-23 or 19-22 nucleotides of the target gene mRNA for silencing.
[002356] [002356] The antisense strand or first strand has a sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, eg, sufficient complementarity to initiate destruction of the desired target mRNA by the RNAi machinery or process.
[002357] [002357] Approximately: As used in this application, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stipulated reference value.
[002358] [002358] In certain embodiments, the term "approximately" or
[002359] [002359] Associated with: As used in this application, the terms "associated with", "conjugated", "bonded", "attached", and "bonded", when used in relation to one or more portions, means that the portions are physically associated or connected to each other, either directly or through one or more additional moieties that serve as a binding agent, to form a structure that is sufficiently stable that the moieties remain physically associated under the conditions in which the structure is used , for example, physiological conditions.
[002360] [002360] An "association" need not be strictly through direct covalent chemical bonding.
[002361] [002361] It may also suggest an ionic or hydrogen bond or hybridization-based connectivity stable enough for the "associated" entities to remain physically associated.
[002362] [002362] Bifunctional: As used in this application, the term "bifunctional" refers to any substance, molecule or moiety that is capable of maintaining at least two functions.
[002363] [002363] Functions can cause the same or a different outcome.
[002364] [002364] The structure that produces the function can be the same or different.
[002365] [002365] Biologically active: As used in this application, the term "biologically active" refers to a characteristic of any substance (eg AAV) that has activity in a biological system and/or organism.
[002366] [002366] For example, a substance that, when administered to an organism, has a biological effect on that organism, is considered biologically active.
[002367] [002367] In particular embodiments, a polynucleotide of the present invention may be biologically active if even a portion p of the polynucleotide is biologically active or mimics an activity considered biologically relevant.
[002368] [002368] Biological system: As used in this application, the term "biological system" refers to a group of organs, tissues, cells, intracellular components, proteins, nucleic acids, molecules (including, but not limited to, biomolecules) that function together to perform a certain biological task in cell membranes, cell compartments, cells, tissues, organs, organ systems, multicellular organisms, or any biological entity.
[002369] [002369] In some embodiments, biological systems are cell signaling pathways comprising intracellular and/or extracellular cell signaling biomolecules.
[002370] [002370] In some embodiments, biological systems comprise growth factor signaling events in the extracellular matrix/cell matrix and/or cell niches.
[002371] [002371] Biomolecule: As used in this application, the term "biomolecule" is any naturally occurring molecule that is amino acid based, nucleic acid based, carbohydrate based or lipid based, among others.
[002372] [002372] Complementary and substantially complementary. As used in this application, the term "complementary" refers to the ability of polynucleotides to base pair with each other.
[002373] [002373] Base pairs are typically formed by hydrogen bonds between —nucleotide units in strands of antiparallel polynucleotides.
[002374] [002374] Complementary polynucleotide strands can form base pairs in the Watson-Crick fashion (eg, A to T, A to U, C to G), or in any other manner that allows for duplex formation.
[002375] [002375] As those skilled in the art know, when using RNA as opposed to DNA, uracil and not thymine is the base that is considered complementary to adenosine.
[002376] [002376] However, when a U is indicated in the context of the present invention, the ability to substitute a T is implied unless otherwise indicated.
[002377] [002377] Perfect complementarity or 100% complementarity refers to the situation where each nucleotide unit of a polynucleotide strand can form a hydrogen bond with a nucleotide unit of a second polynucleotide strand.
[002378] [002378] Less than perfect complementarity refers to the situation in which some, but not all, two-strand nucleotide units can form hydrogen bonds with each other.
[002379] [002379] For example, for two 20-mers, if only two base pairs from each strand can form hydrogen bonds with each other, the polynucleotide strands show 10% complementarity.
[002380] [002380] In the same example, if 18 base pairs in each strand can form hydrogen bonds with each other, the polynucleotide strands have 90% complementarity.
[002381] [002381] As used in this application, the term "sub-complementary" means that the SiRNA has a sequence (eg, in the antisense strand) that is sufficient to bind the desired target mMRNA, and initiate RNA silencing of the target mRNA.
[002382] [002382] Compound: As used in this application, the term "compound" refers to a distinct chemical entity.
[002383] [002383] In some embodiments, a particular compound may exist in one or more isomeric or isotopic forms (including, but not limited to, stereoisinerism, geometric isomers and isotopes).
[002384] [002384] In some embodiments, a compound is presented or used in only one of these ways.
[002385] [002385] In some embodiments, a compound is presented or used as a mixture of two or more of these forms (including, but not limited to, a racemic mixture of stereoisomers).
[002386] [002386] Those skilled in the art are aware that some compounds exist in several of these forms, and exhibit different properties and/or activities (including, but not limited to, biological activities).
[002387] [002387] In such cases, those skilled in the art know how to select or avoid particular forms of the compound for use in accordance with the present invention.
[002388] [002388] For example, compounds containing asymmetrically distributed carbon atoms can be isolated in optically active or racemic forms.
[002389] [002389] Methods on how to prepare optically active forms from optically active starting materials are known in the prior art, such as by resolving racemic mixtures or by stereoselective synthesis.
[002390] [002390] Many geometric isomers of olefins, C=N double bonds, among others may also be present in the compounds described in this application, and all these stable isomers are contemplated in the present invention.
[002391] [002391] Geometric cis and trans isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separate isomeric forms.
[002392] [002392] The compounds of the present invention may also include tautomeric forms.
[002393] [002393] Tautomeric forms result from the exchange of a single bond for an adjacent double bond and the concomitant migration of a proton.
[002394] [002394] Tautomeric forms include prototropic tautomers which are isomeric states of protonation having the same empirical formula and the same total charge.
[002395] [002395] The compounds of the present invention also include all isotopes of the atoms occurring in the intermediate or final compounds.
[002396] [002396] "Isotopes" refers to atoms that have the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
[002397] [002397] For example, isotopes of hydrogen include tritium and deuterium.
[002398] [002398] The compounds and salts of the present invention can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
[002399] [002399] Conserved: As used in this application, the term "conserved" refers to nucleotide or amino acid residues of a polynucleotide sequence or a polypeptide sequence, respectively, which are those that occur unchanged at the same position of two or more sequences being compared.
[002400] [002400] Nucleotides or amino acids that are relatively conserved are those that are conserved among more related sequences than nucleotides or amino acids that appear elsewhere in the sequences.
[002401] [002401] In some embodiments, two or more sequences are said to be "completely conserved" if they are 100% identical to each other.
[002402] [002402] In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other.
[002403] [002403] In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to each other.
[002404] [002404] In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least least 80% identical, at least 90% identical, or at least 95% identical to each other.
[002405] [002405] In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other.
[002406] [002406] Sequence conservation may apply to the full length of an oligonucleotide, polynucleotide or polypeptide or may apply to a portion, region or aspect thereof.
[002407] [002407] In one embodiment, the conserved sequences are not contiguous.
[002408] [002408] Those skilled in the art are able to understand how to achieve alignment when gaps in the continuous alignment are "present between sequences, and align corresponding residues despite insertions or deletions present.
[002409] [002409] In one embodiment, the conserved sequences are not contiguous.
[002410] [002410] Those skilled in the art are able to understand how to achieve alignment when gaps in the continuous alignment are "present between sequences, and align corresponding residues despite insertions or deletions present.
[002411] [002411] Delivery: As used in this application, "delivery" refers to the act or manner of delivering a compound such as a parvovirus, e.g., an AAV and/or a compound, substance, entity, moiety, payload, or payload. from AAV to a target.
[002412] [002412] Such a target may be a cell, tissue, organ, organism, or system (whether biological or production).
[002413] [002413] Delivery agent: As used in this application, "delivery agent" refers to any agent or substance that facilitates, at least in particular, the in vivo and/or in vitro delivery of a polynucleotide and/or a or more substances (including, but not limited to, the compounds and/or compositions of the present invention, e.g., viral particles or expression vectors) to target cells.
[002414] [002414] Destabilized: As used in this application, the term "destabilized", "destabilize", or "region of destabilization" means a region or molecule that is less stable than the initial wild-type or native reference form of the same region or molecule.
[002415] [002415] Detectable Marker: As used in this application, "detectable marker" refers to one or more markers, signals or portions that are attached to, incorporated into, or associated with another entity that is readily detected by methods known in the literature including radiography, fluorescence, chemiluminescence, enzymatic activity, immunological detection by absorbance, among others.
[002416] [002416] Detectable markers can include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, among others.
[002417] [002417] Detectable markers can be located at any position of the entity to which they are attached, embedded or associated.
[002418] [002418] For example, when attached to, incorporated into, or associated with a peptide or protein, they may be in amino acids, in peptides, or in proteins located at the N- or C-termini.
[002419] [002419] Dosage regimen: As used in this application, "dosing regimen" is a physician-determined administration program or regimen of treatment, prophylaxis, or palliative care.
[002420] [002420] Effective Amount: As used in this application, the term "effective amount" of an agent is that amount sufficient to effect beneficial or desired results, for example, upon administration of a single dose or multiple doses to a cell in question. , in curing, alleviating, attenuating or ameliorating one or more symptoms of a disorder, clinical outcomes, and thus an "effective amount" depends on the context in which it is being applied.
[002421] [002421] For example, in the context of administering an agent that treats Parkinson's disease, an effective amount of an agent is, for example, an amount sufficient to effect the treatment, as defined in this application, of Parkinson's disease, compared to the response obtained without agent administration.
[002422] [002422] Encapsulate: As used in this order, the term "encapsulate" means to enclose, envelop, or insert.
[002423] [002423] Genetically engineered: As used in this application, modalities are "genetically engineered" when they are designed to have an attribute or property, whether structural or chemical, that varies from a wild-type or native starting molecule.
[002424] [002424] Thus, agents or entities genetically manipulated are those whose design and/or production include the action of the human hand.
[002425] [002425] Epitope: As used in this application, an "epitope"" refers to a surface or region on a molecule that is capable of interacting with a biomolecule.
[002426] [002426] For example, a protein may contain one or more amino acids, for example an epitope, which interacts with an antibody, for example a biomolecule.
[002427] [002427] In some embodiments, when referring to a protein or a protein module, an epitope may comprise a linear stretch of amino acids or a trudimensional structure formed by coiled amino acid chains.
[002428] [002428] Expression: As used in this application, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing an RNA transcript (e.g., by splicing, editing, forming the 5' cap, and/or processing the 3' end); (3) translating an RNA into a polypeptide or protein; (4) folding a polypeptide or protein; and (5) post-translational modification of a polypeptide or protein.
[002429] [002429] Aspect: As used in this application, an "aspect" refers to a characteristic, a property, or a distinctive element.
[002430] [002430] Formulation: As used in this application, a "formulation" includes at least one polynucleotide and/or compound and/or composition of the present invention (e.g., a vector, an AAV particle, etc.) and a delivery agent. .
[002431] [002431] Fragment: A "fragment", as used in this application, refers to a contiguous portion of a whole.
[002432] [002432] For example, protein fragments may comprise polypeptides obtained by digestion of full-length protein isolated from cultured cells.
[002433] [002433] In some embodiments, a fragment of a protein includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more amino acids.
[002434] [002434] In some embodiments, fragments of an antibody include portions of an antibody subjected to enzymatic digestion or synthesized as such.
[002435] [002435] Functional: As used in this application, a "functional" biological molecule is a biological molecule and/or entity having a structure and form in which it exhibits a property and/or activity by which it is characterized.
[002436] [002436] Gene expression: The term "gene expression" refers to the process by which a nucleic acid sequence undergoes successful transcription and in most cases translation to produce a protein or peptide.
[002437] [002437] For the sake of clarity, when reference is made to the measurement of "gene expression", it should be understood that the measurements may be of the nucleic acid product of transcription, e.g. RNA or mRNA, or the amino acid product of transcription, for example, polypeptides or peptides.
[002438] [002438] Methods for measuring the amount or levels of RNA, mMRNA, polypeptides and peptides are well known in the prior art.
[002439] [002439] Homology: As used in this application, the term "homology" refers to the overall relationship between polymeric molecules, e.g. between nucleic acid molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
[002440] [002440] In some embodiments, polymer molecules are considered "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar.
[002441] [002441] The term "homolog" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
[002442] [002442] According to the invention, two polynucleotide sequences are considered homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% by at least a stretch of at least about 20 amino acids.
[002443] [002443] In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.
[002444] [002444] For polynucleotide sequences less than 60 nucleotides in length, homology is typically determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.
[002445] [002445] In accordance with the invention, two protein sequences are considered homologous if the proteins were at least about 50%, 60%, 70%, 80%, or 90% identical for at least a stretch of at least about about 20 amino acids.
[002446] [002446] In many embodiments, homologous proteins may exhibit a high degree of total homology and a high degree of homology for at least a short stretch of at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acids.
[002447] [002447] In many embodiments, homologous proteins share one or more characteristic sequence elements.
[002448] [002448] As used in this application, the term "characteristic sequence element" refers to a motif present in related proteins.
[002449] [002449] In some embodiments, the presence of such motifs is correlated with a particular activity (such as biological activity).
[002450] [002450] /identity: As used in this application, the global term "identity" between polymeric molecules, e.g. between oligonucleotide and/or polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
[002451] [002451] Calculation of percent identity of two polynucleotide sequences, for example, can be done by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of a first and a second sequence of nucleic acids for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
[002452] [002452] In certain embodiments, the length of an aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the reference sequence length.
[002453] [002453] The nucleotides at the corresponding nucleotide positions are then compared.
[002454] [002454] When a position in the first sequence is coupled by the same nucleotide as in the corresponding position in the second sequence, then the molecules are identical at that position.
[002455] [002455] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, considering the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
[002456] [002456] The comparison of sequences and the determination of the percentage of identity between two sequences can be carried out with the help of a mathematical algorithm.
[002457] [002457] For example, percent identity between two nucleotide sequences can be determined by methods such as those described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds, M Stockton Press, New York, 1991; the entirety of each is incorporated herein by reference.
[002458] [002458] For example, the percentage of identity between two nucleotide sequences can be determined, for example, by means of the Meyers and Miller algorithm (CABIOS, 1989, 4:11-17), which was incorporated into the ALIGN program (version 2.0) using a PAM120 weight residual table, a gap length penalty of 12, and a gap penalty of 4.
[002459] [002459] The percent identity between two nucleotide sequences can alternatively be determined with the help of the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
[002460] [002460] Methods commonly employed to determine percent identity between sequences include, but are not limited to, those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); the entirety of which is incorporated herein by reference.
[002461] [002461] Techniques for determining identity are codified in publicly available computer programs.
[002462] [002462] Exemplary computer software for determining homology between two sequences includes, but is not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)) , BLASTP, BLASTN, and FASTA Altschul, SFetal., J. Molec. Biol., 215, 403 (1990)).
[002463] [002463] Inhibiting the expression of a gene: As used in this application, the expression "inhibiting the expression of a gene" means causing a reduction in the amount of a gene expression product.
[002464] [002464] The expression product can be RNA transcribed from the gene (eg, mRNA) or a polypeptide translated from mRNA transcribed from the gene.
[002465] [002465] Typically, a reduction in the level of mRNA results in a reduction in the level of a polypeptide translated therefrom.
[002466] [002466] The level of expression can be determined by traditional techniques to measure MRNA or protein.
[002467] [002467] In vitro: As used in this application, the term "in vitro" refers to events that occur in an artificial environment, for example, in a test tube or reaction vessel, in cell culture, in a plate of Petri, etc., and not in an organism (eg, animal, plant, or microbe).
[002468] [002468] In vivo: As used in this application, the term "in vivo" refers to events that occur in an organism (eg, animal, plant, or microbe or cell or tissue thereof).
[002469] [002469] /sole: As used in this application, the term "isolated" is synonymous with "separated", but carries with it the inference that the separation was performed by man.
[002470] [002470] In one embodiment, an isolated substance or entity is one that has been separated from at least some of the components with which it was previously associated (either in nature or in an experimental setting).
[002471] [002471] Isolated substances may have varying levels of purity in relation to the substances with which they were associated.
[002472] [002472] Isolated substances and/or entities can be separated to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% , about 80%, about 90%, or more of the other components to which they were initially associated.
[002473] [002473] In some embodiments, the isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,
[002474] [002474] As used in this application, a substance is "pure" if it is substantially free of other components.
[002475] [002475] Substantially isolated: By "substantially isolated" is meant that the compound is substantially separate from the environment in which it was formed or detected.
[002476] [002476] Partial separation may include, for example, a composition enriched in the compound of the present invention.
[002477] [002477] Substantial separation may include compositions containing 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 97%, or at least about 99% by weight of the compound of the present invention, or a salt thereof.
[002478] [002478] Methods for isolating compounds and their salts are routine in the literature.
[002479] [002479] In some embodiments, isolation of a substance or entity includes the disruption of chemical associations and/or bonds.
[002480] [002480] In some embodiments, isolation includes only the separation of components with which the isolated substance or entity was previously combined and does not include such disruption.
[002481] [002481] Modified: As used in this application, the term "modified" refers to an altered state or structure of a molecule or entity of the invention relative to a parent or reference molecule or entity.
[002482] [002482] Molecules can be modified in many ways including chemically, structurally, and functionally.
[002483] [002483] In some embodiments, the compounds and/or compositions of the present invention are modified by the introduction of unnatural amino acids, or unnatural nucleotides.
[002484] [002484] Mutation: As used in this application, the term "mutation" refers to a modification and/or alteration.
[002485] [002485] In some embodiments, the mutations may be modifications and/or alterations in proteins (including peptides and polypeptides) and/or in nucleic acids (including polynucleic acids).
[002486] [002486] In some embodiments, mutations comprise modifications and/or alterations in a protein and/or nucleic acid sequence.
[002487] [002487] Such modifications and/or alterations may comprise the addition, substitution and/or deletion of one or more amino acids (in the case of proteins and/or peptides) and/or nucleotides (in the case of nucleic acids and/or polynucleic acids) .
[002488] [002488] In embodiments where the mutations comprise the addition and/or substitution of amino acids and/or nucleotides, such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues and may include modified amino acids and/or nucleotides.
[002489] [002489] Naturally occurring: As used in this application, "naturally occurring" means existing in nature without artificial aid or human involvement.
[002490] [002490] Non-human vertebrate: As used in this application, a "non-human vertebrate" includes all vertebrates other than Homo sapiens, including wild and domestic species.
[002491] [002491] Examples of non-human vertebrates include, but are not limited to, mammals such as alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama , mule, pig, rabbit, reindeer, water buffalo, and yak.
[002492] [002492] Nucleic Acid: As used in this application, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" refer to any nucleic acid polymer composed of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), or polyribonucleotides (containing D-ribose), or any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases.
[002493] [002493] There is no intentional distinction in length between the terms "nucleic acid", "polynucleotide" and "oligonucleotide"", and these terms will be used interchangeably.
[002494] [002494] These terms refer only to the primary structure of the molecule.
[002495] [002495] Accordingly, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA.
[002496] [002496] Off-target: As used in this application, "off-target" refers to any unintended effect on any one or more targets, genes, and/or cellular transcripts.
[002497] [002497] Open reading frame: As used in this application, "open reading frame" or "ORF" refers to a sequence that does not contain a stop codon in a given reading frame.
[002498] [002498] Operably linked: As used in this application, the term "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, portions, and so on.
[002499] [002499] Particle: As used in this application, a "particle" is a virus comprised of at least two components, a protein capsid and a capsid-enclosed polynucleotide sequence.
[002500] [002500] Patient: As used in this application, "patient" refers to an individual who seeks or needs treatment, requires treatment, is receiving treatment, is to receive treatment, or an individual who is under the care of a trained professional for a particular disease or condition such as, for example, Parkinson's disease.
[002501] [002501] Payload As used in this application, "payload" refers to one or more polynucleotides or polynucleotide regions encoded by or in a viral genome or an expression product of such a polynucleotide or polynucleotide region, for example, a transgene, a polynucleotide encoding a polypeptide or a multi-polypeptide or a modulator nucleic acid or a regulatory acid.
[002502] [002502] Payload construct: As used in this application, "payload construct" is one or more polynucleotide regions encoding or comprising a payload that is flanked on one or both sides by an inverted terminal repeat (ITR) sequence .
[002503] [002503] The payload construct is a template that is replicated in a viral production cell to produce a viral genome.
[002504] [002504] Payload construct vector: As used in this application, "payload construct vector" is a vector encoding or comprising a payload construct construct, and regulatory regions for replication and expression in bacterial cells.
[002505] [002505] Payload construct expression vector: As used in this application, the "payload construct expression vector" is a vector encoding or comprising a construct payload and which further comprises one or more polynucleotide regions encoding or comprising components for viral expression in a viral replication cell.
[002506] [002506] Peptide: As used in this application, "peptide" is 50 or less amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.
[002507] [002507] Pharmaceutically acceptable: The term "pharmaceutically acceptable" is used in this application to denote those compounds, materials, compositions, and/or dosage forms that are, within reasonable medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, consistent with a reasonable risk/benefit ratio.
[002508] [002508] —Pharmaceutically acceptable excipients: The term "pharmaceutically acceptable excipient" as used in this application refers to any ingredient other than the active compounds and/or agents. (e.g. described in this application) present in pharmaceutical compositions and having the properties of being substantially non-toxic and non-inflammatory in a subject such as a patient.
[002509] [002509] In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and/or dissolving active agents.
[002510] [002510] Excipients may include, for example: non-sticks, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), emollients, emulsifiers, fillers (diluents),) film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, absorbents, suspending or dispersing agents, sweeteners, and water of hydration.
[002511] [002511] Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac,
[002512] [002512] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts of the compounds described in this application.
[002513] [002513] As used in this application, "pharmaceutically acceptable salts" refers to derivatives or forms of the disclosed compounds where the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., as generated by reaction of the free base group with a suitable organic acid).
[002514] [002514] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carbolic acids; between others.
[002515] [002515] Representative acid addition salts include salts of acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate , ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerats, among others.
[002516] [002516] Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, among others, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium , methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, among others.
[002517] [002517] The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
[002518] [002518] In some embodiments a pharmaceutically acceptable salt of the present invention may be a synthesized salt prepared from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
[002519] [002519] Generally, such salts can be prepared by reacting the acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
[002520] [002520] Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed, Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), the entirety of each of which is incorporated herein by reference.
[002521] [002521] Pharmaceutically acceptable solvate: The term "pharmaceutically acceptable solvate" as used in this application refers to a crystalline form of a compound of the invention where molecules of a suitable solvent are incorporated into the crystal lattice.
[002522] [002522] A suitable solvent is physiologically tolerable at the dosage administered.
[002523] [002523] For example, solvates can be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof.
[002524] [002524] Examples of suitable solvents are ethanol, water (e.g. mono-, di-, and trihydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone / ( DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, among others.
[002525] [002525] When the solvent is water, the solvate is called a "hydrate".
[002526] [002526] In some embodiments, the solvent incorporated into a solvate is of a type or at a level that is physiologically tolerable by the organism to which the solvate is administered (eg, in a unit dosage form of a pharmaceutical composition).
[002527] [002527] Pharmacokinetics: As used in this application, "pharmacokinetics" refers to any one or more properties of a molecule or compound as it relates to determining the fate of substances administered to a living organism.
[002528] [002528] Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion.
[002529] [002529] This is commonly indicated by ADME where: (A) Absorption is the process of a substance entering the bloodstream; (D) Distribution is the dispersion or dissemination of substances in all body fluids and tissues; (M) Metabolism (or Biotransformation) is irreversible transformation! of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of substances from the body.
[002530] [002530] In rat cases, some drugs accumulate irreversibly in body tissue.
[002531] [002531] Physicochemical: As used in this application, "physical chemistry" means or refers to a physical and/or chemical property.
[002532] [002532] Prevent. As used in this application, the term "prevent" refers to the partial or complete delay of the onset of an infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the course of a particular infection, disease, disorder and/or condition; and/or reducing the risk of developing a pathology associated with the infection, disease, disorder, and/or condition, such as, for example, Parkinson's disease.
[002533] [002533] Prodrug: The present invention also includes prodrugs of the compounds described in this application.
[002534] [002534] As used in this application, "prodrugs" refers to any substance, molecule, or entity that is in a form predisposed to that substance, molecule, or entity to act as a drug upon undergoing chemical or physical change.
[002535] [002535] Prodrugs can be covalently bound or sequestered in some way and that release or are converted to the active drug moiety before, during, or after being administered to a mammalian subject.
[002536] [002536] The preparation and use of prodrugs are discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug
[002537] [002537] Proliferarn As used in this application, the term "proliferate" means to grow, expand, replicate or increase or cause growth, expansion, replication or increase.
[002538] [002538] "Proliferative" means having the ability to proliferate.
[002539] [002539] "Antiproliferative" means having properties contrary or opposite to proliferative properties.
[002540] [002540] Prophylactic: As used in this application, prophylactic refers to a course of action therapeutic or used to prevent spread.
[002541] [002541] Prophylaxis: As used in this application, "prophylaxis" refers to a measure taken to maintain health and prevent the spread of disease.
[002542] [002542] Protein of interest: As used in this application, the terms "proteins of interest" or "desired proteins" include those set forth in this application and fragments, mutants, variants, and alterations thereof.
[002543] [002543] Purified: As used in this application, "purify", "purified", "purification" means to leave substantially pure or free from unwanted components, polluting material, mixture or imperfection.
[002544] [002544] Region: As used in this application, the term "region" refers to a generic zone or area.
[002545] [002545] In some embodiments, when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three-dimensional area, an epitope and/or a cluster of epitopes.
[002546] [002546] In some embodiments, regions comprise terminal regions.
[002547] [002547] As used in this application, the term "terminal region" refers to regions located at the ends or ends of a given agent.
[002548] [002548] When referring to proteins, the terminal regions may comprise N- and/or C-termini.
[002549] [002549] N-termini refer to the end of a protein comprising an amino acid with a free amino group.
[002550] [002550] C-termini refer to the end of a protein comprising an amino acid with a free carboxyl group.
[002551] [002551] The N- and/or C-terminal regions may therefore comprise the N- and/or C-termini as well as neighboring amino acids.
[002552] [002552] In some embodiments, the N- and/or C-terminal regions comprise from about 3 amino acids to about 30 amino acids, from about 5 amino acids to about 40 amino acids, from about 10 amino acids to about 50 amino acids , from about 20 amino acids to about 100 amino acids and/or at least 100 amino acids.
[002553] [002553] In some embodiments, the N-terminal regions can comprise any length of amino acid that includes the N-terminus, but does not include the C-terminus.
[002554] [002554] In some embodiments, the C-terminal regions may comprise any length of amino acid that includes the C-terminus, but does not comprise the N-terminus.
[002555] [002555] In some embodiments, when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three-dimensional area, a secondary structure, or a tertiary structure.
[002556] [002556] In some embodiments, regions comprise terminal regions.
[002557] [002557] As used in this application, the term "terminal region" refers to regions located at the ends or ends of a given agent.
[002558] [002558] When referring to polynucleotides, the terminal regions may comprise 5' and 3' termini.
[002559] [002559] 5' termini refer to the end of a polynucleotide comprising a nucleic acid with a free phosphate group.
[002560] [002560] 3' termini refer to the end of a polynucleotide comprising a nucleic acid with a free hydroxyl group.
[002561] [002561] The 5' and 3' regions may therefore comprise the 5' and 3' termini as well as neighboring nucleic acids.
[002562] [002562] In some embodiments, the 5' and 3' terminal regions comprise from about 9 nucleic acids to about 90 nucleic acids, from about 15 nucleic acids to about 120 nucleic acids, from about 30 nucleic acids to about from 150 nucleic acids, from about 60 nucleic acids to about 300 nucleic acids and/or at least 300 nucleic acids.
[002563] [002563] In some embodiments, the 5' regions can comprise any length of nucleic acid that includes the 5' terminus, but does not include the 3' terminus.
[002564] [002564] In some embodiments, the 3' regions may comprise any length of nucleic acid that includes the 3' terminus, but does not include the 5' terminus.
[002565] [002565] RNA or RNA molecule: As used in this application, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides; the term
[002566] [002566] DNA and RNA can be synthesized naturally, eg by DNA replication and DNA transcription, respectively; or they can be chemically synthesized.
[002567] [002567] The DNA and RNA can be single-stranded (i.e., sSSRNA or sSDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). The term "MRNA" or "messenger RNA", as used in this application, refers to a single-stranded RNA encoding the amino acid sequence of one or more polypeptide chains.
[002568] [002568] RNA Interference: As used in this application, the term "RNA interference" or "RNAi" refers to a sequence-specific regulatory mechanism mediated by RNA molecules that results in inhibition or interference or "silencing" of expression of a corresponding protein-coding gene.
[002569] [002569] Sample: As used in this application, the term "sample" refers to an aliquot, subset or portion taken from a source and/or presented for analysis or processing.
[002570] [002570] In some embodiments, a sample is from a biological source such as a tissue, cell, or component part (e.g., a body fluid, including, but not limited to, blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).
[002571] [002571] In some embodiments, a sample may be or comprise a homogenate, lysate, or extract prepared from an entire organism or a subset of tissues, cells, or component parts thereof, or a fraction or portion thereof, including, but without limitation, for example, plasma, serum, spinal fluid, lymphatic fluid, the outer sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, or organs.
[002572] [002572] In some embodiments, a sample is or comprises a medium, such as a nutrient broth or gel, which may contain cellular components, such as a protein or nucleic acid molecule.
[002573] [002573] In some embodiments, a "primary" sample is an aliquot of the count.
[002574] [002574] In some embodiments, a primary sample is subjected to one or more processing steps (eg, separation, purification, etc.) to prepare a sample for analysis or other use.
[002575] [002575] Self-complementary viral particle. As used in this application, a "self-complementary viral particle" is a particle made up of at least two components, a protein capsid and a polynucleotide sequence encoding a self-complementary genome enclosed in the capsid.
[002576] [002576] Sense strand: As used in this application, the term "the sense strand" or "the second strand" or "the passenger strand" of a siRNA molecule refers to a strand that is complementary to the antisense strand or first strand .
[002577] [002577] The antisense and sense strands of a siRNA molecule are hybridized to form a duplex structure.
[002578] [002578] As used in this application, a "siRNA duplex'" includes a strand of siRNA having sufficient complementarity to a section of about 10-50 nucleotides of the mMRNA of the target gene for silencing and a strand of siRNA having sufficient complementarity to form a duplex with the siRNA strand.
[002579] [002579] Signal sequences: As used in this application, the term "signal sequences" refers to a sequence that can direct transport or location.
[002580] [002580] Single unit dose: As used in this application, "single unit dose" is a dose of any drug administered in one dose/once/one route/single point of contact, ie, a single administration event . In some embodiments, a single unit dose is presented as a discrete dosage form (e.g., a tablet, capsule, patch, filled syringe, vial, etc.).
[002581] [002581] Similarity: As used in this application, the term "similarity" refers to the overall relationship between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
[002582] [002582] The calculation of the percentage of similarity between polymeric molecules between the immune system can be done in the same way as the calculation of the percentage of identity, except that the calculation of the percentage of similarity takes into account conservative substitutions, as is already known in literature.
[002583] [002583] Small/Short Interfering RNA: As used in this application, the term "Short/Small Interfering RNA" or "siRNA!" refers to an RNA molecule (or RNA analogue) comprising between about 5-60 nucleotides (or nucleotide analogues) that is capable of targeting or measuring RNAi.
[002584] [002584] Preferably, a siRNA molecule comprises between about 15-30 nucleotides or nucleotide analogues, more preferably between about 16-25 nucleotides (or nucleotide analogues), even more preferably between about 18-23 nucleotides ( or nucleotide analogues), and most preferably between about 19-22 nucleotides (or nucleotide analogues) (e.g. 19, 20, 21 or 22 nucleotides or nucleotide analogues).
[002585] [002585] The term "short" SiRNA refers to a siRNA comprising 5-23 nucleotides, preferably 21 nucleotides (or nucleotide analogues), for example 19, 20, 21 or 22 nucleotides.
[002586] [002586] The term "long" siRNA refers to a siRNA comprising 24-60 nucleotides, preferably about 24-25 nucleotides, for example 23, 24, 25 or 26 nucleotides.
[002587] [002587] Short siRNAs may, in some cases, include less than 19 nucleotides, e.g. 16, 17 or 18 nucleotides, or as little as 5 nucleotides, as long as the shorter siRNA retains its ability to mediate RNAi.
[002588] [002588] Likewise, long siRNAs can, in some cases, include more than 26 nucleotides, for example, 27, 28, 29, 30, 35, 40, 45, 50, 55, or even 60 nucleotides, as long as the longer siRNA retains its ability to mediate RNAi or missing translational repression for further processing, eg enzymatic processing, for a short siRNA.
[002589] [002589] SiRNAs can be single-stranded RNA molecules (ss-SiRNAs) or double-stranded RNA molecules (ds-SIiRNAs) comprising a sense strand and an antisense strand that hybridize to form a duplex structure called a SIiRNA duplex.
[002590] [002590] Fractionated Dose: As used in this application, a "fractional dose" is the division of a single unit dose or total daily dose into two or more doses.
[002591] [002591] Stable: As used in this application "stable" refers to a compound or entity that is sufficiently robust to survive isolation from a reaction mixture at a useful degree of purity, and preferably capable of formulation into an agent. effective therapeutic.
[002592] [002592] Stabilized: As used in this application, the term "stabilize", "stabilized", "stabilized region" means to leave or become stable.
[002593] [002593] In some embodiments, stability is measured relative to an absolute value.
[002594] [002594] In some embodiments, stability is measured relative to a reference compound or entity.
[002595] [002595] Subject: As used in this application, the term "subject" or "patient" refers to any organism to which a composition according to the invention can be administered, for example, for experimental, diagnostic, prophylactic, and /or therapeutic.
[002596] [002596] Typical individuals include animals (eg, mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
[002597] [002597] In some embodiments, the individual may be an infant, an infant, or a child under 12 years of age.
[002598] [002598] In some embodiments, the individual may be in the womb.
[002599] [002599] Substantially: As used in this application, the term "substantially" refers to the qualitative condition of presenting a full or near-total extent or degree of a characteristic or property of interest.
[002600] [002600] The expert in the biological arts knows that biological and chemical phenomena rarely, if ever, come to an end and/or proceed to completion or achieve or avoid an absolute result.
[002601] [002601] The term "substantially" is therefore used in this application to capture the potential lack of wholeness inherent in many biological and chemical phenomena.
[002602] [002602] Substantially Equal: As used in this application when referring to time differences between doses, the term means plus/minus 2%.
[002603] [002603] Substantially Simultaneously: As used in this application and when referring to a plurality of doses, the term typically means within about 2 seconds.
[002604] [002604] Suffering from: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or exhibits one or more symptoms of a disease, disorder, and/or condition such as, for example, Parkinson's disease .
[002605] [002605] Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition has not yet been diagnosed with and/or may not have symptoms of the disease, disorder, and/or condition, but harbors a tendency to develop a disease or its symptoms.
[002606] [002606] In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (e.g., cancer) may be characterized by one or more of the following events: (1) a genetic mutation associated with the development of the disease , disorder, and/or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acids associated with the disease, disorder, and/or condition; (4) habits and/or lifestyle associated with the development of the disease, disorder, and/or condition; (5) family history of the disease, disorder, and/or condition; and (6) exposure and/or infection with a microbe associated with the development of the disease, disorder, and/or condition.
[002607] [002607] In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
[002608] [002608] In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
[002609] [002609] Systematic release: As used in this application, the term "systematic release" refers to the release profile of a pharmaceutical composition or compound that adjusts to a release rate over a specific period of time.
[002610] [002610] Synthetic: The term "synthetic" means produced, prepared, and/or manufactured by man.
[002611] [002611] The synthesis of the polynucleotides or polypeptides or other molecules of the present invention can be chemical or enzymatic.
[002612] [002612] Vectorization: As used in this application, "vectorization"" means the process of creating and selecting a nucleic acid sequence that will hybridize to a target nucleic acid and induce an effect of interest.
[002613] [002613] Target Cells: As used in this application, "target cells" refers to any one or more cells of interest.
[002614] [002614] Cells can be found in vitro, in vivo, in situ or in the tissue or organ of an organism.
[002615] [002615] The organism may be an animal, preferably a mammal, more preferably a human, and even more preferably a patient.
[002616] [002616] Therapeutic agent: The term "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or produces a desired biological and/or pharmacological effect.
[002617] [002617] Therapeutically effective amount: As used in this application, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to an individual suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, ameliorate symptoms, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and /or condition such as, for example, Parkinson's disease.
[002618] [002618] In some embodiments, a therapeutically effective amount is presented in a single dose.
[002619] [002619] In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses.
[002620] [002620] Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of a dosage regimen. .
[002621] [002621] Therapeutically effective outcome: As used in this application, the term "therapeutically effective outcome" means an outcome that is sufficient in an individual suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
[002622] [002622] Total Daily Dose: As used in this order, a "Total Daily Dose" is an amount given or prescribed in a 24-hour period.
[002623] [002623] It can be administered as a single unit dose.
[002624] [002624] Transfection: As used in this application, the term "transfection" refers to methods for introducing exogenous nucleic acids into a cell.
[002625] [002625] Transfection methods include, but are not limited to, chemical methods, physical treatments, and lipid or cationic mixtures.
[002626] [002626] Treat As used in this application, the term "treat" refers to partially or completely alleviating, ameliorating, recovering, alleviating, delaying onset, inhibiting progression, reducing the severity, and/or reducing the incidence of one or more more symptoms or aspects of a particular infection, disease, disorder, and/or condition.
[002627] [002627] For example, "treating" cancer may refer to inhibiting the survival, growth, and/or spread of a tumor.
[002628] [002628] Treatment may be administered to an individual who shows no signs of a disease, disorder, and/or condition and/or to an individual who only shows early signs of a disease, disorder, and/or condition for the purpose of reduce the risk of developing a pathology associated with the disease, disorder, and/or condition, such as, for example, Parkinson's disease.
[002629] [002629] Unmodified: As used in this application, "unmodified" refers to any substance, compound, or molecule before it has been altered in any way.
[002630] [002630] Unmodified may, but not always, refer to the wild-type or native form of a biomolecule or entity.
[002631] [002631] Molecules or entities can undergo a series of modifications whereby each modified substance, compound, molecule or entity can serve as an "unmodified" starting molecule for a subsequent modification.
[002632] [002632] Vector. As used in this application, a "vector" is any molecule or moiety that transports, transduces or otherwise acts as a carrier of a heterologous molecule.
[002633] [002633] The vectors of the present invention may be produced recombinantly and may be based on and/or may comprise parental or reference sequences from adeno-associated virus (AAV).
[002634] [002634] Such AAV parent or reference sequences may function as an original, secondary, tertiary or subsequent sequence for vector construction.
[002635] [002635] In non-limiting examples, such AAV parent or reference sequences may comprise any one or more of the following sequences: a polynucleotide sequence encoding a polypeptide or multi-polypeptide, this sequence may be wild-type or modified from the wild-type and that sequence may encode the full- or partial-length sequence of a protein, protein domain, or one or more subunits of a protein; a polynucleotide comprising a modulator or regulatory nucleic acid, this sequence being wild-type or modified from wild-type; and a transgene which may or may not be modified from a wild-type sequence.
[002636] [002636] These AAV sequences can function as the "donor" sequence of one or more codons (at the nucleic acid level) or amino acids (at the polypeptide level) or as "acceptor" sequences of one or more codons (at the nucleic acid) or amino acids (at the polypeptide level).
[002637] [002637] Viral construct vector: As used in this application, a "viral construct vector" is a vector comprising one or more polynucleotide regions encoding or comprising the Rep and or Cap protein.
[002638] [002638] Viral construct expression vector: As used in this application, the "viral construct expression vector" is a vector comprising one or more polynucleotide regions encoding or comprising Rep and or Cap which further comprises one or more polynucleotide regions encoding or comprising components for viral expression in a viral replication cell.
[002639] [002639] Viral genome: As used in this application, the "viral genome" is a polynucleotide encoding at least one inverted terminal region (ITR), at least one regulatory sequence, and at least one useful carpha.
[002640] [002640] The viral genome is distributed by replication of a payload construct and a payload construct expression vector.
[002641] [002641] A viral genome encodes at least one copy of the payload construct. EQUIVALENTS AND SCOPE.
[002642] [002642] Those skilled in the art will recognize, or will be able to determine, with no more than routine experimentation, many equivalents of the specific embodiments according to the invention described in this application.
[002643] [002643] It is not intended that the scope of the present invention be limited to the above Description, but rather that it is as set out in the appended claims.
[002644] [002644] In the claims, articles such as "a", "an" and "the" may indicate one or more than one, unless otherwise indicated or evident from the context.
[002645] [002645] Claims or descriptions that include "or" among one or more members of a group are considered satisfied if one, more than one, or all members of the group are present, employed, or otherwise relevant to a given product or process, unless otherwise indicated or evident from the context.
[002646] [002646] The invention includes embodiments in which exactly one member of the group is present, employed, or is in some way relevant to a given product or process.
[002647] [002647] The invention includes embodiments in which more than one or all of the members are present, employed, or otherwise relevant to a given product or process.
[002648] [002648] It is also noted that the term "comprising" is open and allows, but does not require, the inclusion of additional elements or steps.
[002649] [002649] When the term "comprising" is used in this application, the term "consisting of" is therefore also covered and disclosed.
[002650] [002650] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as that commonly known to those skilled in the art to which this invention pertains.
[002651] [002651] In this report methods and materials for use in the present invention are described; other suitable methods and materials known in the art may also be used.
[002652] [002652] Where ranges are shown, their limits are included.
[002653] [002653] Furthermore, it is understood that unless otherwise indicated or evident to the contrary from the context and knowledge of one skilled in the art, values that are expressed as ranges may assume any specific value or subrange within the values specified in different embodiments of the invention to within one tenth of the lower limit of the range, unless clearly indicated otherwise by the context.
[002654] [002654] Furthermore, it is understood that any particular embodiment of the present invention that falls within the prior art should be explicitly excluded from any one or more of the claims.
[002655] [002655] Since such modalities are considered known to the person skilled in the art, they should be excluded even if the exclusion is not explicitly stated in this application.
[002656] [002656] Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded by the same; any method of production; any method of use, etc.) may be excluded from any one or more of the claims, for whatever reason, whether or not it is related to the existing state of the art.
[002657] [002657] All cited sources, e.g. references, publications, databases, database entries, and state of the art cited in this application, are incorporated in this application by way of reference, although not expressly indicated in the quoted excerpts .
[002658] [002658] In cases of conflicting information from a cited source and the present application, the information in the present application prevails.
[002659] [002659] Section and table titles are not limiting. EXAMPLES Example 1. Payload drawings: AADC polynucleotides.
[002660] [002660] AADC polynucleotides are designed to comprise at least one nucleic acid sequence encoding an AADC protein.
[002661] [002661] Once designed, the sequence is constructed or synthesized or inserted into a plasmid or vector and delivered to a cell or organism.
[002662] [002662] Suitable plasmids or vectors are any plasmid or vector that transduces or transfects the target cell.
[002663] [002663] Adeno-associated virus (AAV) particles may be used.
[002664] [002664] The administration results in the processing of the AADC polynucleotide to generate the AADC protein that alters the etiology of the disease, in this case Parkinson's disease.
[002665] [002665] In a non-limiting example, plasmids that contain an AADC polynucleotide of the invention have a CMV promoter and encode AADC.
[002666] [002666] In some embodiments the open reading frame of the MGRNA of the AADC protein has codons optimized.
[002667] [002667] AADC polynucleotides, listed ITR to ITR, suitable for use in AAV particles include those in Table 2. Table 2. AADC polynucleotides ITR to ITR [conto san
[002668] [002668] The start and end positions of various regions of the AADC polynucleotides are shown and are relative to the AADC ITR to AADC ITR polynucleotides described in Table 2.
[002669] [002669] In the Table, ITR means inverted terminal repeat, MCS means multiple cloning site, CMV means cytomegalovirus, lei means immediate early 1, hBglobin means human beta-globin, AADC means region encoding the AADC polynucleotide, and poly(A) stands for polyadenylation signal.
[002670] [002670] AADC polynucleotides are designed to comprise at least one nucleic acid sequence encoding an AADC protein.
[002671] [002671] Once designed, the sequence is constructed or synthesized or inserted into a plasmid or vector and delivered to a cell or organism.
[002672] [002672] Suitable plasmids or vectors are any plasmid or vector that transduces or transfects the target cell.
[002673] [002673] Adeno-associated virus (AAV) particles may be used.
[002674] [002674] Administration results in the processing of the AADC polynucleotide to generate the AADC protein that alters the etiology of the disease, in this case Parkinson's disease. Example 3. Administration of AAV particles.
[002675] [002675] AAV particles are infused into the substantia nigra, and in particular the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) of patients with Parkinson's disease and identified as eligible for treatment by methods known in the state of the technique.
[002676] [002676] One method of administration contemplated for use in the methods described in this application is real-time convection-enhanced delivery (RCD) of AAV particle compositions by co-infusion of gadoteridol (a magnetic resonance (MR) contrast agent) and magnetic resonance imaging (MRI) T1 or T2, which can predict areas of subsequent AADC gene expression. As described by Richardson, et al., 2011, the accuracy of cannula placement and the distribution of the initial infused material can be safely determined by saline infusion without significantly altering the subsequent delivery of the tracer agent (Richardson, et al., 2011). ., 2011, Neurosurgery, 69(1):154-163 ), T2 RCD provides the detection of convectionally enhanced intraparenchymal delivery in the uninjured brain and can predict the subsequent delivery of a transgene after infusion of AAV particles.
[002677] [002677] Subjects undergo saline infusion/T2 acquisition, immediately followed by gadoteridol infusion/T1 acquisition into the putamen and brain stem.
[002678] [002678] Distribution volumes and spatial patterns are analyzed.
[002679] [002679] Gadoteridol and AAV-encoded AADC are co-fused under alternating T2/T1 acquisition in the thalamus, and the hyperintense areas are compared with areas of subsequent transgene expression.
[002680] [002680] Volume of distribution to volume infusion ratios are expected to be similar between saline and gadoteridol RCDs.
[002681] [002681] Spatial overlap should be well correlated between T2 and T1 images.
[002682] [002682] The second infused material will follow a spatiotemporal pattern similar to that of the first, filling the target area before developing extra-target distribution.
[002683] [002683] Areas of AADC expression should be well correlated with areas of both T1 and T2 hyperintensity observed during RCD (Richardson, et al, 2011, Neurosurgery, 69(1):154-163).
[002684] [002684] Convection-enhanced delivery (CED) of macromolecules directly into the brain parenchyma has been known for over two decades.
[002685] [002685] CED is a term that designates the use of a pressure gradient for massive flow in the brain parenchyma, i.e., convection of macromolecules in the interstitial fluid induced by infusion of a solution through a cannula placed directly into the target structure.
[002686] [002686] This method allows therapeutic agents to be distributed homogeneously through large volumes of brain tissue by bypassing the blood-brain barrier and overcoming simple diffusion (Richardson, et al, 2011, Stereotact. Funct Neurosurg. 89:141–151).
[002687] [002687] Salegio, et al. recently demonstrated the distribution of nanoparticles of different sizes, including micelles (-15 nm in size), AAV (-20-25 nm) and liposomes (-65 nm), in the CNS of rodents and NHPs (Salegio et al., 2014, Frontiers in Neuroanatomy, vol 8, article 9: pp. 1-8).
[002688] [002688] Single injections cannot reach the perivascular system, and special infusion cannulae are required, allowing constant pressures to be exerted from the cannula tip so that the interstitial hydrostatic pressure is exceeded and the infusion material passes into tissue.
[002689] [002689] Single needles generate significant reflux; therefore, reflux resistant cannulas were developed to counteract this tendency.
[002690] [002690] The advent of platform for MRI-guided convection-enhanced infusions further refined the understanding of the mechanics of perivascular flow, and demonstrated that the perivascular distribution of liposomes was linear with time, the slope of the curve increased in the regions myelinated, and stopping the infusion prevented further expansion of the volume of distribution.
[002691] [002691] (Richardson, et al. 2011, Stereotact. Funct.Neurosurg. 89:141—151; Salegio et al., 2014, Frontiers in Neuroanatomy, vol. 8, article 9: pp. 1-8).
[002692] [002692] Intraparenchymal injections of rAAV are known to result in robust but relatively local transduction.
[002693] [002693] Such local distribution methods are advantageous when attempting gene therapy for neurological disorders that result from neuropathology that is localized to a specific anatomical region or anatomical circuit such as in the case of Parkinson's disease.
[002694] [002694] However, in treatments that require broader transduction of the SCN, intraparenchymal injections are impractical.
[002695] [002695] Treatment of neurological disorders attributable to inborn errors of metabolism and/or to single gene defects, or to those affecting spinal cord motor neurons, may result in large-scale transduction of the brain or spinal cord, respectively.
[002696] [002696] The development of less invasive trans-BBB delivery methods for vectors is an extremely important challenge.
[002697] [002697] Numerous attempts to use molecules known to interact with various active (probably receptor-mediated) transport mechanisms to transport proteins across the BBB have been reported with varying results.
[002698] [002698] Due to the large number of AAV serotypes available, one or more serotypes may bind to a cell entry receptor capable of transporting the AAV capsid across the BBB (Manfredsson, et al., 2009, "AAV9: a potential blood-brain barrier buster." Molecular Therapy 17(3):403-405 ). Vector infusion and stereotaxic infusion
[002699] [002699] A stereotaxic approach can be used for the surgical delivery of AADC polynucleotides.
[002700] [002700] Although individuals with AADC deficiency lack epinephrine and norepinephrine, these patients must maintain stable blood pressure and heart rate during surgery.
[002701] [002701] There should be no significant intracerebral haemorrhages on postoperative computed tomography (CT) or MRI images.
[002702] [002702] The needle traces, as shown in the MRI images, should indicate accurate injection into the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA).
[002703] [002703] Patients will be released from the hospital about a week after surgery (Hwu, WL, et a/., 2012. Gene therapy for aromatic L-amino acid decarboxylase deficiency. Sci. Transl. Med. Vol. 4, 134ra61 ).
[002704] [002704] Subjects indicated for treatment receive the AAV vector composition, delivered safely to the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) by bilateral infusions, or alternatively, intrastriatally (into the nucleus caudate and putamen), or in the subthalamic nucleus (STN), for example, optionally via the FDA-approved SMARTFLOW® neuroventricular cannula (SurgiVision, Inc.) specifically designed for clinical application, with or without the aid of the CLEARPOINT® system to assist neurosurgeons to direct and observe the distribution of the therapeutic agent in the brain (see, for example, San Sebastian, et al., 2014, Mol. Ther. Methods Clin. Dev. 3: 14049; see, for example, Feng and Maguire- Zeiss, 2010, CNS Drugs 24(3):177-192 ).
[002705] [002705] For example, during surgery, two target points are determined in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) that are sufficiently separated from each other in dorsolateral directions and identified on a magnetic resonance image.
[002706] [002706] A drill hole is drilled into each side of the cranial bone, through which the vector is injected into target points via the two-track insertion route.
[002707] [002707] The solution containing the AAV vector is prepared at a concentration of 1.5 x 1012 vector genomes/ml, and 50 µl per dot of the solution is injected at 1 µl/min; each patient receives 3 x 10 11 vector genomes of the AAV vector construct.
[002708] [002708] AAV2 neutralizing antibody titers are determined by measuring the B-galactosidase activities in HEK293 cells transduced with 5 x 10 8 vector genomes/cell of AAV2 vectors expressing B-galactosidase at various dilutions of serum. PET.
[002709] [002709] The level of AADC expression in the substantia nigra is assessed on PET images with FMT six days before surgery and one and six months after gene transfer.
[002710] [002710] All patients stopped taking dopaminergic medications 18 hours before PET and took 2.5 mg/kg carbidopa orally one hour before FMT injection.
[002711] [002711] Thereafter, 0.12 mCi/kg of FMT in saline was infused into an antecubital vein, and a sequence of dynamic acquisitions was obtained within 90 minutes.
[002712] [002712] The PET and MRI data are co-registered with a fusion processing program (Syntegra; Philips, Amsterdam, The Netherlands) to produce the fusion images.
[002713] [002713] Radioactivities within the volumes of interest traced in the nigrostriatal pathway are calculated between 80 and 90 minutes after the tracer injection.
[002714] [002714] A change in FMT uptake in the nigrostriatal pathway from baseline to 24 weeks is assessed using the substantia nigra to striatal ratio of radioactivity. Statistical analysis.
[002715] [002715] Values at baseline and 6 months after gene transfer are compared using Student's t test (paired analysis).
[002716] [002716] A two-sided P value < 0.05 is considered to indicate significant differences.
[002717] [002717] Two-way analysis of variance with Bonferroni correction of P-values is used for short-term response to levodopa.
[002718] [002718] The safety and tolerability of bilateral administration of AAV vector compositions using real-time image-guided infusion into the brain of individuals with Parkinson's disease can be monitored for up to or after 9 months after surgery.
[002719] [002719] Wide coverage of target areas (substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA)) and wide distribution of AADC protein in the striatum should be achieved without inducing adverse effects.
[002720] [002720] Changes in growth and motor skills: Patients should gain weight and show improvement in their motor scores after gene transfer within one year of treatment.
[002721] [002721] Weight will be measured 3 to 6 months after gene transfer.
[002722] [002722] All patients should initially have raw scores of zero on the Alberta Infantile Motor Scale (AIMS) and very low raw scores on the Peabody Developmental Motor Scale, Second Edition (PDMS-II).
[002723] [002723] After gene transfer, all patients should show continued increases in their raw scores on these two scales, which indicates that their motor functions have improved.
[002724] [002724] The Comprehensive Developmental Inventory for Infants and Toddlers (CDIIT) covers both cognitive and motor development.
[002725] [002725] All patients should have low raw CDIIT scores prior to gene transfer, and the subsequent increase in scores demonstrates improvement in both motor and cognitive functions. Subjective improvements after gene transfer.
[002726] [002726] To document symptoms that are more difficult to quantify, patients' spouses, guardians or caregivers are asked to complete a questionnaire at the end of the study.
[002727] [002727] Symptoms of oculogyric crises should subside, and eye deviations and sleep disruptions, for example, are some mild symptoms of oculogyric crises that may remain after gene therapy.
[002728] [002728] Individuals may experience greater emotional stability, and/or some improvement in sweating and hyperthermia (a common manifestation of body temperature instability in hot weather).
[002729] [002729] There should be no detectable abnormality in heart rate variability assessed by 24-hour Holter monitoring either before or after gene transfer.
[002730] [002730] Prior to gene therapy, bedridden patients with little spontaneous movement may experience less severe ptosis (drooping upper eyelid) one to two weeks after gene transfer.
[002731] [002731] According to previous studies, dyskinesia can occur one month after gene transfer, but when a reduction in dyskinesia is observed, motor development should begin (Hwu, WL, et al, 2012. Gene therapy for aromatic L-amino acid decarboxylase deficiency. Sci. Transl. Med. Vol. 4, 134ra61).
[002732] [002732] Individuals may experience greater head control after three months, sit with support after six to nine months, rise from prone position after three months, and hold trinkets and stand with support after sixteen months after gene transfer, for example.
[002733] [002733] Anti-ANAV2 antibodies should be negative in patients prior to gene therapy, and titers may increase slightly after gene transfer. PET scans and CSF analysis
[002734] [002734] PET scans and CSF analyzes are performed for treated patients.
[002735] [002735] Six months after gene transfer, PET scans should reveal that uptake of 6-fluorodopa (FDOPA) increases from baseline at the combined treatment sites (right and left).
[002736] [002736] CSF analysis should reveal increases in homovanillic acid (HVA, a metabolite of dopamine) and 5-hydroxyindoleacetic acid (HIAA, a metabolite of serotonin) levels.
[002737] [002737] However, L-DOPA and 3-O-methyldopa levels may remain elevated (Hwu, WL, et al., 2012. Gene therapy for aromatic L-amino acid decarboxylase deficiency. Sci. Transl. Med. Vol. 4, 134ra61). Example 4. Administration of AADC Polynucleotides.
[002738] [002738] The AAV particle compositions are infused into the putamen of Parkinson's disease patients via the methods of administration described in Example 3.
[002739] [002739] The dose, number of patients and volume are shown in Table 4.
[002740] [002740] During the course of the study the safety and tolerability of infusion of recombinant adeno-associated virus (AAV) vector compositions containing AADC polynucleotides were evaluated in human patients diagnosed with Parkinson's disease.
[002741] [002741] Patients were assessed before surgery and monthly for six months after surgery, using multiple measures, including the Generalized Dystonia Scale (GDS) (see Comella, et al., 2003, Movement Disorders, 18( 3):303-312), the L-DOPA challenge test, UPDRS scores, motor status diaries, and laboratory tests.
[002742] [002742] Using diaries that separate the day into half-hour segments, caregivers of patients will record their mobility during the four days prior to admission and for an additional four days at six months after admission to the study site.
[002743] [002743] Caregivers of patients are trained to classify the individual's condition as sleeping, immobile, mobile without bothersome dyskinesias, or mobile with bothersome dyskinesias.
[002744] [002744] The total number of hours spent in each of these categories is calculated, and differences between baseline and six-month scores are compared between groups.
[002745] [002745] Short-term response to levodopa is assessed at baseline and 6 months after gene transfer; subjects take 100 mg of levodopa orally with 25 mg of benserazide after 20 hours without dopaminergic medication.
[002746] [002746] Motor symptoms based on GDS and plasma concentrations of levodopa are assessed at baseline and minutes, 1, 2, 3, and 4 hours after ingestion of levodopa (see, for example, Muramatsu, et al., 2010 , "A phase | study of aromatic L-amino acid decarboxylase gene therapy for Parkinson's disease." Mol. Ther. 18:1731—1735).
[002747] [002747] While the present invention has been described to some extent and with some particularity with respect to the various embodiments described, it should not be limited to any such details or embodiments of any particular embodiment, but should be interpreted with reference to the appended claims. in order to provide the broadest possible interpretation of such claims in view of the state of the art and, therefore, effectively cover the desired scope of the invention.
[002748] [002748] All publications, patent applications, patents, and other references mentioned in this application are hereby incorporated in their entirety by reference.
[002749] [002749] In case of conflict, this specification, including definitions, prevails.
[002750] [002750] In addition, section titles, materials, methods, and examples are illustrative only and are not intended to be limiting.

权利要求:
Claims (34)
[1]
1. An aromatic L-amino acid decarboxylase (AADC) polynucleotide, characterized in that it comprises a region of AADC sequence that has at least 95% identity to SEQ ID NO: 979.
[2]
2. AADC polynucleotide according to claim 1, characterized in that the AADC sequence region comprises a promoter region, an enhancer region, a multiple cloning site (MCS) region and a polyadenylation signal region ( pulley)).
[3]
3. AADC polynucleotide according to claim 2, characterized in that the AADC sequence region comprises at least one 5' inverted terminal repeat (ITR) region and a 3' ITR region.
[4]
4. AADC polynucleotide according to claim 3, characterized in that one or more of the 5' ITRs are located 5' in relation to the MCS region and one or more of the 3' ITRs are located 3' in relation to the signal of pulley).
[5]
5. AADC polynucleotide according to claim 3, characterized in that the AADC sequence region comprises a first exon region, a first intron region, a second intron region and a second exon region.
[6]
6. AADC polynucleotide according to claim 5, characterized in that the enhancer region and the promoter region are derived from CMV.
[7]
7. AADC polynucleotide according to claim 6, characterized in that the first exon region is immediate early exon 1 (ie1) or fragments thereof, the first intron region is intron 1 ie1i or fragments of same, the second intron region is intron 2 of human beta-globin (hBglobin) or fragments thereof and the second exon region is exon 3 hBgalobin or fragments thereof.
[8]
8. AADC polynucleotide according to claim 2, characterized in that the poly(A) signal is derived from human growth hormone.
[9]
9. Plasmid or vector characterized in that it encodes the AADC polynucleotide as defined in any one of claims 1 to 8.
[10]
10. Plasmid or vector according to claim 9, characterized in that an adeno-associated virus (AAV) or is derived from it.
[11]
11. Recombinant AAV virus, characterized in that it comprises the AAV plasmid or vector as defined in claim 10.
[12]
12. Recombinant AAV virus according to claim 11, characterized in that it comprises a capsid serotype selected from the group consisting of AAV2, PHP.B, PHP.A, AAV1, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3 , AAV4, AAV4-4, AAVS, AAV6, AAVG6.1, AAVG6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAVO, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9 .45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVIO, AAV1I1, AAV12, AAVI1IG;3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42 -2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAVA42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15 , AAVA42-aa, AAVA43-1, AAV43-12, AAVA43-20, AAV43-21, AAV43-23, AAVA43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223 .2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7/rh.48, AAV1-8/rh.49, AAV2-15/rh.62, AAV2-3/rh.61 , AAV2-4/rh.50, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-11/rh.53, AAVA4 - 8/111.64, AAV4-9/rh.54, AAV4-19/rh.55, AAV5-3/rh.57, AAV5-22/rh.58,
AAV7.3/hu.7, —AAV1I6.8/hu.10, AAV1I6.12/hu11, AAV29.3/bb1, AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu .40, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54 , AAV145.6/hu.55, AAV161.10/hu.60, AAV161.6/hu.61, AAV33.12/hu.17, AAV33.4/hu.15, AAV33.8/hu.16, AAV52 /hu.19, AAV52.1/hu.20, AAV58.2/hu.25, AAVA3S.3, AAVASA, AAVAS.5, AAVAS.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3 , AAVF5, AAVH2, AAVrh.72, AAVh.8, AAVrh.68, AAVrh.70, AAVPpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh .47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVHG6, AAVLKO3, AAVH-1/hu.1, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh .38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5R1, AAVCy.2, AAVcy.3, AAVcy4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy .5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.1o, AAVhu.11 , AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, A AVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu. 41, AAVhu.42, AAVhu.43, AAVhu44, AAVhud44R1, AAVhu.44R 2, AAVhu.44R3, —AAVhu45, AAVhu46, AAVhu47, AAVhu.4ô, AAVhu.48R1, AAVhu.48R2, AAVhu48R3, AAVhu.49, AAVhu .51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67 , AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVIh. 17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh46, AAVIrh48 AAVIrh481, AAVIh48.1.2,
AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh. 64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8.R, AAVrh8R mutant ASSGR, AAVrIh8R R533A mutant, AMAV, BAAV, goat AAV, bovine AAV, AAVhE1.1, AAVRERN.5, AAVRER1.14, AAVhEr1. 8, AAVREr.16, AAVREr1.18, AAVREr.35, AAVRErI.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVRER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LKO1, AAV-LKO2, AAV-LKO3, AAV-LKO4, AAV-LKOS, AAV-LKO6, AAV-LKO7, AAV-LKO8, AAV-LKO9, AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAECA4, AAV-PAECS6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-MIRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10- 8, AAV SM 100-3, AAV SM 100-10, B NP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAVA4-8/rh.64, AAVLG-9/hu .39, AAVB5A4.5/hu.23, AAV54.2/hu.22, AAV5A4.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAVA46.2/hu.28 , AAVA46.6/hu.29, AAV128.1/hu.43, true-type AAV (ttAAV), UPENN AAV 10, Japanese AAV serotypes 10, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-
6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-P1, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt- P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV
CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-H1, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-NA, AAV CKd-N9, AAV CLg-F1, AAV CLg-F2, AAV CLg-F3, AAV CLg-FA4, AAV CLQg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLv1-1, AAV Clv1-10, AAV CLv1-2, AAV CLv-12, AAV CLv1-3, AAV CLv-13, AAV CLv1-4, AAV Clv1-7, AAV Clv1-8, AAV Clv1-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLV-R1, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-
8.6, AAV CSp-87, AAV CSp-8.8, AAV CSp-89, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/ HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVFIG6/HSC16, AAVF1I7/HSC17, AAVF2/HSC 2, AAVF3/HSC3, — AAVF4/HSC4, — AAVFE5/HSC5, — AAVF6/HSCG6, AAVF7/HSC7 , AAVF8/HSC8, AAVF9/HSC9, PHP.B (AAV-PHP.B), PHP.A (AAV.PHP.A) G2B-26, G2B-13, TH1.1-32, TH1.1-35, AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT- - AAVPHP.B-NOT, AAVPHP.B-EGS, —AAVPHP.B-SGN,
AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, —AAVPHP .B-TTP, —AAVPHP.S/G2A12, AAVG2A15/G2A3, AAVG2BA, and/or AAVG2B5, and variants thereof.
[13]
13. Recombinant AAV virus according to claim 12, characterized by the fact that the capsid serotype is AAV 2.
[14]
14. Recombinant AAV virus according to claim 12, characterized in that the capsid serotype is AAVrh10.
[15]
15. Recombinant AAV virus according to claim 12, characterized in that the capsid serotype is AAV9 (hu14).
[16]
16. Recombinant AAV virus according to claim 12, characterized in that the capsid serotype is AAV-DJ.
[17]
17. Recombinant AAV virus according to claim 12, characterized in that the capsid serotype is AAV9. 47.
[18]
18. Recombinant AAV virus according to claim 12, characterized in that the capsid serotype is AAV-DJ8.
[19]
19. AADC polynucleotide according to claim 1, characterized in that the AADC sequence region has at least 99% identity with SEQ ID NO: 979.
[20]
20. A pharmaceutical composition characterized in that it comprises an adeno-associated virus (AAV) particle, said AAV particle comprising an AAV capsid and a vector genome, said vector genome comprising at least one AADC sequence region with at least 95% identity to SEQ ID NO: 900.
[21]
21. Pharmaceutical composition according to claim 20, characterized in that the AADC sequence region has at least 99% identity with SEQ ID NO: 979.
[22]
22. Pharmaceutical composition according to claim 20, characterized in that at least 70% of the AAV particles contain the genome of a vector.
[23]
23. Method for reducing periods of dyskinesia in an individual, characterized in that it comprises administering to said individual the pharmaceutical composition as defined in claims 20 to 22.
[24]
24. Method according to claim 23, characterized in that the reduction in periods of dyskinesia is shown by the improvement of rest time and motor fluctuations by at least 30%.
[25]
25. Method according to claim 24, characterized in that the improvement lasts at least 6 hours.
[26]
26. Method according to any one of claims 23 to 25, characterized in that the individual has Parkinson's disease.
[27]
27. Method for improving the sleep-wake cycle of an individual, characterized in that it comprises administering to said individual the pharmaceutical composition as defined in claims 20 to 22.
[28]
28. The method of claim 27, characterized in that the subject's amount of rapid eye movement (REM) sleep is decreased relative to the subject's amount of REM sleep prior to administration of the pharmaceutical composition.
[29]
29. Method according to claim 27, characterized in that the amount of non-REM sleep (NREM) is increased in relation to the amount of REM sleep of the individual before the administration of the pharmaceutical composition.
[30]
30. Method according to any one of claims 27 to 29, characterized in that the individual has Parkinson's disease.
[31]
31. A method for treating a sleep disorder in a subject, characterized in that it comprises administering to said subject the pharmaceutical composition as defined in claims 20 to 22.
[32]
32. Method according to claim 31, characterized in that the sleep disorder is insomnia.
[33]
33. Method according to any one of claims 31 or 32, characterized in that the individual has Parkinson's disease.
[34]
34. Method for increasing the level of AADC protein in an individual, characterized in that it comprises the administration of the pharmaceutical composition as defined in claims 20 to 22..
“ | | "THE - :
J

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

优先权:

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申请号 | 申请日 | 专利标题

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US201762520084P| true| 2017-06-15|2017-06-15|

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US62/520,084|2017-06-15|

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US201762554155P| true| 2017-09-05|2017-09-05|

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US62/554,155|2017-09-05|

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PCT/US2018/037437|WO2018232055A1|2017-06-15|2018-06-14|Aadc polynucleotides for the treatment of parkinson's disease|

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