capsids of adeno-associated virus variants and methods of using them

专利摘要:
Variant adeno-associated virus (AAV) capsid proteins are provided here that have one or more modifications in the amino acid sequence relative to a parental AAV capsid protein, which, when present in an AAV virion, confer greater infectivity to one or more types of muscle cells when compared to muscle cell infectivity by an AAV virion comprising the unmodified parental AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof that comprise a variant AAV capsid protein that is described here, methods of producing these capsid proteins and rAAV virions and methods for using these capsid proteins and rAAV virions in research and clinical practice, for example, in providing nucleic acid sequences for one or more muscle cells for the treatment of muscle disorders and diseases.
公开号:BR112020005436A2
申请号:R112020005436-4
申请日:2018-09-19
公开日:2020-09-24
发明作者:David H. KIRN；Melissa KOTTERMAN；David Schaffer
申请人:4D Molecular Therapeutics Inc.；
IPC主号:

专利说明:

[001] [001] This patent application claims the benefit of Provisional U.S. Patent Application Serial No. 62 / 560,901, filed on September 20, 2017, the full disclosure of which is incorporated herein by reference. FIELD OF THE INVENTION
[002] [002] The invention disclosed here relates generally to the field of adeno-associated virus (AAV) virions that comprise variant capsid proteins and to the production of these variant capsids using directed evolution techniques. BACKGROUND OF THE DISCLOSURE
[003] [003] Muscle is associated with a variety of serious genetic disorders. Muscle is the target tissue in gene therapy for many diseases of muscular dystrophy and can also be exploited as a biological factory to produce secretion factors to treat a systemic disease. The provision of therapeutic genes for muscle tissue in humans is arguably the most urgent unmet need in the treatment of muscle-related diseases.
[004] [004] One approach to carry out gene delivery directed to muscle is gene-based adeno-associated virus (AAV) therapy, in which a recombinant adeno-associated virus (rAAV) is used to supply a gene for one or more more muscle cells, for example, to replace a missing gene, to correct a defective dominant gene or to provide a template for ongoing protein therapy. Although clinical AAV-based gene therapy is progressively successful, it is still fraught with flaws in the properties of viral vectors, including, for example, targeting the desired muscle cells with high efficiency. Consequently, there is a need in the art for new AAV variants with superior transduction capabilities that will provide more efficient gene-based delivery to muscle cells for the treatment of disease. There is a need in the art for such AAV variants that exhibit an enhanced muscle transduction profile - in some cases broadly, in other cases preferably for certain types of muscle cells - when compared to wild-type AAVs or variants of AAV that are known in the art.
[005] [005] The naturally occurring AAV is a single-stranded DNA virus that contains three open reading frames, rep, cap and aap. The first gene, rep, encodes four proteins necessary for genome replication (Rep78, Rep68, Rep52 and Rep40), the second, cap, expresses three structural proteins (VP1-3) that come together to form the viral capsid and the third expresses the assembly activation protein (AAP) that is essential for the assembly of the capsid. AAV is dependent on the presence of a helper virus, such as an adenovirus or a herpesvirus, for active replication. In the absence of a helper virus, AAV establishes a latent state in which its genome is maintained episomally or integrated into the host chromosome at the AAVS1 locus.
[006] [006] The techniques of directed evolution in vitro and in vivo can be used to select variants of AAV that offer an improvement over current gene supply vectors based on AAV. Such directed evolution techniques are known in the art and described, for example, in the publication PCT WO 2014/194132 and in Kotterman & Schaffer (Nature Review Genetics, AOP, published online
[007] [007] AAV variants have been disclosed, for example, in U.S. Patent Numbers 9,193,956; 9; 186; 419; 8,632,764; 8,663,624;
[008] [008] All documents and references cited here and in the referenced patent documents are hereby incorporated by reference. SUMMARY OF THE INVENTION
[009] [009] Here are provided variant adeno-associated virus (AAV) capsid proteins that have one or more modifications in the amino acid sequence in relation to a parental AAV capsid protein, which, when present in an AAV virion, confer greater infectivity of one or more types of muscle cells when compared to the infectivity of muscle cells by an AAV virion comprising an unmodified parental AAV capsid protein. Also provided are recombinant AAV virions and pharmaceutical compositions thereof which comprise a variant AAV capsid protein as described here, methods of producing capsid proteins and variant rAAV virions and methods for using these capsid proteins and rAAV virions in research and clinical practice, for example, in providing nucleic acid sequences for one or more muscle cells for the treatment of disorders and diseases.
[0010] [0010] In some aspects of the disclosure, variant adeno-associated virus (AAV) capsid proteins are provided, these variant AAV capsid proteins having one or more modifications in the amino acid sequence in relation to a parental AAV capsid, which , when present in an AAV virion, confer greater infectivity to one or more types of muscle cells (for example, skeletal muscle cells and / or cardiac muscle cells) when compared to the infectivity of muscle cells by an AAV virion comprising a parental AAV capsid protein that does not include modification of the amino acid sequence. In related aspects of the disclosure, variant AAV capsid proteins, when present in an AAV virion, also provide greater resistance to neutralization by anti-AAV antibodies.
[0011] [0011] In some aspects of the disclosure, recombinant AAV virions (rAAV) are provided, these rAAV virions comprising a variant capsid protein which is described here, in which the rAAV virions exhibit greater infectivity of one or more cell types muscle (e.g. skeletal muscle cells and / or cardiac muscle cells) in relation to muscle cell infectivity by an AAV virion comprising a corresponding unmodified parental AAV capsid protein. In some embodiments, the rAAV virion exhibits greater infectivity of all muscle cells compared to the AAV virion which comprises the parental AAV capsid protein. In other embodiments, the rAAV virion exhibits greater infectivity for certain types of muscle cells, but not others related to the AAV virion which comprises the parental AAV capsid protein. Put another way, the rAAV virion exhibits greater infectivity which is preferable for certain types of muscle cells, but not for others, for example, rAAV demonstrates a preferentially greater infectivity of one or more cell types selected from skeletal muscle fibroblasts , skeletal muscle satellite cells, cardiac fibroblasts, cardiac progenitor cells, smooth muscle cells and / or diaphragm muscle cells, but does not show greater infectivity of all cell types.
[0012] [0012] In some embodiments, the rAAV virion comprises a heterologous nucleic acid. In some of these embodiments, the heterologous nucleic acid encodes an RNA that encodes a polypeptide. In other of these embodiments, the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, for example, the heterologous nucleic acid sequence is an RNA interference agent, a guide RNA for a nuclease etc.
[0013] [0013] Pharmaceutical compositions comprising the target infectious rAAV virions and a pharmaceutically acceptable carrier are also provided here.
[0014] [0014] Also provided is the use of a rAAV virion comprising a variant capsid protein which is described here in a method of delivering a heterologous nucleic acid to a target cell (such as a cardiomyocyte) through contact of the target cell with the rAAV virion. In some embodiments, the target cell is in vivo, such as in the heart of an individual in need of treatment for cardiovascular disease. In other embodiments, the target cell is in vitro.
[0015] [0015] Methods of treating and / or preventing a disease (e.g., a cardiac or skeletal muscle disorder) are also provided by administering to an individual in need of this treatment an efficient amount of rAAV virions comprising a protein of the variant capsid as described herein or a pharmaceutical composition comprising an efficient amount of the rAAV virions.
[0016] [0016] An isolated nucleic acid is also provided which comprises a sequence encoding a variant AAV capsid protein which is described herein and a host cell comprising the isolated nucleic acid. In still other embodiments, the isolated nucleic acid and / or the isolated host cell comprises rAAV.
[0017] [0017] In some respects, the variant AAV capsid protein comprises an insertion of approximately 5 amino acids to approximately 20 amino acids (a "heterologous peptide" or a "peptide insertion") in the GH-loop of the capsid protein, relative to a corresponding parental AAV capsid protein, where the variant capsid protein, when present in an AAV virion, confers greater infectivity of a muscle cell compared to the infectivity of a muscle cell by an AAV virion comprising the protein of the corresponding parental AAV capsid. In some embodiments, the peptide essentially comprises or consists of a sequence selected from the group consisting of NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), GNLTKGN (SEQ ID NO: 15) NO: 16), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19), DNTVTRS (SEQ ID NO: 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 25), ASDSTKA (SEQ ID NO: 26), LANKIQRTDA (SEQ ID NO: : 27), LANKTTNKDA (SEQ ID NO: 28), LATNKIGVTA (SEQ ID NO: 29), LAGNLTKGNA (SEQ ID NO: 30), LANTVKLSTA (SEQ ID NO: 31), LASNTVKAIA (SEQ ID NO: 32), LAASNITKAA (SEQ ID NO: 33), LADNTVTRSA (SEQ ID NO: 34), LANKISAKDA (SEQ ID NO: 35), LANQDYTKTA (SEQ ID NO: 36), LATNKIGVTS (SEQ ID NO: 37), LATNKIGVTA (SEQ ID NO: 38), LAQADTTKNA (SEQ ID NO: 39), LATNRTSPDA (SEQ ID NO: 40), LASNTTQKTA (SEQ ID NO: 41) and LAASDSTKAA (SEQ ID NO: 42). In some preferred embodiments, the peptide essentially comprises or consists of a sequence selected from the group consisting of NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), LANKIQRTDA (SEQ ID NO: 27), LANKTTNKDA (SEQ ID NO: 28), LATNKIGVTA (SEQ ID NO: 29) and LATNKIGVTS (SEQ ID NO: 37).
[0018] [0018] In some respects, the variant AAV capsid protein comprises one or more amino acid substitutions in relation to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in an AAV virion, confers greater infectivity of a muscle cell compared to the infectivity of a muscle cell by an AAV virion comprising the corresponding parental AAV capsid protein.
[0019] [0019] In some embodiments, a variant AAV capsid protein is disclosed which comprises a P363L substitution in relation to AAV2 and optionally further comprising an E347K and / or V708I substitution in relation to AAV2.
[0020] [0020] In related aspects, the variant AAV capsid protein comprises a peptide insert and one or more amino acid substitutions in relation to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in a virion of AAV, confers greater infectivity of a muscle cell compared to the infectivity of a muscle cell by an AAV virion that comprises the corresponding parental AAV capsid protein. In various embodiments, a variant AAV capsid protein is provided that comprises a peptide insert and a V708I substitution over AAV2, where the peptide insert is optionally selected from the group consisting of NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), GNLTKGN (SEQ ID NO: 16), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19), DNTVTRS (SEQ ID NO: 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT ( SEQ ID NO: 25), ASDSTKA (SEQ ID NO: 26), LANKIQRTDA (SEQ ID NO: 27), LANKTTNKDA (SEQ ID NO: 28), LATNKIGVTA (SEQ ID NO: 29), LAGNLTKGNA (SEQ ID NO: 30) ), LANTVKLSTA (SEQ ID NO: 31), LASNTVKAIA (SEQ ID NO: 32), LAASNITKAA (SEQ ID NO: 33), LADNTVTRSA (SEQ ID NO: 34), LANKISAKDA (SEQ ID NO: 35), LANQDYTKTA (SEQ ID NO: 36), LATNKIGVTS (SEQ ID NO: 37), LATNKIGVTA (SEQ ID NO: 38), LAQADTTKNA (SEQ ID NO: 39), LATNRTSPDA (SEQ ID NO: 40), LASNTTQKTA (SEQ ID NO: 41) and LAASDSTKAA (SEQ ID NO: 42), preferably from the group consisting of NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), LANKIQRTDA (SEQ ID NO: 27), LANKTTNKDA (SEQ ID NO: 28), LATNKIGVTA (SEQ ID NO: 29) and LATNKIGVTS (SEQ ID NO: 37). In various embodiments, a variant AAV capsid protein is provided that comprises a peptide insert and a P363L substitution with respect to AAV2, where the peptide insert is optionally selected from the group consisting of GNLTKGN (SEQ ID NO: 16), LAGNLTKGNA (SEQ ID NO: 30), QADTTKN (SEQ ID NO: 23) and LAQADTTKNA (SEQ ID NO: 39).
[0021] [0021] In some embodiments, a variant AAV capsid protein is disclosed which comprises the heterologous peptide LANKIQRTDA (SEQ ID NO: 27) and a V708I substitution with respect to AAV2 and which optionally further comprises an A593E and / or S109T substitution and / or T330A and / or R588M in relation to AAV2. In other embodiments, a variant AAV capsid protein comprising the heterologous peptide LANKIQRTDA (SEQ ID NO: 27) and an A35P substitution with respect to AAV2 is disclosed. In other embodiments, a variant AAV capsid protein is disclosed which comprises the heterologous peptide LANKIQRTDA (SEQ ID NO: 27) and amino acid substitutions N312K, N449D, N551S, I698V and L735Q in relation to AAV2 and which optionally further comprises a substitution V708I over AAV2.
[0022] [0022] In some embodiments, a variant AAV capsid protein is disclosed which comprises the heterologous peptide LANKTTNKDA (SEQ ID NO: 28) and a V708I substitution with respect to AAV2 and which optionally further comprises a S109T and / or W694C substitution and / or W606C in relation to AAV2. In other embodiments, a variant AAV capsid protein comprising the heterologous peptide LANKTTNKDA (SEQ ID NO: 28) and an I698V substitution with respect to AAV2 is disclosed. In other embodiments, a variant AAV capsid protein is disclosed which comprises the heterologous peptide LANKTTNKDA (SEQ ID NO: 28) and amino acid substitutions N312K, N449D, N551S, I698V and L735Q in relation to AAV2 and which optionally further comprises a substitution V708I over AAV2.
[0023] [0023] In some embodiments, a variant AAV capsid protein is disclosed which comprises the heterologous peptide LATNKIGVTA (SEQ ID NO: 29) and a V708I substitution with respect to AAV2 and which optionally further comprises an N449K and / or G222S substitution in relation to AAV2. In other embodiments, a variant AAV capsid protein is disclosed which comprises the heterologous peptide LATNKIGVTA (SEQ ID NO: 29) and amino acid substitutions N312K, N449D, N551S, I698V and L735Q in relation to AAV2 and which optionally further comprises a substitution V708I over AAV2.
[0024] [0024] In some embodiments, a variant AAV capsid protein is disclosed which comprises a heterologous peptide which is described here and a P363L substitution with respect to AAV2.
[0025] [0025] Methods for manufacturing and / or supplying an rAAV comprising a variant AAV capsid are also disclosed here. In addition, kits are provided here that comprise an rAAV that comprises a variant AAV capsid that is disclosed here and for use in the methods described here.
[0026] [0026] In other embodiments, the AAV virion comprising the capsid protein variant in the preceding paragraphs may incorporate any of the preceding or subsequently disclosed modalities. Indeed, it is considered that certain features of the invention, which are, for clarity, described in the context of separate modalities, can also be provided in combination in a single modality. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination. All combinations of the modalities that pertain to the invention are specifically included by the invention and are disclosed here only as if each and every combination were individually and explicitly disclosed. In addition, all subcombination of the various modalities and their elements are also specifically included by the invention and are disclosed here only as if each and every such subcombination were individually and explicitly disclosed here.
[0027] [0027] The Summary of the Invention is not intended to define the claims nor is it intended to limit the scope of the invention in any way.
[0028] [0028] The invention is best understood from the detailed description below when read in association with the attached drawings. The patent or patent application file contains at least one drawing made in color. Copies of this patent or patent application publication with colored drawing (s) will be provided by the Office upon application and payment of the necessary fee. It is emphasized that, according to common practice, the various characteristics of the drawings are not to scale. On the contrary, the dimensions of the various characteristics are expanded or reduced arbitrarily for clarity. The following figures are included in the drawings.
[0029] [0029] Figure 1 represents modalities of a directed evolution methodology. Step (a) represents the production of a viral capsid library that comprises combinations of DNA mutation techniques and cap genes. Step (b) represents the packaging of the viruses so that each viral particle is composed of a mutant capsid that surrounds the cap gene that encodes this capsid and purified. The capsid library is then placed under selective pressure in vitro or in vivo. In this aspect of directed evolution technology, tissues or cellular material of interest is collected to isolate variants of
[0030] [0030] Figure 2 shows the PCR amplification of viral genomes from cardiac and skeletal muscle tissues in a representative round of selection. The bands inside the red boxes represent the successful amplification of the viral genomes.
[0031] [0031] Figures 3A-3C show the frequency of motifs within the sequencing analysis. Fig. 3A provides the Round 4 sequencing analysis for the selective pressure of the intravenous delivery to the cardiac tissue. Fig. 3B provides the sequencing analysis for Round 2 for the selective pressure of the intravenous delivery in the presence of neutralizing antibodies to the cardiac tissue. Fig. 3C provides the sequencing analysis for Round 3 for the selective pressure of intravenous delivery to skeletal muscle tissue. Figure 3A shows 57.40% of LANKIQRTDA Reason, 16.96% of LANKTTNKDA Reason, 7.32% of Reason A593E, 7.32% of Other, 4.88% of Reason V708I and 4.88% of LASNTVKAIA Reason . Figure 3B shows 21.14% of Other, 20.33% of Reason LAQADTTKNA, 15.45% of Reason LANKTTNKDA, 15.45% of Reason LAASNITKAA, 15.45% of Reason for Chimera AAV6 / AAV5 and 12.20 % of Reason
[0032] [0032] Figures 4A-4C Fig. 4A is a three-dimensional model representative of AAV2 that contains a random heptamer after amino acid 587 and a V708I substitution. Fig. 4B is a three-dimensional model representative of the chimera AAV6 / AAV5 which contains substitutions V229I, A490T and A581T (corresponding to the amino acid sequence shown as SEQ ID NO: 62). Fig. 4C is a three-dimensional model representative of AAV2 that contains a P363L replacement.
[0033] [0033] Figure 5 provides an alignment of SEQ ID NOS: 1-11 of wild-type AAV that shows amino acid locations between and along wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B and AAV4 -10.
[0034] [0034] Figures 6A-6E provide data on the transduction of human cardiomyocytes in vitro by the recombinant AAV virus comprising the capsid of the new variant LANKIQRTDA + V708I of the AAV, the capsid of the new variant LANKTTNKDA + V708I of the new AAV and the capsid of the new LATNKIGVTA + V708I variant, each expressing a GFP transgene under the control of the CAG promoter. Figure 6A: Cells that have been differentiated into cardiomyocytes from a human pluripotent stem cell line have been infected with | new AAV variant LANKIQRTDA + V708I.CAG.GFP, new variant LANKTTNKDA + V708I from AAV.CAG.GFP, new variant of AAV LATNKIGVTA + V708I.CAG.GFP or wild-type controls AAV1.CAG.GFP, AAV2.CAG.GFP and AAV9.CAG.GFP in MOIs of 20, 100, 500 and 2500. Immunofluorescence imaging of cell cultures 6 days after infection in all MOIs it demonstrates that capsids of the new variant of AAV transduce cardiomyocytes better than capsules of wild type AAV1, AAV2 or AAV9. Figure 6B: Quantification of the percentage of GFP-positive cardiomyocytes in each culture by flow cytometry reveals that capsids of the new AAV variant cause a significant dose-dependent improvement in the number of cells transduced compared to AAV1, AAV2 or AAV9 capsids from wild type. * p <0.05 Figures 6C-6D: Quantification of the amount of GFP in each culture by Western blot reveals that the new variants of AAV cause a significant improvement in the expression of the transgene in relation to the AAV1, AAV2 or AAV9 type capsids. wild. NT = not transduced. Figure 6E: The cells that were differentiated into cardiomyocytes from a human pluripotent stem cell line were infected with a new variant of AAV LANKIQRTDA + V708I.CAG.GFP, new variant LANKTTNKDA + V708I from AAV.CAG.GFP, new variant of AAV LATNKIGVTA + V708I.CAG.GFP or AAV1.CAG.GFP, AAV2.CAG.GFP and AAV9.CAG.GFP wild type controls. Imaging by immunofluorescence of cell cultures on days 1, 2, 3 and 5 after infection in an MOI of 500 demonstrates that the capsules of the new AAV variant transduce cardiomyocytes better and begin to express the GFP transgene earlier than AAV1, AAV2 or AAV9 wild type capsids.
[0035] [0035] Figures 7A-E provide data on the transduction of human cardiomyocytes in vitro by the recombinant AAV virus comprising the AAV6 / AAV5 chimera capsid of the new AAV variant of SEQ ID NO: 62, which expresses a GFP transgene under control of the CAG promoter. Figure 7A: The cells that were differentiated into cardiomyocytes from a human pluripotent stem cell line were infected with the AAV6 / AAV5 chimera capsid of the new AAV variant or wild-type controls AAV1.CAG.GFP, AAV8.CAG.GFP and AAV9.CAG.GFP in MOIs of 100, 500 and 2500. Imaging by immunofluorescence of cell cultures 6 days after infection in all MOIs demonstrates that the capsid of the new variant of AAV transduces cardiomyocytes better than capsids from AAV1, AAV8 or AAV9 wild type. Figure 7B: Quantification of the percentage of GFP-positive cardiomyocytes in each culture by flow cytometry reveals that the capsid of the new AAV variant causes a significant dose-dependent improvement in the number of cells transduced compared to the capsids of AAV1, AAV8 or AAV9 of the wild type. * p <0.05 Figures 7C-7D: Quantification of the amount of GFP in each culture by Western blot reveals that the new variant of AAV causes a significant improvement in the expression of the transgene in relation to AAV1, AAV8 or AAV9 type capsids. wild. vehicle = not transduced. Figure 7E: Cells that have been differentiated into cardiomyocytes from a human pluripotent stem cell line have been infected with the AAV6 / AAV5 chimera capsid of the new AAV variant or the wild type control AAV8.CAG.GFP. Imaging by immunofluorescence of cell cultures on days 3, 4, 5, and 6 after infection in a 2500 MOI demonstrates that the capsules of the new AAV variant transduce cardiomyocytes better and begin to express the GFP transgene earlier than the wild-type AAV8 capsid.
[0036] [0036] Figures 8A-C provide data on the transduction of human skeletal myofibers in vitro by the recombinant AAV virus comprising the capsid of the new variant LANKIQRTDA + V708I of the AAV, the capsid of the new variant LANKTTNKDA + V708I of the AAV and the capsid of the AAV6 / AAV5 chimera of the new variant of AAV, each expressing a GFP transgene under the control of the CAG promoter. Figure 8A: Cells that have been differentiated into skeletal myofibers from human primary myoblasts have been infected with the new variant of AAV LANKIQRTDA + V708I.CAG.GFP, the new variant LANKTTNKDA + V708I from AAV.CAG.GFP, the chimera AAV6 / AAV5 the new variant of AAV.CAG.GFP or wild-type controls AAV8.CAG.GFP and AAV9.CAG.GFP in MOIs of 100, 500 and 2500. Immunofluorescence imaging of cell cultures 7 days after infection in all MOIs demonstrate that capsids from the new AAV variant transduce skeletal myofibers better than wild-type AAV8 or AAV9 capsids. Figure 8B: Quantification of the percentage of GFP-positive skeletal myofibers in each culture by flow cytometry reveals that capsids of the new AAV variant cause a significant dose-dependent improvement in the number of cells transduced compared to AAV8 or AAV9 capsids from wild type. * p <0.05 Figure 8C: The cells that were differentiated into skeletal myofibers from human primary myoblasts were infected with the new variant of the AAV LANKIQRTDA + V708I.CAG.GFP, the chimera AAV6 / AAV5 of the new variant of AAV.CAG .GFP or AAV8.CAG.GFP and AAV9.CAG.GFP wild type controls. Imaging by immunofluorescence of cell cultures on days 2-7 after infection in a MOI of 2500 demonstrates that the capsules of the new variant of AAV transduce skeletal myofibers better and begin to express the GFP transgene earlier than capsids from AAV8 or AAV9 wild type.
[0037] [0037] Figures 9A-B provide data on the transduction of human muscle progenitor cells in vitro by the recombinant AAV virus comprising the capsid of the new variant LANKIQRTDA + V708I of the AAV, the capsid of the new variant LANKTTNKDA + V708I of the AAV and the capsid of the AAV6 / AAV5 chimera of the new AAV variant, each expressing a GFP transgene under the control of the CAG promoter. Figure 9A: Cells that have been differentiated into muscle progenitor cells from a human pluripotent stem cell line have been infected with the new AAV variant LANKIQRTDA + V708I.CAG.GFP, the new variant LANKTTNKDA + V708I from AAV.CAG.GFP, the AAV6 / AAV5 chimera of the new variant of AAV.CAG.GFP or the wild-type control AAV9.CAG.GFP in an MOI of 500. Immunofluorescence imaging of cell cultures 6 days after infection in all MOIs shows that the capsids of the new variant of AAV transduce muscle progenitor cells better than wild-type AAV9. Figure 9B: Quantification of the percentage of GFP-positive muscle progenitor cells in each culture by flow cytometry reveals that the capsids of the new variant of AAV cause a significant improvement in the number of cells transduced compared to wild-type AAV9. * p <0.05
[0038] [0038] Figures 10A-B provide data on the magnitude of improvement in the transduction of human cardiomyocytes and human skeletal myofibers in vitro by the recombinant AAV virus comprising the capsid of the new variant LANKIQRTDA + V708I of the AAV, the capsid of the new variant LANKTTNKDA + AAV V708I and the AAV6 / AAV5 chimera capsid of the new AAV variant, each expressing a GFP transgene under the control of the CAG promoter. Figure 10A: Amount of increase in transduction of human cardiomyocytes by the new AAV capsid variants compared to wild-type AAV8 and AAV9, the serotypes most widely used in clinical applications for muscle diseases. Figure 10B: Amount of increase in the transduction of human skeletal myofibers by the new AAV capsid variants compared to wild-type AAV8 and AAV9.
[0039] [0039] Figures 11A-B provide data on the transduction of mouse tissue in vivo by the recombinant AAV virus comprising the capsid of the new variant LANKIQRTDA + V708I of the AAV that expresses a luciferase transgene under the control of the CAG promoter. The mice were given a single intravenous injection through the caudal vein of 2x1011 viral genomes per animal. Figure 11A: Live imaging of luciferase on day 14 (left) and day 28 (right) after administration demonstrates that the capsid of the new variant LANKIQRTDA + V708I from AAV can transduce mouse cells in vivo. Figure 11B: Luciferase activity in the heart, diaphragm and quadriceps 56 days after administration demonstrates that the capsid of the new variant LANKIQRTDA + V708I from AAV can transduce mouse cardiac and skeletal muscle in vivo.
[0040] [0040] Figures 12A-B provide data on the transduction of skeletal muscle of non-human primates in vivo by the recombinant AAV virus that comprises the capsid of the new variant
[0041] [0041] Before the present methods and compositions are described, it is to be understood that this invention is not limited to a particular method or composition described and thus may vary. It should also be understood that the terminology used here is only for the purpose of describing particular modalities and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0042] [0042] The invention disclosed here is illustrated in the figures and in the description. However, although particular modalities are illustrated in the figures, there is no intention to limit the invention to the specific modality or to the illustrated and / or disclosed modalities. Instead, the invention disclosed herein is intended to cover all modifications, alternative and equivalent constructions that are within the spirit and scope of the invention. Thus, the figures are intended to be illustrative and not restrictive.
[0043] [0043] When a range of values is provided, it is understood that each intermediate value, up to the tenth of the unit of the lower limit unless the context clearly determines otherwise, between the upper and lower limits of this range is also specifically disclosed. Each minor range between any given value or intermediate value in a given range and any other determined or intermediate value in this given range is covered within the invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the range and each range in which any, none or both of the limits are included in the lower ranges is also covered within the invention, subject to any limit specifically excluded in the given range . When the determined range includes one or both of the limits, bands that exclude either or both of these included limits are also included in the invention.
[0044] [0044] Unless otherwise stated, all technical and scientific terms used here have the same meaning as is commonly understood by a person skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned here are incorporated here as a reference to disseminate and describe the methods and / or materials in association with those mentioned in the publications. It is understood that the present disclosure replaces any disclosure of an incorporated publication to the extent that there is a contradiction.
[0045] [0045] As will be apparent to those skilled in the art after reading this disclosure, each of the individual modalities described and illustrated here has distinct components and characteristics that can be easily separated or combined with the characteristics of any of the various other modalities without depart from the scope or spirit of the present invention. Any method mentioned can be performed in the order of the events mentioned or in any other order that is logically possible.
[0046] [0046] It is noted that as used here and in the appended claims, the forms in the singular "one", "one", "o" and "a" include referents in the plural unless the context clearly determines otherwise. Thus, for example, reference to "a recombinant AAV virion" includes a large number of these virions and reference to "muscle cell" includes reference to one or more muscle cells and equivalents of those known to those skilled in the art and so on. It is further noted that claims can be designed to exclude any optional elements. Accordingly, it is intended that this statement serves as an antecedent basis for the use of exclusive terminology as "only," "only" and similar in association with the citation of the elements of claim or the use of a "negative" limitation.
[0047] [0047] Page15 The publications discussed here are provided solely for disclosure before the filing date of this patent application. Nothing herein should be construed as an admission that the present invention is not entitled to precede this publication by virtue of the previous invention. In addition, the publication dates provided may differ from the actual publication dates which may need to be independently confirmed. DEFINITIONS
[0048] [0048] The adeno-associated virus is a non-pathogenic parvovirus composed of a 4.7-kb single-stranded DNA genome inside an icosahedral non-enveloped capsid. The genome contains three open reading frames (ORF) flanked by inverted terminal repetitions (ITR) that function as the origin of viral replication and the packaging signal. The ORF rep encodes four structural nonproteins that play a role in viral replication, transcription regulation, site specific integration and virion assembly. The ORF cap encodes three structural proteins (VP 1-3) that combine to form a 60-mer viral capsid. Finally, an ORF present as an alternative reading frame within the cap gene produces the assembly-activating protein (AAP), a viral protein that locates AAV capsid proteins in the nucleolus and acts in the capsid's motive process.
[0049] [0049] There are several naturally occurring serotypes (“wild-type”) and more than 100 known variants of AAV, each of which differs in relation to the amino acid sequence, particularly within the hypervariable regions of the capsid proteins and so in relation to its properties of gene supply. No AAV was associated with any human disease, making recombinant AAV attractive for clinical applications.
[0050] [0050] For the purposes of the disclosure presented here, the terminology “AAV” is an abbreviation for adeno-associated virus, including, without limitation, the virus itself and its derivatives. Unless otherwise stated, the terminology refers to all subtypes or serotypes and to both replication and recombinant competent forms. The term “AAV” includes, without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV -7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrh10), poultry AAV, bovine AAV , Canine AAV, goat AAV, equine AAV, primate AAV, non-primate AAV and sheep AAV. "Primate AAV" refers to AAV that infects primates, "Non-primate AAV" refers to AAV that infects non-primate mammals, "Bovine AAV" refers to AAV that infects bovine mammals, etc.
[0051] [0051] The genomic sequences of various AAV serotypes, as well as the sequences of native terminal repeats (TRs), Rep proteins and capsid subunits are known in the art. These strings can be found in the literature or in public databases such as GenBank. See, for example, GenBank Accession Numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1 (AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6) , NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AAO88208 (AAVrh10); the disclosures of which are incorporated herein as a reference for the instruction of AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virology 45: 555; Chiorini et al. (1998) J. Virology 71: 6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73: 939; Xiao et al. (1999) J. Virology 73: 3994; Muramatsu et al. (1996) Virology 221: 208; Shade et. al. (1986) J. Virol. 58: 921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33: 375-383; international patent publications WO 00/28061, WO 99/61601, WO 98/11244; and Pat. No. 6,156,303.
[0052] [0052] The sequences of cap proteins (capsids) that exist naturally associated with AAV serotypes are known in the art and include those disclosed here as AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3A (SEQ ID NO: 3), AAV3B (SEQ ID NO: 4), AAV4 (SEQ ID NO: 5), AAV5 (SEQ ID NO: 6), AAV6 (SEQ ID NO: 7), AAV7 (SEQ ID NO: 8) , AAV8 (SEQ ID NO: 9), AAV9 (SEQ ID NO: 10), AAV10 (SEQ ID NO: 11) and AAVrh10 (SEQ ID NO: 12). The terms "variant AAV capsid protein" or "AAV variant" refer to an AAV capsid protein that comprises an amino acid sequence that includes at least one modification or substitution (including deletion, insertion, point mutation, etc. .) in relation to AAV capsid protein sequences that exist naturally or "wild type", for example, as shown in SEQ ID NOs: 1-12 here. A variant AAV capsid protein can have approximately 80% identity or more with the amino acid sequence of a wild type capsid protein, for example, 85% identity or more, 90% identity or more or 95% identity or more with the amino acid sequence of the wild-type capsid protein, for example, 98% or 99% identity with the wild-type capsid protein. A variant AAV capsid protein may not be a wild type capsid protein.
[0053] [0053] For the purposes of the disclosure presented here, "AAV virion" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsulated AAV polynucleotide.
[0054] [0054] For the purposes of the disclosure presented here, the terminology “rAAV” is an abbreviation that refers to recombinant adeno-associated virus. “Recombinant”, when applied to a polynucleotide means that the polynucleotide is the product of several combinations of cloning, restriction or ligation steps and other procedures that result in a construction that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle that comprises a recombinant polynucleotide. The terms include replicas of the original polynucleotide construct and progeny of the original virus construct, respectively.
[0055] [0055] The term "rAAV vector" encompasses rAAV virions (i.e., rAAV viral particles) (e.g., an infectious rAAV virion), which by definition include an rAAV polynucleotide; and also encompasses polynucleotides that encode rAAV (for example, a single-stranded polynucleotide that encodes rAAV (ss-rAAV); a double-stranded polynucleotide that encodes rAAV (ds-rAAV), for example, plasmids that encode rAAV; and the like) .
[0056] [0056] If an AAV virion comprises a heterologous polynucleotide (i.e., a polynucleotide that is not a wild-type AAV genome, for example, a transgene that must be supplied to a target cell, an RNAi agent or an agent CRISPR that must be supplied to a target cell etc.), this is typically referred to as a "recombinant AAV virion (rAAV)" or a "rAAV viral particle." In general, the heterologous polynucleotide is flanked by at least one and usually by two, inverted terminal repeat sequences (ITRs) of the AAV.
[0057] [0057] The term "packaging" refers to a series of intracellular events that results in the assembly and encapsidation of an AAV particle. The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode adeno-associated virus replication and encapsidation proteins. rep and cap from AAV are referred to here as AAV “packaging genes”.
[0058] [0058] The term "helper virus" for AAV refers to a virus that allows an AAV (for example, wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of these AAV helper viruses are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. Adenoviruses cover a number of different subgroups, although subgroup C type 5 Adenovirus is the most commonly used. Numerous adenoviruses of human, non-human mammals and birds are known and are available from depositories such as ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); which are also available from depositaries such as the ATCC.
[0059] [0059] The terminology "helper virus function (s)" refers to the function (s) encoded in a helper virus genome that allows AAV replication and packaging (in association with other requirements) replication and packaging described here). As described here, "helper virus function" can be provided in a variety of ways, including by providing the helper virus or by providing, for example, polynucleotide sequences that encode the requirement function (s) for a producing cell in trans. For example, a plasmid or other expression vector comprising nucleotide sequences that encode one or more adenoviral proteins is transfected into a producer cell along with an rAAV vector.
[0060] [0060] The terminology virus or “infectious” viral particle is one that comprises a viral capsid competently assembled and is capable of delivering a polynucleotide component into a cell for which the viral species is tropic. The term does not necessarily imply any ability to replicate the virus. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11: S337 (which describes an infectious TCID50 titration assay); and Zolotukhin et al. (1999) Gene Ther. 6: 973. See also the Examples.
[0061] [0061] The term "tropism" as used here refers to the preferential targeting of a virus (for example, an AAV) to cells of a particular host species or to particular cell types within a host species. For example, a virus that can infect cells in the heart, lungs, liver, and muscle has a more comprehensive (that is, greater) tropism than a virus that can infect only lung and muscle cells. Tropism can also include dependence on a virus for particular types of host cell surface molecules. For example, some viruses can infect only cells with surface glycosaminoglycans, while other viruses can infect only cells with sialic acid (such dependencies can be tested using several cell lines deficient in particular classes of molecules as potential host cells for viral infection) . In some cases, the tropism of a virus describes the relative preferences of the virus. For example, a first virus may be able to infect all types of cells, but it is much more successful in infecting cells with surface glycosaminoglycans. A second virus can be considered to have a tropism similar (or identical) to that of the first virus if the second virus also prefers the same characteristics (for example, the second virus is also more successful in infecting cells with surface glycosaminoglycans) , even if the transduction efficiencies are not similar. For example, the second virus could be more efficient than the first virus in infecting each type of cell supplied tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have a similar tropism (or identical to that of the first virus. In some embodiments, the tropism of a virion comprising a target variant AAV capsid protein is not altered in relation to a naturally occurring virion. In some embodiments, the tropism of a virion comprising a target variant AAV capsid protein is expanded (i.e., expanded) in relation to a naturally occurring virion. In some embodiments, the tropism of a virion comprising a target variant AAV capsid protein is reduced compared to a naturally occurring virion.
[0062] [0062] The terminology "replication-competent" virus (for example, a replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of replicating in an infected cell ( that is, in the presence of a helper virus or helper virus functions). In the case of AAV, replication competency often requires the presence of functional AAV packaging genes. In general, the rAAV vectors that are described here are incompetent with respect to replication in mammalian cells (especially in human cells) due to the lack of one or more AAV packaging genes. Typically, these rAAV vectors do not have any AAV packaging gene sequences in order to minimize the possibility that replication-competent AAV is generated by recombination between the AAV packaging genes and a received rAAV vector. . In many embodiments, preparations of rAAV vectors that are described here are those that contain little if any replication-competent AAV (rcAAV, also referred to as RCA) (for example, less than approximately 1 rcAAV per 102 rAAV particles, less approximately 1 rcAAV per 104 rAAV particles, less than approximately 1 rcAAV per 10 rAAV particles, less than approximately 1 rcAAV per 1012 rAAV particles or no rcAAV).
[0063] [0063] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or the like. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be disrupted by non-nucleotide components. If present, changes in the structure of the nucleotide can be transmitted before or after assembly of the polymer. The term polynucleotide, as used here, refers interchangeably to single and double-stranded molecules. Unless otherwise specified or required, any embodiment presented here that comprises a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or intended to constitute the double-stranded form.
[0064] [0064] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that, when aligned, the percentage of bases or amino acids is the same when the two sequences are compared. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using computer methods and programs, including BLAST, available across the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available from the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are the alignment programs that allow gaps (spaces) in the sequence. Smith-Waterman is a type of algorithm that allows gaps in the alignments of sequences. See Meth. Mol. Biol. 70: 173-187 (1997). In addition, the GAP program using the Needleman and Wunsch alignment method can be used to align the sequences. See J. Mol. Biol. 48: 443-453 (1970).
[0065] [0065] The term "gene" refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene may contain an open reading frame that is capable of encoding a gene product. An example of a gene product is a protein, which is transcribed and translated from the gene. Another example of a gene product is an RNA, for example, a functional RNA product, for example, an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transporter RNA (tRNA), a non-coding RNA (ncRNA) , a guide RNA for nucleases, etc., which is transcribed but not translated.
[0066] [0066] The terminology "product of gene expression" or "gene product" is a molecule that results from the expression of a particular gene, as defined above. Products of gene expression include, for example, a polypeptide, an aptamer, an interfering RNA, a messenger RNA (mRNA), an rRNA, a tRNA, a noncoding RNA (ncRNA) and the like.
[0067] [0067] The term "siRNA agent" ("small interfering RNA" or "short interfering" (or siRNA)) is a duplex of RNA nucleotides that is targeted to a gene of interest (a "target gene"). A "RNA duplex" refers to the structure formed by the complementary pairing between two regions of an RNA molecule, forming a region of double stranded RNA (dsRNA). SiRNA is "targeted" to a gene in which the nucleotide sequence of the duplex portion of siRNA is complementary to a nucleotide sequence of the target gene.
[0068] [0068] The terminology "antisense RNA" encompasses RNA that is complementary to a product of gene expression. For example, an antisense RNA targeting a specific mRNA is an RNA-based agent (or it may be a modified RNA) that is complementary to the mRNA, where the hybridization of the antisense RNA to the mRNA alters the expression of the mRNA (for example , by changing the RNA stability, changing the RNA translation, etc.). Also included in "antisense RNA" are nucleic acids that encode an antisense RNA.
[0069] [0069] In relation to “CRISPR / Cas9 agents”, the term “CRISPR” covers short palindromic repetitions interspersed regularly in CRISPR-associated clusters / systems (Cas) that evolved to provide bacteria and archaea with adaptive immunity against viruses and plasmids through use of CRISPR RNAs (crRNAs) to guide the silencing of invasive nucleic acids. The Cas9 protein (or equivalent and / or functional variant thereof, that is, a protein similar to Cas9) naturally contains endonuclease DNA activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA ( also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA ("sgRNA")). Thus, the Cas9 or Cas9-like protein associates with an RNA that directs to DNA (whose term covers both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 protein or similar to Cas9 and guides the protein to a target nucleic acid sequence.
[0070] [0070] If the Cas9 or Cas9-like protein maintains its natural enzymatic function, it will cleave the target DNA to create a double-stranded break, which can lead to genome alteration (ie editing: deletion, insertion (when a polynucleotide donor is present), substitution, etc.), thereby altering gene expression. Some Cas9 variants (whose variants are covered by the term similar to Cas9) have been altered in such a way that they have reduced DNA cleavage activity (in some cases, they cleave a single strand instead of both strands of the target DNA, whereas in other cases, they have been severely reduced to no DNA cleavage). Cas9-like proteins with reduced DNA cleavage activity (even less DNA cleavage activity) can still be targeted to target DNA to block RNA polymerase activity. Alternatively, the Cas9 or Cas9-like protein can be modified by fusing the VP64 transcriptional activation domain with the Cas9 protein and co-supplying the fusion protein with an MS2-P65-HSF1 helper protein and a single guide RNA comprising aptamers of MS2 RNA in the tetra-loop and rod-loop to form a Synergistic Activation Mediator complex (Cas9-SAM) in the cell that activates transcription. Thus, enzymatically inactive Cas9-like proteins can be targeted to a specific location in a target DNA by a targeting RNA for DNA in order to block or activate the transcription of the target DNA. The term "CRISPR / Cas9 agents" as used herein encompasses all forms of CRISPR / Cas9 that are described above or that are known in the art.
[0071] [0071] Detailed information on CRISPR agents can be found, for example, in (a) Jinek et. al., Science. 2012 Aug 17; 337 (6096): 816-21: "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell. 2013 Feb 28; 152 (5): 1173-83: "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression" and (c) US Patent Application number 13 / 842,859 and PCT Patent Application number PCT / US13 / 32589 ; of which all are incorporated here as a reference in their entirety. Thus, the term "CRISPR agent" as used herein encompasses any agent (or nucleic acid encoding such an agent), which comprises naturally occurring and / or synthetic sequences, which can be used in the Cas9-based system (for example, a protein Cas9 or similar to Cas9; any component of a DNA targeting RNA, for example, a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc .; a donor polynucleotide; and the like).
[0072] [0072] By "zinc finger nucleases" (ZFNs) is meant artificial DNA endonucleases generated by the fusion of a zinc finger DNA binding domain with a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this allows zinc finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit the target DNA in the cell (for example, the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol. Ther. 2012 Feb; 20 (2): 329-38; Bibikova et al. Science. 2003 May 2; 300 (5620): 764; Wood et al. Science. 2011 Jul 15; 333 (6040): 307; Ochiai et al. Genes Cells. 2010 Aug; 15 (8): 875-85; Takasu et. al., Insect Biochem Mol Biol. 2010 Oct; 40 (10): 759-65; Ekker et al, Zebrafish 2008 Summer;
[0073] [0073] The terminology "Nuclease effector similar to the transcription activator" or "TALEN" agents refers to effector Nucleases similar to the transcription activator (TALENs). TALENs are artificial DNA endonucleases generated by fusing a TAL effector DNA binding domain (Similar to the transcription activator) with a DNA cleavage domain. TALENs can quickly be engineered to bind to virtually any desired DNA sequence and when introduced into a cell, TALENs can be used to edit the target DNA in the cell (for example, the cell's genome) by inducing cell breaks. double filament. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul 7; 29 (8): 731-4; Wood et al. Science. 2011 Jul 15; 333 (6040): 307; Tesson et al. Nat Biotechnol. 2011 Aug 5; 29 (8): 695-6; and Huang et. al., Nat Biotechnol. 2011 Aug 5; 29 (8): 699-700; of which everyone is incorporated here as a reference for their teaching in relation to TALENs. The term "TALEN agent" encompasses a TALEN and / or a polynucleotide that comprises a nucleotide sequence that encodes a TALEN.
[0074] [0074] The terminology "control element" or "control sequence" refers to a sequence of nucleotides involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation or degradation of the polynucleotide. Regulation can affect the frequency, speed or specificity of the process and can be of an intensifying or inhibiting nature. Control elements known in the art include, for example, transcriptional regulating sequences such as promoters and enhancers. A promoter is a region of DNA capable under certain conditions to bind to RNA polymerase and initiate transcription of a coding region usually located downstream (in the 3 'direction) of the promoter. Promoters can act ubiquitously, that is, active in many types of cells, for example, CAG or CMV promoters; or specific for tissues or cells, for example, the promoter can be tissue specific for expression in cardiomyocytes.
[0075] [0075] The terminology "operatively linked" or "operationally linked" refers to a juxtaposition of genetic elements, in which the elements are in a relationship that allows them to operate as expected. For example, a promoter is operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. There may be intermediate residues between the promoter and the coding region as long as this functional relationship is maintained.
[0076] [0076] The term "expression vector" encompasses a vector that comprises a polynucleotide region that encodes a polypeptide of interest and is used to carry out the expression of the protein in a desired target cell. An expression vector can also comprise control elements operably linked to the coding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operationally linked for expression is sometimes referred to as an "expression cassette", of which a large number is known and is available in the art or can be easily constructed from of components that are available in the art.
[0077] [0077] The term "heterologous" means derived from a genotypically distinct entity from that of the rest of the entity with which it is being compared. For example, a polynucleotide introduced using genetic engineering techniques into a plasmid or a vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence with which it is not naturally found linked is a heterologous promoter, so, for example, an rAAV that includes a heterologous nucleic acid sequence that encodes a heterologous gene product is an rAAV that includes a polynucleotide not normally included in a naturally occurring wild-type AAV and the encoded heterologous gene product is a gene product not normally encoded by a naturally occurring wild-type AAV.
[0078] [0078] The terminologies "genetic modification" and "genetic modification" (and their grammatical variants), are used here interchangeably to refer to a process in which a genetic element (for example, a polynucleotide) is introduced into a cell without mitosis or meiosis. The element can be heterologous to the cell, it can be an additional copy or an improved version of an element already present in the cell. Genetic alteration can be carried out, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, precipitation with calcium phosphate or contact with a polynucleotide-liposome complex. Genetic alteration can also be carried out, for example, by transduction or infection with a DNA or RNA virus or a viral vector. Usually, the genetic element is introduced into a chromosome or minicromosome in the cell; but any change that changes the phenotype and / or genotype of the cell and its progeny is included in this term.
[0079] [0079] Under the aspect of cell modification, the terminology "genetically modified" or "transformed" or "transfected" or "transduced" by exogenous DNA (for example, through a recombinant virus) refers to when such DNA was introduced in the cell. The presence of exogenous DNA results in permanent or transient genetic alteration. The transforming DNA may or may not be integrated (covalently linked) into the cell's genome. A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.
[0080] [0080] As used here, a cell is said to have been altered, transduced, genetically modified or transformed “stably” with a genetic sequence if the sequence is available to perform its function during prolonged cell culture in vitro and / or over an extended period of time in vivo. Generally, such a cell is altered “in an inherited manner” (genetically modified) by the fact that a genetic alteration is introduced that can also be inherited by the progeny of the altered cell.
[0081] [0081] The terms "polypeptide", "peptide" and "protein" are used here interchangeably to refer to polymers of amino acids of any length. The terms also cover a polymer of amino acids that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation or conjugation to a labeling component. Polypeptides such as antiangiogenic polypeptides, neuroprotective polypeptides and the like, when discussed in the context of providing a gene product to an individual mammal and its compositions, refer to the respective intact polypeptide or any genetically engineered fragment or derivative thereof, which maintains its function desired biochemistry of the intact protein. Similarly, references to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides and other such nucleic acids for use in providing a gene product to an individual mammal (which may be referred to as "transgenes" that will be supplied to a recipient cell ), include polynucleotides that encode the intact polypeptide or any genetically engineered fragment or derivative that has the desired biochemical function.
[0082] [0082] As used herein, a plasmid, nucleic acid, vector, virus, virion, host cell, protein or other "isolated" substance refers to a preparation of the substance devoid of at least part of the others components that may also be present when the substance or a similar substance occurs naturally or from which it is initially prepared. Thus, for example, an isolated substance can be prepared using a purification technique to enrich it starting from a mixture of origin. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured against a second potentially interfering substance present in the source mixture. Increasing enrichment of the modalities of this dissemination are increasingly isolated. A plasmid, nucleic acid, vector, virus, host cell or other isolated substance is purified in some embodiments, for example, from approximately 80% to approximately 90% pure, at least approximately 90% pure, at least approximately 95 % pure, at least approximately 98% pure or at least approximately 99% or more, pure.
[0083] [0083] As used here, the terms "treatment", "treat" and the like, refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or a symptom of it and / or it can be therapeutic in terms of a partial or complete cure for a disease and / or an adverse effect that can be attributed to the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease (and / or the symptoms caused by the disease) from occurring in an individual who may be predisposed to the disease or may be at risk of acquiring the disease, but has not yet been diagnosed as having it; (b) inhibiting the disease (and / or the symptoms caused by the disease), that is, interrupting its development; and (c ) alleviate the disease (and / or the symptoms caused by the disease), that is, cause the disease to regress (and / or symptoms caused by the disease), that is, improve the disease and / or one or more symptoms of the disease. , compositions and targeting methods can be targeted for the treatment of muscle disease. Non-limiting methods for assessing muscle disease and treatment include measuring therapeutic protein production (e.g. muscle biopsy followed by immunohistochemistry or serum sampling followed by ELISA or enzyme activity assays), measure heart failure symptoms (for example, the New York Heart Association Functional Classification or Minnesota Living With Heart Failure Questionnaire), cardiac functional capacity (for example, the 6-minute walk test or consumption maximum oxygen), biomarker analysis (eg, brain N-terminal pro-hormone natriuretic peptide), left ventricular function / remodeling (eg, left ventricular ejection fraction or left ventricular end systolic volume), strength muscle (for example, the Medical Research Council Scales Clinical Investigation of Duchenne Dystrophy, handshake dynamometry or maximum weight lifting), muscle function (for example, the Vignos Scale, Timed Function Tests, the Hammersmith Motor Ability Score, timed rise from floor, walk tests, Motor Function Measure Scale, North Star Ambulatory Assessment, 9 Hole Peg Test or Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders), symptoms of muscle diseases (for example, Neuromuscular Symptoms Score or Clinical Global Impressions), mitochondrial function (for example, 31P magnetic resonance spectroscopy), assessments based on quality of life questionnaires, results reported by the patient or daily activity.
[0084] [0084] The terms "individual", "host", "individual" and "patient"
[0085] [0085] In some embodiments, the individual is a human being who has previously been naturally exposed to AAV and as a result carries anti-AAV antibodies (ie, AAV neutralizing antibodies). In some embodiments, the individual is a human being who has previously received the administration of an AAV vector (and as a result can carry anti-AAV antibodies) and needs new vector administration for the treatment of a different condition or for the treatment additional condition. Based on positive results in clinical tests involving gene supply with AAV, for example, to the liver, muscle and retina - all tissues affected by neutralizing antibodies against this vehicle - there are many therapeutic applications / targets for diseases.
[0086] [0086] The term "efficient amount" as used here is an amount sufficient to achieve beneficial or desired clinical results. An efficient amount can be administered in one or more administrations. For the purposes of this disclosure, an efficient amount of a compound (for example, an infectious rAAV virion) is an amount that is sufficient to palliate, improve, stabilize, reverse, prevent, reduce or delay the prevention of (and / or symptoms associated with) a particular disease state (for example, a muscle disease). Consequently, an efficient amount of an infectious rAAV virion is an amount of the infectious rAAV virion that is capable of efficiently delivering a heterologous nucleic acid to a target cell (or target cells) in the individual. Efficient amounts can be determined preclinically, for example, through detection in the cell or tissue of the gene product
[0087] [0087] The term "muscle cell" or "muscle tissue" refers here to a cell or a group of cells derived from muscle of any type, including, without limitation, skeletal muscle, cardiac muscle, smooth muscle (e.g. digestive tract, bladder and blood vessels) and diaphragm muscle. These muscle cells can be differentiated or undifferentiated such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts. Since muscle tissue is readily accessible to the circulatory system, a protein produced and secreted by muscle cells and tissues in vivo will logically enter the bloodstream for systemic benefit, thereby providing prolonged therapeutic levels of protein secretion from the muscle.
[0088] [0088] The term “directed evolution” refers to a capsid engineering methodology, in vitro and / or in vivo, which mimics natural evolution through iterative rounds of genetic diversification and selection processes, thus accumulating beneficial mutations that progressively improves the function of a biological molecule. Targeted evolution often involves an in vivo method referred to as “biopanning” (bioselection) for selecting AAV variants from a library whose variants have a more efficient level of infectivity for a cell or tissue type of interest. DETAILED DESCRIPTION
[0089] [0089] Adeno-associated viruses (AAVs) are a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within an non-enveloped capsid. The viral genome of a naturally occurring AAV has 2 inverted terminal repeats (ITR) - which function as the origin of viral replication and the packaging signal - flanking 2 primary open reading frames (ORF): rep (which encodes proteins that act viral replication, transcription regulation, site specific integration and virion assembly) and cap. The cap ORF encodes 3 structural proteins that combine to form a 60-mer viral capsid. Many naturally occurring AAV variants and serotypes have been isolated and none have been associated with the disease in humans.
[0090] [0090] Recombinant versions of AAV can be used as delivery vectors, in which a marker or therapeutic gene of interest is inserted between the ITRs in place of rep and cap. Vectors have been shown to transduce both dividing and non-dividing cells in vitro and in vivo and can result in stable expression of transgenes for years in post-mitotic tissue. See, for example, Knipe DM, Howley PM. Fields ’Virology. Lippincott Williams & Wilkins, Philadelphia, PA, USA, 2007; Gao G-P, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci U S A 2002; 99: 11854–9; Atchison RW, Casto BC, Hammon WM. Adenovirus- Associated Defective Virus Particles. Science 1965; 149: 754–6; Hoggan MD, Blacklow NR, Rowe WP. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics. Proc Natl Acad Sci U S A 1966; 55: 1467–74; Blacklow NR, Hoggan MD, Rowe WP. Isolation of adenovirus-associated viruses from man. Proc Natl Acad Sci U S A 1967; 58: 1410–5; Bantel-Schaal U, zur Hausen H. Characterization of the DNA of a defective human parvovirus isolated from a genital site. Virology 1984; 134: 52–63; Mayor HD, Melnick JL. Small deoxyribonucleic acid-containing viruses
[0091] [0091] Recombinant AAV (referred to here simply as “AAV”) has produced promising results in an increasing number of clinical trials. However, there are impediments in gene supply that can limit the use of AAV, such as the immune response against capsid, low transduction of certain tissues, an inability to deliver targeted to specific cell types and a relatively low load capacity. In many situations, there is insufficient mechanistic knowledge to efficiently enable rational design with the ability to improve AAV. As an alternative, directed evolution has emerged as a strategy to create new variants of AAV that satisfy specific biomedical needs. Targeted evolution strategies use genetic diversification and selection processes to enable the accumulation of beneficial mutations that progressively improve the function of a biological molecule. In this process, the wild-type AAV cap genes are diversified through various approaches to create large genetic libraries that are packaged to generate libraries of viral particles and selective pressure is then applied to isolate new variants that can overcome barriers to gene supply. . Importantly, the mechanistic basis underlying a gene supply problem does not need to be known for targeted evolution to work, which can thus accelerate the development of improved vectors.
[0092] [0092] Typically, the variants disclosed here were generated through the use of an AAV library and / or libraries. Such a library or AAV libraries are generated by mutating the cap gene, the gene that encodes the structural proteins of the AAV capsid, by a series of directed evolution techniques known and readily available to the person skilled in the art in the field of genome engineering. viral. See, for example, Bartel et al. Am. Soc. Gene Cell Ther. 15th Annu. Meet. 20, S140 (2012); Bowles, D. et al. J. Virol. 77, 423-432 (2003); Gray et al. Mol. Ther. 18, 570-578 (2010); Grimm, D. et al. J. Virol. 82, 5887-5911; Koerber, J. T. et al. Mol. Ther. 16, 1703-1709 (2008); Li W. et al. Mol. Ther. 16, 1252-1260 (2008); Koerber, J. T. et al. Methods Mol. Biol. 434, 161-170 (2008); Koerber, J. T. et al. Hum. Gene Ther. 18, 367-378 (2007); and Koerber, J. T. et al. Mol. Ther. 17, 2088-2095 (2009). These techniques, without limitation, are as follows: i) Error-prone PCR (error-prone PCR) to introduce random point mutations into the open reading frame (ORF) of AAV cap at a rate that can be modified predetermined; ii) Viral recombination in vitro or in vivo or “DNA shuffling” (DNA shuffling) to generate random chimeras of AAV cap genes to produce a gene library with numerous AAV serotypes; iii) Random peptide insertions at defined capsid sites through the binding of degenerate oligonucleotides to the cap ORF; iv) Defined insertions of peptide coding sequences in random locations of the AAV cap ORF using transposon mutagenesis; v) Replacement of AAV capsid surface loops by libraries of peptide sequences designed by bioinformatics based on the conservation level of each amino acid position among the natural and variant AAV serotypes to generate loop-swap libraries; vi) Random amino acid substitution in the degeneration positions among the AAV serotypes to generate libraries of ancestral variants (Santiago-Ortiz et al., 2015); and a combination of these techniques.
[0093] [0093] DNA shuffling ("DNA shuffling") generates chimeras that combine their parenting properties in unique and often beneficial ways; however, some may be unable to be packaged, which in fact reduces the diversity of the library. The concentration of library diversity in specific region (s) of the capsid is achieved through peptide insertion techniques such as, without limitation, iii-iv) above. The diversity of the library is also concentrated in the specific region (s) of the capsid in techniques such as v) above and this concentration is directed to numerous hypervariable regions, which are on the exposed handles on the surface, of the AAV capsid. Although many of the techniques generate variant capsids with only a small area of the mutated capsid, these techniques can be paired with additional mutagenesis strategies to modify the capsid.
[0094] [0094] Once the AAV library or libraries are generated, the viruses are then packaged, so that each AAV particle is comprised of a mutant capsid surrounding a cap gene encoding that capsid, and are purified. The library variants are then subjected to the in vitro and / or in vivo selective pressure techniques known and readily available to the person skilled in the art in the field of AAV. See, for example, Maheshri, N. et al. Nature Biotech. 24, 198-204 (2006); Dalkara, D. et al. Sci. Transl. Med. 5, 189ra76 (2013); Lisowski, L. et al. Nature. 506, 382-286 (2013); Yang, L. et al. PNAS. 106, 3946-3951 (2009); Gao, G. et al. Mol. Ther. 13, 77-87 (2006); and Bell, P. et al. Hum. Gene. The R. 22, 985-997 (2011). For example, without limitation, AAV variants can be selected using i) affinity columns in which elution of different fractions provides variants with altered binding properties; ii) primary cells - isolated from tissue samples or immortal cell lines that mimic the behavior of cells in the human body - that produce AAV variants with greater efficiency and / or tissue specificity; iii) animal models - that mimic a clinical gene therapy environment - that produce AAV variants that have successfully infected the target tissue; iv) human xenograft models that produce AAV variants that have infected grafted human cells; and / or a combination of these selection techniques.
[0095] [0095] Once the viruses are selected, they can be recovered using known techniques such as, without limitation, adenovirus-mediated replication, PCR amplification, Next Generation sequencing and cloning and the like. The virus clones are then enriched through repeated rounds of selection techniques and the AAV DNA is isolated to retrieve the selected cap var genes of interest. These selected variants can be subjected to further modification or mutation and thus serve as a new starting point for additional selection steps to iteratively increase the performance of the AAV virus. However, in certain cases, successful capsids have been generated without further mutation.
[0096] [0096] The AAV variants disclosed here have been generated at least in part through the use of in vivo directed evolution methodology, such as the techniques described above, involving the use of primate cardiac and skeletal muscle checks after intravenous administration. Thus, the capsids of the AAV variants disclosed here comprise one or more modifications in the amino acid sequence that confer more efficient transduction of primate muscle cells than a corresponding parental AAV capsid protein. As used herein, a "corresponding parental AAV capsid protein" refers to an AAV capsid protein of the same wild type or variant AAV serotype as the target variant AAV capsid protein, but which does not understand the one or more modifications in the amino acid sequence of the target variant AAV capsid protein. In particular embodiments, an AAV comprising a variant AAV capsid protein that is described here has systemic tropism by the cardiac muscle and / or by various groups of skeletal muscles throughout the body after systemic or tissue-directed administration.
[0097] [0097] In some embodiments, the target variant AAV capsid protein comprises a heterologous peptide of approximately 5 amino acids to approximately 20 amino acids inserted by covalent bond in the GH loop or in loop IV of an AAV capsid protein, relative to a corresponding parental AAV capsid protein. By "GH loop" or loop IV, of the AAV capsid protein is meant the solvent accessible portion referred to in the art as the GH loop or loop IV, of the AAV capsid protein. For the GAV loop / loop IV of the AAV capsid, see, for example, van Vliet et al. (2006) Mol. Ther. 14: 809; Padron et al. (2005) J. Virol. 79: 5047; and Shen et al. (2007) Mol. Ther. 15: 1955. Thus, for example, the insertion site may be within approximately amino acids 411-650 of an AAV VP1 capsid protein. For example, the insertion site can be within AAV1 VP1 amino acids 571-612, AAV2 VP1 amino acids 570-611, within AAV3A VP1 amino acids 571-612, within AAV3B VP1 amino acids 571-612 , within amino acids 569-610 of VP1 of AAV4, within amino acids 560-601 of VP1 of AAV5, within amino acids 571 to 612 of VP1 of AAV6, within amino acids 572 to 613 of VP1 of AAV7, within amino acids 573 to 614 of VAV1 of AAV8, within amino acids 571 to 612 of VP1 of AAV9 or within amino acids 573 to 614 of VP1 of AAV10 or the corresponding amino acids of any variant thereof. Those skilled in the art would know, based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, where an insertion site "corresponding to the AAV2 amino acids" would be on a capsid protein of any given AAV serotype. See also Figure 6 for an alignment of SEQ ID NOS: 1-11 of the wild type AAV that provides amino acid locations between and along the wild type serotypes (naturally occurring AAV1, AAV2, AAV3A, AAV3B and AAV4-10 .
[0098] [0098] In certain embodiments, the insertion site is a unique insertion site between two adjacent amino acids located between amino acids 570-614 of VP1 of any wild type AAV serotype or AAV variant, for example, the insertion site is between two adjacent amino acids located at amino acids 570-610, amino acids 580-600, amino acids 570-575, amino acids 575-580, amino acids 580-585, amino acids 585-590, amino acids 590-600 or amino acids 600-614, from VP1 of any AAV serotype or variant. For example, the insertion site can be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589 or amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579 or amino acids 579 and 580. The insertion site can be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599 or amino acids 599 and
[0099] [0099] In some embodiments, a peptide insert disclosed here has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids or 20 amino acids. In another embodiment, a peptide insert disclosed here comprises 1 to 4 spacer amino acids at the amino terminus (N-terminus) and / or the carboxyl terminus (C terminus) of any of the peptide inserts disclosed herein. Examples of spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T) and proline (P). In certain embodiments, a peptide insert comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, a peptide insert comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus.
[00100] [00100] The peptide inserts disclosed here have not been described and / or previously inserted into an AAV capid. Without wishing to be limited to theory, the presence of any of the disclosed peptide inserts can act to reduce the affinity of the variant's capsid for heparin sulfate, which could alter the extracellular or intracellular steps within the viral transduction pathway. In addition, the peptide insertion motifs disclosed here can confer greater transduction of muscle cells (e.g., cardiomyocytes) by adding a cell surface receptor binding domain.
[00101] [00101] In some preferred embodiments, the insertion peptide comprises an amino acid sequence of any of the formulas below.
[00102] [00102] In some respects, an insertion peptide can be a peptide 7 to 10 amino acids in length, of Formula 1a: Y1Y2X1X2X3X4X5X6X7Y3 Where each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro X1 is selected from Ala, Asn, Thr, Gly, Ser, Ala, Gln and Asp X2 is selected from Lys, Asn, Thr, Ser, Ala and Gln X3 is selected from Ile, Thr, Lys, Leu , Val, Asn, Asp and Arg X4 is selected from Gln, Thr, Ile, Lys, Val, Ser and Tyr
[00103] [00103] In certain embodiments, the Formula 1a insertion peptide comprises an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), GNLTKGN ( SEQ ID NO: 16), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19), DNTVTRS (SEQ ID NO: 20), NKISAKD (SEQ ID NO: 21) ), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 25) and ASDSTKA (SEQ ID NO: 26). In other embodiments, the Formula 1a insertion peptide does not comprise an amino acid sequence selected from NKTTNKD (SEQ ID NO: 14), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24) and NQDYTKT (SEQ ID NO: 24) NO: 22).
[00104] [00104] In other respects, an insertion peptide can be a peptide of 7 to 10 amino acids in length, of Formula 1b: Y1Y2X1X2X3X4X5X6X7Y3 Where each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro X1 is selected from Thr and Asn X2 is selected from Asn and Lys X3 is selected from Lys, Ile and Thr X4 is selected from Ile, Gln and Thr X5 is selected from Gly, Arg and Asn
[00105] [00105] In certain embodiments, the Formula 1b insertion peptide comprises an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and TNKIGVT (SEQ ID NO: 15). In other embodiments, the Formula 1a insertion peptide does not comprise the amino acid sequence NKTTNKD (SEQ ID NO: 14).
[00106] [00106] In other respects, an insertion peptide can be a peptide of 7 to 10 amino acids in length, of Formula 1c Y1Y2X1X2X3X4X5X6X7Y3 In which each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser , Thr, Pro X1 is selected from Thr and Asn X2 is selected from Asn and Lys X3 is selected from Lys and Ile X4 is selected from Ile and Gln X5 is selected from Gly and Arg X6 is selected from Val and Thr X7 is selected from Thr and Asp
[00107] [00107] In certain embodiments, the Formula 1c insertion peptide comprises an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13) and TNKIGVT (SEQ ID NO: 15).
[00108] [00108] In other respects, an insertion peptide can be a peptide of 7 to 10 amino acids in length, of Formula 1d:
[00109] [00109] In certain embodiments, the Formula 1d insertion peptide comprises the amino acid sequence TNKIGVT (SEQ ID NO: 15).
[00110] [00110] In other embodiments, the insertion peptide comprises an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and TNKIGVT (SEQ ID NO: 15). In related embodiments, the insertion peptide comprises an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13) and TNKIGVT (SEQ ID NO: 15).
[00111] [00111] In some embodiments, the insertion peptide comprises an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), GNLTKGN (SEQ ID NO: : 16), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19), DNTVTRS (SEQ ID NO: 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 25) and ASDSTKA (SEQ ID NO: 26).
[00112] [00112] In other preferred embodiments, the insertion peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of an amino acid sequence selected from NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), GNLTKGN (SEQ ID NO: 16), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19) , DNTVTRS (SEQ ID NO: 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 24) NO: 25) and ASDSTKA (SEQ ID NO: 26). In some of these modalities, the insertion peptide is selected from the group consisting of: LANKIQRTDA (SEQ ID NO: 27), LANKTTNKDA (SEQ ID NO: 28), LATNKIGVTA (SEQ ID NO: 29), LAGNLTKGNA (SEQ ID NO: 30), LANTVKLSTA (SEQ ID NO: 31), LASNTVKAIA (SEQ ID NO: 32), LAASNITKAA (SEQ ID NO: 33), LADNTVTRSA (SEQ ID NO: 34), LANKISAKDA (SEQ ID NO: 35), LANQDYTKTA ( SEQ ID NO: 36), LATNKIGVTS (SEQ ID NO: 37), LATNKIGVTA (SEQ ID NO: 38), LAQADTTKNA (SEQ ID NO: 39), LATNRTSPDA (SEQ ID NO: 40), LASNTTQKTA (SEQ ID NO: 41) ) and LAASDSTKAA (SEQ ID NO: 42).
[00113] [00113] In some embodiments, the AAV capsid protein target variant does not include any other changes in the amino acid sequence other than a peptide insertion of approximately 5 amino acids to approximately 20 amino acids in the GH loop or in the IV loop. For example, in some embodiments, the target variant AAV capsid protein comprises a peptide insert that comprises an amino acid sequence selected from the group consisting of NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), TNKIGVT (SEQ ID NO: 15), GNLTKGN (SEQ ID NO: 16), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19), DNTVTRS (SEQ ID NO: 19) : 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 25), ASDSTKA (SEQ ID NO: 26), LANKIQRTDA (SEQ ID NO: 27), LANKTTNKDA (SEQ ID NO: 28), LATNKIGVTA (SEQ ID NO: 29), LAGNLTKGNA (SEQ ID NO: 30), LANTVKLSTA (SEQ ID NO: 31),
[00114] [00114] In other embodiments, the AAV capsid protein target variant, in addition to the fact that it comprises a peptide insert, for example, which is disclosed here or which is known in the art, in the GH loop, comprises from approximately 1 to approximately 100 amino acid substitutions or deletions, for example, 1 to approximately 5, from approximately 2 to approximately 4, from approximately 2 to approximately 5, from approximately 5 to approximately 10, from approximately 10 to approximately 15, from approximately 15 to approximately 20 , from approximately 20 to approximately 25, from approximately 25-50, from approximately 50-100 amino acid substitutions or deletions compared to the parental AAV capsid protein. Thus, in some embodiments, a target variant capsid protein comprises an amino acid sequence that has a sequence identity of 85% or more, 90% or more, 95% or more or 98% or more, for example, or 99 % identity with that of the corresponding parent AAV capsid, for example, a wild type capsid protein which is shown in SEQ ID NOs: 1-12.
[00115] [00115] In an additional embodiment, one or more amino acid substitutions occur in the amino acid residue (s) 35, 109, 195, 213, 222, 229, 312, 319, 330, 333, 347 , 363, 427, 447, 449, 453, 490, 527, 551, 581, 585, 588, 593, 606, 649, 651, 694, 698, 708 and / or 735 of the AAV2 VP1 capsid protein which are numbered before insertion of the corresponding peptide or amino acid residue (s) from another AAV capsid protein. In some of these modalities, one or more amino acid substitutions are selected from the group consisting of A35P, S109T, P195L, D213N, G222S, V229I, N312K,
[00116] [00116] In a preferred embodiment, a variant AAV capsid protein is provided which comprises a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and TNKIGVT (SEQ ID NO: 15) and b) one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another parental serotype of AAV (that is, other than AAV2), in which the substituted amino acid (s) does not occur naturally in the corresponding positions: A35P, S109T, P195L, D213N, G222S, V229I , N312K, A319T, T330A, A333S, E347K, P363L, A427D, V447F, N449D, N449K, G453R, A490T, K527Q, N551S, A581T, Y585S, R588M, A593E, W606C, IQ6, V6, E6 and a combination of them. In some modalities, one or more amino acid substitutions are selected from the group consisting of: V708I, V708I + A593E, V708I + S109T, V708I + T330A, A35P, V708I + R588M, V708I + W606C, V708I + W694C, I698V, N3 + N449D + N551S + I698V + L735Q, N312K + N449D + N551S + I698V + V708I + L735Q, V708I + N449K and V708I + G222S. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid, between amino acids 587 and 588 of the AAV2 capsid, between amino acids 588 and 589 of the AAV3A capsid, AAV3B, AAV9 or AAV10, among the amino acids 589 and 590 of the AAV7 capsid, between amino acids 590 to 591 of the AAV1 capsid, AAV6 or AAV8, between amino acids 584 and 585 of the AAV4 capsid or between amino acids 575 and 576 of the AAV5 capsid.
[00117] [00117] In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or that comprises, essentially consists of or consists of the amino acid sequence LANKIQRTDA (SEQ ID NO: 27) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and further comprises a substitution of amino acid V708I at residue 708 in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) and optionally further comprises an A593E and / or S109T and / or T330A and / or R588M substitution with respect to AAV2 or the corresponding substitutions in another parental AAV serotype, in which the substituted amino acid (s) does not occur ( m) naturally in the corresponding position. In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or that comprises, essentially consists of or consists of the amino acid sequence LANKIQRTDA (SEQ ID NO: 27) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and further comprises a substitution of amino acid A35P at residue 35 with respect to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another parental AAV serotype. The variant AAV capsid can have at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or at least approximately 99% or more, of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2 or the corresponding parental AAV capsid. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that is at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98%
[00118] [00118] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or that comprises, essentially consists of or consists of the amino acid sequence LANKIQRTDA (SEQ ID NO: 27) between amino acids 587 and 588 of the AAV2 capsid protein or the corresponding position in the capsid protein of another AAV serotype and comprises an amino acid substitution N312K compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2 ) or the corresponding substitution in another parental AAV serotype and optionally further comprises (i) amino acid substitutions N449D, N551S, I698V and L735Q or (ii) N449D, N551S, I698V, L735Q and V708I compared to the amino acid sequence of the AAV2 or the corresponding substitutions in another parental AAV serotype. The variant AAV capsid can have at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or more, amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLKFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYF PSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTD TPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSE YSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEK TSVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANKIQRTDARQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL
[00119] [00119] In another embodiment, a variant AAV capsid protein is provided which comprises a) a peptide insert located between amino acids 588 and 589 of VP1 of AAV3A, AAV3B, AAV9 or
[00120] [00120] In yet another embodiment, the variant capsid protein comprises a) a peptide insert which comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or which comprises, essentially consists of or consists of the amino acid sequence LANKIQRTDA (SEQ ID NO : 27) between amino acids 587 and 588 of the AAV2 capsid and b) a substitution of valine for amino acid isoleucine at amino acid 708 compared to the amino acid sequence of AAV2 and is otherwise identical to the amino acid sequence of SEQ ID NO: 2 .
[00121] [00121] In yet another embodiment, the variant capsid protein comprises a) a peptide insert that comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or that comprises, essentially consists of or consists of the amino acid sequence LANKIQRTDA (SEQ ID NO : 27) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANKIQRTDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00122] [00122] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence TNKIGVT (SEQ ID NO: 15) or which comprises, essentially consists of or consists of the amino acid sequence LATNKIGVTA (SEQ ID NO: 29) or LATNKIGVTS (SEQ ID NO: 37) between amino acids 587 and 588 of the AAV2 capsid or the corresponding position in the capsid protein of another AAV serotype and comprises an amino acid substitution V708I compared to the amino acid sequence of AAV2 or the corresponding substitution in another parental AAV serotype and optionally further comprising an N449K and / or G222S substitution in relation to AAV2 or the corresponding substitution in the capsid protein of another parental AVV serotype, in which the substituted amino acids do not naturally occur in the corresponding position . In another preferred embodiment, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence TNKIGVT (SEQ ID NO: 15) or that comprises, essentially consists of or consists of the amino acid sequence LATNKIGVTA (SEQ ID NO: 29) or LATNKIGVTS (SEQ ID NO: 37) between amino acids 587 and 588 of the AAV2 capsid or the corresponding position in the capsid protein of another AAV serotype and comprises amino acid substitutions N312K, N449D, N551S, I698V and L735Q and optionally V708I compared to amino acid sequence of AAV2 or the corresponding substitution (s) in another AAV parental serotype, where the substituted amino acid (s) does not occur naturally in the corresponding position. The variant AAV capsid can have at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or more, amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLATNKIGVTARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00123] In another embodiment, a variant AAV capsid protein is provided which comprises a) a peptide insert located between amino acids 588 and 589 of VP1 of AAV3A, AAV3B, AAV9 or AAV10, between amino acids 589 and 590 of AAV7, between amino acids 590 to 591 of AAV1, AAV6 or AAV8, between amino acids 584 and 585 of AAV4 or between amino acids 575 and 576 of AAV5, the peptide insert comprising an amino acid sequence selected from TNKIGVT (SEQ ID NO: 15), LATNKIGVTA (SEQ ID NO: 29) and LATNKIGVTS (SEQ ID NO: 37) and b) a replacement of valine for isoleucine at amino acid 709 of AAV3A or AAV3B, a replacement of alanine for isoleucine at position 709 of AAV1 or AAV6, a replacement of asparagine for isoleucine at amino acid 707 of AAV4 or amino acid 709 for AAV9 or a substitution of threonine for isoleucine at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a substitution of glutamine for isoleucine at amino acid 697 of AAV5. In preferred embodiments, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence TNKIGVT (SEQ ID NO: 15) or that comprises, essentially consists of or consists of the amino acid sequence LATNKIGVTA (SEQ ID NO: 29) or LATNKIGVTS ( SEQ ID NO: 37) between amino acids 587 and 588 of the AAV2 capsid and comprises a substitution of valine for amino acid isoleucine at amino acid 708 (V708I) compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions and preferably has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or more of the amino acid sequence with the total length of the amino acid sequence shown in SEQ ID NO: 2.
[00124] [00124] In yet another embodiment, the variant capsid protein comprises a) a peptide insert that comprises the amino acid sequence TNKIGVT (SEQ ID NO: 15) or that comprises, essentially consists of or consists of the amino acid sequence LATNKIGVTA (SEQ ID NO : 29) or LATNKIGVTS (SEQ ID NO: 37) between amino acids 587 and 588 of the AAV2 capsid and b) a substitution of valine for amino acid isoleucine at amino acid 708 compared to the amino acid sequence of AAV2 and is otherwise identical to amino acid sequence of SEQ ID NO: 2.
[00125] [00125] In yet another embodiment, the variant capsid protein comprises a) a peptide insert that comprises the amino acid sequence TNKIGVT (SEQ ID NO: 15) or that comprises, essentially consists of or consists of the amino acid sequence LATNKIGVTA (SEQ ID NO : 29) or LATNKIGVTS (SEQ ID NO: 37) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids:
[00126] [00126] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDD SRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYL KYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPG KKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQ PPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDR VITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHC HFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTV QVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQY LYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTS ADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIF GKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLATNKI GVTARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSP LMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWE
[00127] [00127] In another preferred embodiment, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence NKTTNKD (SEQ ID NO: 14) or LANKTTNKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid and further comprises an amino acid substitution V708I at residue 708 with respect to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another parental serotype of AAV and optionally further comprising an amino acid substitution S109T and / or W694C and / or W606C compared to the amino acid sequence of AAV2 or the corresponding substitution in another parental serotype of AAV, in which the substituted amino acid (s) does not occur naturally in the corresponding position. In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence NKTTNKD (SEQ ID NO: 14) or that comprises, essentially consists of or consists of the amino acid sequence LANKTTNKDA
[00128] [00128] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or which comprises, essentially consists of or consists of the amino acid sequence LANKTTNKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid protein or the corresponding position in the capsid protein of another AAV serotype and comprises an N312K amino acid substitution compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2 ) or the corresponding substitution in another parental AAV serotype and optionally further comprises amino acid substitutions N449D, N551S, I698V and L735Q and optionally V708I compared to the amino acid sequence of the AAV2 capsid or the corresponding substitutions in another parental AAV serotype. The variant AAV capsid can have at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or more, amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO: 2. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the following amino acid sequence: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLKFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYF PSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTD TPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSE YSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEK TSVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANKTTNKDARQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGL
[00129] [00129] In another embodiment, a variant AAV capsid protein is provided which comprises a) a peptide insert located between amino acids 588 and 589 of AAV3A VP1, AAV3B, AAV9 or AAV10, between amino acids 589 and 590 of AAV7, between amino acids 590 to 591 of AAV1, AAV6 or AAV8, between amino acids 584 and 585 of AAV4 or between amino acids 575 and 576 of AAV5, the peptide insert comprising an amino acid sequence selected from NKTTNKD (SEQ ID NO: 14) and LANKTTNKDA (SEQ ID NO: 28) and b) a substitution of valine for isoleucine at amino acid 709 of AAV3A or AAV3B, a substitution of alanine for isoleucine at position 709 of AAV1 or AAV6, a substitution of asparagine for isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9 or a substitution of threonine for isoleucine in amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a substitution of glutamine for isoleucine in amino acid 697 of AAV5 and is optionally otherwise identical to any uer one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or which comprises, essentially consists of or consists of the amino acid sequence LANKTTNKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid and b) a substitution of valine for amino acid isoleucine at amino acid 708 compared to the amino acid sequence of AAV2, where the variant capsid protein comprises from 2 to 5, from 5 to 10 or from 10 to 15 amino acid substitutions.
[00130] [00130] In yet another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or which comprises, essentially consists of or consists of the amino acid sequence LANKTTNKDA (SEQ ID NO : 28) between amino acids 587 and 588 of the AAV2 capsid and b) a substitution of valine for amino acid isoleucine at amino acid 708 compared to the amino acid sequence of AAV2 and is otherwise identical to the amino acid sequence of SEQ ID NO: 2 .
[00131] [00131] In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence NKTTNKD (SEQ ID NO: 14) or that comprises, essentially consists of or consists of the amino acid sequence LANKTTNKDA (SEQ ID NO: 28) between the amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANKTTNKDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00132] [00132] In other embodiments, a variant AAV capsid protein is provided which comprises a) a peptide insert in the GH loop of the capsid protein, wherein the peptide insert comprises a selected amino acid sequence from GNLTKGN (SEQ ID NO: 16 ), NTVKLST (SEQ ID NO: 17), SNTVKAI (SEQ ID NO: 18), ASNITKA (SEQ ID NO: 19), DNTVTRS (SEQ ID NO: 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 25) and ASDSTKA (SEQ ID NO: 26) and b) one or more of the amino acid substitutions then compared to the AAV2 amino acid sequence (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype (ie, other than AAV2), where the substituted amino acid (s) does not occur naturally in the corresponding positions: A35P, S109T, P195L, D213N, G222S, V229I, N312K, A319T, T330A, A333S, E347K, P363L, A427D, V447F, N449D, N449K, G453R, A490T, K5 A581T, Y585S, R588M, A593E, W606C, K649E, R651H, W694C, I698V, V708I, L735Q and a combination thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: V708I, S109T, R651H, A319T, P195L, P363L, I698V, D213N, G453R and a combination of them. In some preferred embodiments, the one or more amino acid substitutions include at least one amino acid substitution V708I and / or P363L or the corresponding substitution in another parental AAV serotype. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or the corresponding position in the capsid protein of another AAV serotype.
[00133] [00133] In some embodiments, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or which comprises, essentially consists of or consists of the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and further comprises one or more of the following amino acid substitutions in relation to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or corresponding replacement in another parental AAV serotype, in which the substituted amino acid (s) does not occur naturally in the corresponding positions: V708I, V708I + S109T, R651H, A319T + P195L, P363L, P363L + V708I . In some embodiments, the variant AAV capsid comprises (i) a peptide insert comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or which comprises, essentially consists of or consists of the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a V708I substitution in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another capsid of AAV and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In other embodiments, the variant AAV capsid comprises (i) a peptide insert comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or which comprises, essentially consists of or consists of the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a P363L substitution with respect to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another capsid of AAV and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In other embodiments, the variant AAV capsid comprises (i) a peptide insert comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or which comprises, essentially consists of or consists of the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) an R651H substitution with respect to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another capsid of AAV and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence GNLTKGN (SEQ ID NO: 16) or that comprises, essentially consists of or consists of the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAGNLTKGNARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00134] [00134] In some embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence NTVKLST (SEQ ID NO: 17) or that comprises, essentially consists of or consists of the amino acid sequence LANTVKLSTA (SEQ ID NO : 31) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) an amino acid substitution of V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) corresponding in another parental AAV serotype, in which the substituted amino acid (s) does not occur naturally in the corresponding positions and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or it is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence NTVKLST (SEQ ID NO: 17) or that comprises, essentially consists of or consists of the amino acid sequence LANTVKLSTA (SEQ ID NO: 31) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANTVKLSTARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00135] [00135] In some embodiments, the variant AAV capsid comprises (i) a peptide insert comprising the SNTVKAI amino acid sequence (SEQ ID NO: 18) or which comprises, essentially consists of or consists of the LASNTVKAIA amino acid sequence (SEQ ID NO : 32) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or corresponding substitution in another parental AAV serotype and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the sequence of the capsid protein corresponding parental AAV. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence SNTVKAI (SEQ ID NO: 18) or that comprises, essentially consists of or consists of the amino acid sequence LASNTVKAIA (SEQ ID NO: 32) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLASNTVKAIARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00136] [00136] In some embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence ASNITKA (SEQ ID NO: 19) or that comprises, essentially consists of or consists of the amino acid sequence LAASNITKAA (SEQ ID NO : 33) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or corresponding substitution in another parental AAV serotype and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the sequence of the capsid protein corresponding parental AAV. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence ASNITKA (SEQ ID NO: 19) or that comprises, essentially consists of or consists of the amino acid sequence LAASNITKAA (SEQ ID NO: 33) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLAASNITKAARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00137] [00137] In some embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence DNTVTRS (SEQ ID NO: 20) or that comprises, essentially consists of or consists of the amino acid sequence LADNTVTRSA (SEQ ID NO : 34) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or corresponding substitution in another parental AAV serotype and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the sequence of the capsid protein corresponding parental AAV. In other embodiments, the variant AAV capsid comprises (i) a peptide insert comprising the DNTVTRS amino acid sequence (SEQ ID NO: 20) or which comprises, essentially consists of or consists of the LADNTVTRSA amino acid sequence (SEQ ID NO: 34) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) an amino acid substitution I698V with respect to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another parental serotype of AAV, in which the substituted amino acid does not occur naturally in the corresponding position and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO : 2 or the corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence DNTVTRS (SEQ ID NO: 20) or that comprises, essentially consists of or consists of the amino acid sequence LADNTVTRSA (SEQ ID NO: 34) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLADNTVTRSARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00138] [00138] In some embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence NKISAKD (SEQ ID NO: 21) or that comprises, essentially consists of or consists of the amino acid sequence LANKISAKDA (SEQ ID NO : 35) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or corresponding substitution in another parental AAV serotype and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the sequence of the capsid protein corresponding parental AAV. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence NKISAKD (SEQ ID NO: 21) or that comprises, essentially, or consists of the amino acid sequence LANKISAKDA (SEQ ID NO: 35) between amino acids 587 and 588 of the AAV2 capsid and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLANKISAKDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK HPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKR
[00139] [00139] In some embodiments, the variant AAV capsid comprises (i) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO: 22) or which comprises, essentially consists of or consists of the amino acid sequence LANQDYTKTA (SEQ ID NO : 36) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or corresponding substitution in another parental AAV serotype and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the sequence of the capsid protein corresponding parental AAV. In other embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence NQDYTKT (SEQ ID NO: 22) or which comprises, essentially consists of or consists of the amino acid sequence LANQDYTKTA (SEQ ID NO: 36) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) an amino acid substitution I698V with respect to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another parental serotype of AAV (ie, other than AAV2), in which the substituted amino acid does not occur naturally in the corresponding position and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence NQDYTKT (SEQ ID NO: 22) or that comprises, essentially consists of or consists of the amino acid sequence LANQDYTKTA (SEQ ID NO: 36)
[00140] [00140] In some embodiments, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO: 23) or which comprises, essentially consists of or consists of the amino acid sequence LAQADTTKNA (SEQ ID NO: 39) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and further comprises one or more of the following amino acid substitutions in relation to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or corresponding substitutions in another parental AAV serotype, in which the substituted amino acid (s) does not occur naturally in the corresponding positions: V708I, D213N, P363L, G453R.
[00141] [00141] In some embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence TNRTSPD (SEQ ID NO: 24) or that comprises, essentially consists of or consists of the amino acid sequence LATNRTSPDA (SEQ ID NO : 40) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or corresponding substitution in another parental AAV serotype and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the sequence of the capsid protein corresponding parental AAV. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLATNRTSPDARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00142] [00142] In some embodiments, the variant AAV capsid comprises a peptide insert that comprises the amino acid sequence SNTTQKT (SEQ ID NO: 25) or that comprises, essentially consists of or consists of the amino acid sequence LASNTTQKTA (SEQ ID NO: 41) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADA EFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEH SPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGL GTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRT WALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDW QRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEY QLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFP SQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEY SWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT NVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLASNTTQKTARQAA TADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLK
[00143] [00143] In some embodiments, the variant AAV capsid comprises (i) a peptide insert that comprises the amino acid sequence ASDSTKA (SEQ ID NO: 26) or that comprises, essentially consists of or consists of the amino acid sequence LAASDSTKAA (SEQ ID NO : 42) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid and (ii) a substitution of amino acid V708I in relation to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) the substitution corresponding in another parental AAV serotype, in which the substituted amino acid (s) does not occur naturally in the corresponding positions and comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or it is otherwise identical to the amino acid sequence of SEQ ID NO: 2 or the corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insert that comprises the amino acid sequence ASDSTKA (SEQ ID NO: 26) or that comprises,
[00144] [00144] In several respects, a variant AAV capsid protein is provided that comprises one or more amino acid substitutions with respect to a corresponding parental AAV capsid protein, wherein the variant capsid protein, when present in a virion of AAV, confers greater infectivity of a muscle cell (for example, a skeletal or cardiac muscle cell) compared to the infectivity of a muscle cell by an AAV virion that comprises the corresponding parental AAV capsid protein.
[00145] [00145] In some embodiments a variant AAV capsid protein comprises an amino acid substitution at amino acid 363 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (ie is, other than AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence that is at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or at least approximately 99% or more, identity of amino acid sequences with the total length of the amino acid sequence shown in SEQ ID NO 2 and comprises an amino acid substitution at amino acid 363 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2). In some preferred embodiments, a variant AAV capsid protein comprises an amino acid substitution P363L compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), the AAV3A capid (SEQ ID NO: 3) or the capsid AAV3B (SEQ ID NO: 4); or an amino acid substitution P364L compared to the amino acid sequence of the AAV1 capsid (SEQ ID NO: 1) or the AAV6 capsid (SEQ ID NO: 7); or a P354L amino acid substitution compared to the amino acid sequence of the AAV4 capsid (SEQ ID NO: 5) or the AAV5 capsid (SEQ ID NO: 6); or an amino acid substitution P365L compared to the amino acid sequence of the AAV7 capsid (SEQ ID NO: 8) or the AAV9 capsid (SEQ ID NO: 10); or an amino acid substitution P366L compared to the amino acid sequence of the AAV8 capsid (SEQ ID NO: 9) or the AAV10 capsid (SEQ ID NO: 11). In some preferred embodiments, the variant capsid protein comprises a substitution
[00146] [00146] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 593 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (ie is, other than AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 593 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) and is at least approximately 85%, at least approximately 90%, at least at least approximately 95%, at least approximately 98% or at least approximately 99% or more, of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO 2 or is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the variant capsid protein comprises a substitution of glycine amino acid for glutamate at amino acid 594 compared to the amino acid sequence of AAV1, AAV3A, AAV6 or AAV9 or at amino acid 583 of AAV5 or in amino acid 596 from AAV8 or AAV10 or an arginine amino acid substitution by glutamate at amino acid 594 from AAV3B or an aspartate amino acid substitution p or glutamate at amino acid 592 of AAV4 or a substitution of glutamine amino acid for glutamate at position 595 of AAV7. In other embodiments, the variant capsid protein comprises an A593E amino acid substitution compared to the AAV2 amino acid sequence and does not comprise one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence: I19V, V369A, K26R, N215D, G355S, V46A and S196P.
[00147] [00147] In other embodiments, a variant AAV capsid protein comprises an amino acid substitution at amino acid 708 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding position in another AAV parental serotype (ie is, other than AAV2) in which the substituted amino acid does not occur naturally in the corresponding position. Preferably, the rAAV virion does not comprise a substitution of proline for serine at amino acid 250 compared to AAV2 or a corresponding amino acid in another parental AAV serotype. In some embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 708 compared to the AAV2 capsid amino acid sequence (SEQ ID NO: 2) and is at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or at least approximately 99% or more, of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO 2 or is otherwise identical to SEQ ID NO: 2 . In preferred embodiments, the variant capsid protein comprises a substitution of valine for isoleucine (V708I) at amino acid 708 compared to the amino acid sequence of the AAV2 capsid and is at least approximately 85%, at least approximately 90%, at least approximately 95 %, at least approximately 98% or at least approximately 99% or more, of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO 2 or is otherwise identical to the amino acid sequence of SEQ ID NO : 2, where the variant capsid protein does not comprise a P250S amino acid substitution. In some embodiments, the variant capsid protein comprises a substitution of valine for isoleucine at amino acid 709 of AAV3A or AAV3B, a substitution of alanine for isoleucine at position 709 of AAV1 or AAV6, a substitution of asparagine for isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9 or a substitution of threonine for isoleucine in amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10 or a substitution of glutamine for isoleucine in amino acid 697 of AAV5. In related embodiments, the variant capsid protein comprises an amino acid substitution V708I compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions and wherein the variant capsid protein does not comprise a P250S amino acid substitution. In other embodiments, the variant capsid protein comprises an amino acid substitution V708I and also comprises an amino acid substitution A333S and / or S721L compared to the amino acid sequence of AAV2. In other related embodiments, the variant capsid comprises an amino acid substitution V708I and also comprises an amino acid substitution A333S and / or S721L compared to the amino acid sequence of AAV2 and is at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% or at least approximately 99% or more, of amino acid sequence identity with the total length of the amino acid sequence shown in SEQ ID NO 2 or is otherwise identical to the amino acid sequence of SEQ ID NO: 2.
[00148] [00148] In other embodiments, a variant AAV capsid protein comprises an amino acid sequence of at least 85%, at least 90%, at least 95% or at least 98% identical to a selected wild-type AAV capsid sequence from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12 and also comprises i) one or more amino acid substitutions selected from the group consisting of A35P, D213N , A319T + P195L, P363L,
[00149] [00149] In some embodiments, a variant AAV capsid protein is an ancestral capsid protein that comprises one or more peptide inserts and / or amino acid substitutions that are described here. An ancestral capsid protein means an evolutionary ancestor of a capsid protein that is found in nature today, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11 , AAV12, AAV13, which is generated in silico through random amino acid substitution in degeneration positions between AAV capsid proteins that are found in nature today.
[00150] [00150] In other embodiments, a variant AAV capsid protein is a chimera comprising amino acids 130-725 of the AAV5 capsid (SEQ ID NO: 6) or an amino acid sequence of at least 90%, at least 95% or at least 98% identical to the same.
[00151] [00151] In some respects, a variant AAV capsid protein is a chimera comprising (i) amino acids 1-129 of AAV6 (SEQ ID NO: 7) or an amino acid sequence of at least 90%, at least 95% or at least 98% identical to the same and (ii) amino acids 130- 725 of AAV5 (SEQ ID NO: 6) or an amino acid sequence of at least 90%, at least 95% or at least 98% identical to the same and which further comprises V229I, A490T and A581T and optionally amino acid substitutions V447F or Y585S in relation to the AAV5 sequence (SEQ ID NO: 6). In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLP GYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHAD AEFQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDD HFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAG GGGPLGDNNQGADGVGNASGDWHCDSTWMGDRIVTKSTRTWVLPSYNN HQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNY WGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVG NGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLR TGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQ FNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFTTTNRMEL EGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNM LITSESETQPVNRVAYNVGGQMATNNQSSTTAPTTGTYNLQEIVPGSVWME RDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITS
[00152] [00152] In other respects, a variant AAV capsid protein is a chimera comprising (i) amino acids 1-61 of AAV2 (SEQ ID NO: 2) or an amino acid sequence of at least 90%, at least 95% or at least 98% identical to the same, (ii) amino acids 62- 129 of AAV6 (SEQ ID NO: 7) or an amino acid sequence of at least 90%, at least 95% or at least 98% identical to the same and (iii) amino acids 130-725 of AAV5 (SEQ ID NO: 6) and further comprising amino acid substitutions V229I, A490T and A581T with respect to the AAV5 sequence (SEQ ID NO: 6). In some embodiments, the variant AAV capsid has an amino acid sequence that has at least approximately 85%, at least approximately 90%, at least approximately 95%, at least approximately 98% sequence identity or is 100% identical to the sequence following amino acids: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPG YKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAE FQERLQEDTSFGGNLGRAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHF PKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGG GPLGDNNQGADGVGNASGDWHCDSTWMGDRIVTKSTRTWVLPSYNNHQ YREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYW GFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGN GTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRT GNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQF NKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFTTTNRMELE GASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLI TSESETQPVNRVAYNVGGQMATNNQSSTTAPTTGTYNLQEIVPGSVWMER DVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSF
[00153] [00153] The AAV variants disclosed here were generated through the use of in vivo directed evolution that involves the use of primate cardiac and skeletal muscle selections after intravenous administration.
[00154] [00154] Diseases that can be treated using a variant rAAV vector or virion and / or method disclosed herein include, but are not limited to, monogenic diseases, complex diseases and traumatic injuries. Examples of monogenic diseases include, but are not limited to, muscular dystrophies such as Duchenne, Becker, congenital (including, but not limited to Bethlem myopathy, Ullrich muscular dystrophy, Fukuyama muscular dystrophy, Muscular dystrophy with merosin deficiency with integrin deficiency and Walker-Warburgh syndrome), distal (including, but not limited to Gowers-Laing, Miyoshi and Nonaka), Emery-Dreifuss, facio-scapulohumeral, with waist, myotonic and muscular dystrophies; congenital myotonia and congenital paramytonia; myotubular myopathy; centronuclear myopathy; myofibrillar myopathy, related to desmin; anemia; Andersen-Tawil syndrome; Nemaline myopathy; Brody's disease; lysosomal storage disorders such as alpha-mannosidosis, aspartylglucosaminuria, beta-mannosidosis, cystinosis, Farber's disease, fucosidosis, Gaucher disease, galactosialidosis, gangliosidosis (including, but not limited to AB variant, activator deficiency, beta-galactosidase deficiency , Fabry's disease, Sandhoff's disease and Schindler's disease), glycogen storage disorders (including, but not limited to Andersen's disease, Cori's disease, Danon's disease, Forbes disease, defective glucose-6-phosphate, disease Hers deficiency, lactate dehydrogenase A deficiency, Pompe disease, Tarui disease and von Gierke disease), infantile free sialic acid storage disease, lysosomal acid lipase deficiency, Krabbe disease, Metachromatic Leukodystrophy, mucopolysaccharidoses (including, but not limited to hyaluronidase deficiency, Hunter syndrome, Hurler syndrome, Hurler-Scheie syndrome, Maroteaux-Lamy syndrome, Morquio syndrome, s Sanfilippo syndrome, Scheie syndrome and Sly syndrome), mucolipidosis (including, but not limited to Sialidosis, I cell disease, mucolipidine 1 deficiency and Psuedy-Hurler polydistrophy), multiple sulfase deficiency, Niemann-Pick disease , neuronal keroid lipofuscinosis (including, but not limited to, Batten-Spielmeyer-Vogt disease, congenital Cathepsin D deficiency, German / Serbian Late Childhood, Jansky-Bielschowsky's disease, Kufs disease, late childhood, late childhood variant, Northern Epilepsy , Santavuori-Haltia and Turkish Late Childhood Disease), pycnodysostosis, Salla's disease, Saposina B deficiency, Tay-Sach's disease and Wolman's disease; metabolic disorders such as adenosine monophosphate deaminase deficiency, alkaptonuria, carnitine deficiency, carnitine palmityl transferase deficiency, Hartnup disorder, homocystinuria, maple syrup urine disease, myophosphorylase deficiency, phosphofuctokinase deficiency, phosphoglycerate deficiency phosphoglycerate mutase, phosphorylase deficiency and Tangier disease; Friedreich's ataxia; ataxia telangiectasia; ataxia with vitamin E deficiency; periodic paralysis, such as Gamstorp's disease and periodic hypokalemic paralysis; mitochondrial diseases such as Barth's syndrome, Kearns-Sayre syndrome, mitochondrial myopathy, mitochondrial encephalopathy lactic acidosis and stroke-like episodes, myoclonic epilepsy with red broken fibers and Pearson's syndrome; familial hypertrophic cardiomyopathies; dilated cardiomyopathies; familial congenital heart disease, such as familial aortic valve disease and left ventricular noncompaction with congenital heart defects; familial arrhythmias, such as Anderson's periodic cardiodysrhythmic paralysis, atrial septal defects with AV conduction defects, Brugada syndrome, cardiac conductance defect, catecholaminergic polymorphic ventricular tachycardia and congenital heart blocks; familial vascular disorders, such as arterial tortuosity syndrome, autosomal dominant cerebral arteriopathy with sobcortical infarctions and leukoencephalopathy, dominant recessive cerebral arteriopathy with sobcortical infarcts and leukoencephalopathy, family-type aortic aneurysm, Marfan's syndrome, contractual Ehlersdenitis syndrome, arachnid contract Beals, Loeys-Dietz syndrome and elastic pseudoxanthoma; arrhythmogenic right ventricular cardiomyopathy; familial arrhythmogenic right ventricular dysplasia; Naxos disease; no left ventricular compression; familial atrial fibrillation; familial ventricular tachycardia; familial Wolff-Parkinson-White syndrome; long QT syndromes; short QT syndromes; syndromes of the sick sinus; lipoprotein diseases, such as abetalipoproteinemia and lipoprotein lipase deficiency; alpha-1 antitrypsin deficiency; deficiency of coagulation factor VIII (hemophilia A) or deficiency of coagulation factor IX (hemophilia B); thalassemia; progressive ossifying fibrodysplasia; laminopathies; Huntington's disease; congenital myasthenic syndromes; Progeria Hutchinson-Gilford syndrome; Noonan syndrome; disproportionate myopathy of the congenital fiber type; congenital fibrosis of the extraocular muscles; minicore myopathy; undulating muscle disease; Schwartz-Jampel syndrome; tubular aggregate myopathy; and myopathy with zebra-patterned bodies. Examples of complex diseases include, but are not limited to, heart / cardiovascular disease (for example, congestive heart failure, myocardial infarction, angina, coronary artery disease, ischemic heart disease, cardiomyopathy); cancer; diabetes; and infection. Examples of traumatic injuries include, but are not limited to, viral infection of the muscle, muscle laceration; and muscle contusion. In preferred embodiments, a variant rAAV vector or virion and / or method disclosed herein is used to treat Fabry disease, Friedreich ataxia, Duchenne muscular dystrophy, Becker muscular dystrophy, Pompe disease, myophosphorylase deficiency, muscular dystrophy facio-scapulohumeral, muscular dystrophy of waists or myotonic dystrophy.
[00155] [00155] In another embodiment, a variant capsid disclosed herein comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product such as, without limitation, an interfering RNA, a long non-coding RNA, a short non-coding RNA, an antisense RNA, an aptamer, a polypeptide, a secreted antibody, a single chain antibody, a VHH domain, a soluble receptor, an affibody, a knottin, a DARPin, a centurin, a chaperone, a nuclease specific to the site that causes knock-down specific to the gene function site or a nuclease specific to the modified site that causes specific activation to the transcription gene.
[00156] [00156] A virion variant of rAAV disclosed herein comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes a gene product. In some embodiments, the gene product is an antisense RNA, a microRNA (miRNA), a short hairpin RNA (shRNA) or a small interfering RNA (siRNA) or a precursor or mimic. In some embodiments, the gene product is a long non-coding RNA. In some embodiments, the gene product is a short non-coding RNA. In some embodiments, the gene product is an antisense RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a secreted antibody. In some embodiments, the gene product is a single chain antibody. In some embodiments, the gene product is a VHH domain. In some embodiments, the gene product is a soluble receptor. In some embodiments, the gene product is an affibody. In some embodiments, the gene product is a knottin. In some embodiments, the gene product is a DARPin. In some embodiments, the gene product is a centurine. In some embodiments, the gene product is a chaperone. In some embodiments, the gene product is a site-specific nuclease that causes site-specific knock-down of gene function.
[00157] [00157] Uses of the gene product include, but are not limited to, increasing the level of a factor in a cell, increasing the level of a factor in a neighboring or distant cell by secreting a factor, reducing the level of a factor in a cell or reducing the level of a factor in a neighboring or distant cell by secreting a factor. The gene product can be designed to supplement the level of a defective or absent gene product, to reduce the level of a defective or absent gene product, to introduce a new support gene product, to supplement the level of a support gene product, to reduce the level of a blocking gene product or either reducing the level of a blocking gene product or introducing or supplementing the level of a support gene product.
[00158] [00158] The gene products provided by the target AAV variants can be used to alter the level of gene products or the activity of gene products directly or indirectly linked to muscle diseases and trauma. Skeletal, cardiac or smooth muscle transduced with AAV target variants can also be used as a biological factory to produce and secrete therapeutic proteins for the treatment of in trans diseases in distant organs. Genes whose gene products are directly or indirectly linked to genetic diseases include, for example, genes that encode any of the following gene products: dystrophin including mini- and micro-dystrophins (DMD; for example, GenBank Accession Number NP_003997.1; SEQ ID NO: 64); titin (TTN); titin cap (TCAP) -sarcoglycan -sarcoglycan -sarcoglycan (SGCG) or - sarcoglycan (SGCD); alpha-1-antitrypsin (A1-AT); myosin 6 heavy chain (MYH6); myosin 7 heavy chain (MYH7); myosin 11 heavy chain (MYH11); myosin 2 light chain (ML2); myosin 3 light chain (ML3); myosin 2 light chain kinase 2 (MYLK2); myosin C binding protein (MYBPC3); desmin (DES); dinamine 2 (DNM2); laminin laminate A / C (LMNA); blade B (LMNB); blade B receiver (LBR); dysferlin (DYSF); emerin (EMD); insulin; blood clotting factors, including, but not limited to, factor VIII and factor IX; erythropoietin (EPO); lipoprotein lipase (LPL); Ca2 ++ - Sarcoplasmic reticulum ATPase (SERCA2A), calcium binding protein A1 S100 (S100A1); myotubularin (MTM); DM1 protein kinase (DMPK; for example, GenBank Accession Number NG_009784.1; SEQ ID NO: 65); glycogen phosphorylase L (PYGL); muscle-associated glycogen phosphorylase (PYGM; for example, GenBank Accession Number NP_005600.1; SEQ ID NO: 66); glycogen synthase 1 (GYS1); glycogen synthase 2 (GYS2); -galactosidase A (GLA; for example, GenBank Accession Number NP_000160.1; SEQ ID NO: 67); -N- acetylgalactosaminidase (NAGA); -acid glycosidase (GAA; for example, GenBank Accession Number NP_000143.2; SEQ ID NO: 68), sphingomyelinase phosphodiesterase 1 (SMPD1); lysosomal acid lipase (LIPA); type I collagen chain (COL1A1); type I collagen chain (COL1A2); type 1 collagen chain 1 (COL3A1); type 1 collagen chain 1 (COL5A1); type V collagen chain (COL5A2); type 1 collagen chain 1 (COL6A1); type VI collagen chain (COL6A2); type VI collagen chain (COL6A3); pro-collagen-lysine 2-oxoglutarate 5-dioxigenase (PLOD1); lysosomal acid lipase (LIPA); frataxin (FXN; for example, GenBank Accession Number NP_000135.2; SEQ ID NO: 69); myostatin (MSTN); -N-acetyl hexosaminidase A (HEXA); β-N-acetylhexosaminidase B (HEXB); - glucocerebrosidase (GBA); adenosine monophosphate deaminase 1 (AMPD1); -globin (HBB); iduronidase (IDUA); 2-sulfate iduronate (IDS); troponin 1 (TNNI3); troponin T2 (TNNT2); troponin C (TNNC1); tropomyosin 1 (TPM1); tropomyosin 3 (TPM3); N-acetyl- glycosaminidase (NAGLU); N-sulfoglucosamine sulfohydrolase (SGSH); heparan-glucosaminide N-acetyltransferase (HGSNAT); integrin (IGTA7); glucosamine (N-acetyl) -6-sulfatase (GNS) integrin; galactosamine (N-acetyl) -6-sulfatase (GALNS); β-galactosidase (GLB1); β-glucuronidase (GUSB); hyaluronoglycosaminidase 1 (HYAL1); acid ceramidase (ASAH1); galactosylcermidase (GALC); cathepsin A (CTSA); cathepsin D (CTSA); cathepsin K (CTSK); ganglioside GM2 activator (GM2A); arylsulfatase A (ARSA); arylsulfatase B (ARSB); enzyme that generates formylglycine (SUMF1); neuraminidase 1 (NEU1); N-acetylglucosamine-1-phosphate transfer-acetylglucosamine-1-phosphate transferase β (GNPTB); N-acetylglucosamine-1-phosphate transferase γ (GNPTG); mucolipine-1 (MCOLN1); intracellular carrier NPC 1 (NPC1); intracellular carrier NPC 2 (NPC2); ceroid lipofuscinosis 5 (CLN5); ceroid lipofuscinosis 6 (CLN6); ceroid lipofuscinosis 8 (CLN8); palmitoyl protein thioesterase 1 (PPT1); tripeptidyl peptidase 1 (TPP1); battenin (CLN3); family of heat shock protein DNAJ 40 member C5 (DNAJC5); contains the master facilitator 8 superfamily domain (MFSD8); mano and 2B member 1 (MAN2B1); mannosidase β (MANBA); aspartylglucosaminidase -L-fucosidase (FUCA1); cystinosine, lysosomal cysteine transporter (CTNS); sialin; 2 member 10 solute carrier family (SLC2A10); 17 member 5 solute carrier family (SLC17A5); family of 6 member solute carrier 19 (SLC6A19); 22 member 5 solute carrier family (SLC22A5); 37 member 4 solute carrier family (SLC37A4); membrane protein associated with lysosome 2 (LAMP2); subunit voltage-dependent sodium channel 4 (SCN4A); voltage-dependent sodium channel β subunit 4 (SCN4B); subunit voltage-dependent sodium channel 5 (SCN5A); subunit voltage-dependent sodium channel 4 (SCN4A); voltage-dependent calcium channel subunit voltage-dependent calcium channel subunit phosphoglycerate kinase 1 (PGK1); phosphoglycerate mutase 2 (PGAM2); amylglycosidase, 4- glucanotransferase (AGL); voltage-dependent potassium channel member of the ISK-related subfamily 1 (KCNE1); voltage-dependent potassium channel member of the ISK 2-related subfamily (KCNE2); voltage-dependent potassium channel subfamily J member 2 (KCNJ2); voltage-dependent potassium channel subfamily J member 5 (KCNJ5); voltage-dependent potassium channel subfamily H member 2 (KCNH2); voltage-dependent potassium channel member of the subfamily similar to KQT 1 (KCNQ1); cyclic nucleotide dependent potassium channel activated with hyperpolarization 4 (HCN4); voltage-dependent chloride channel 1 (CLCN1); carnitine palmitoyltransferase 1A (CPT1A); ryanodine 1 receptor (RYR1); ryanodine 2 receptor (RYR2); bridge integrator 1 (BIN1); LARGE xylosyl- and glucuronyltransferase 1 (LARGE1); anchoring protein 7 (DOK7); fucutin (FKTN); fukutin-related protein (FKRP); selenoprotein N (SELENON); protein O-mannosyltransferase 1 (POMT1); protein O-mannosyltransferase 2 (POMT2); mannose N-acetylglucosaminyltransferase linked to protein O 1 (POMGNT1); mannose N-acetylglucosaminyltransferase linked to protein O 2 (POMGNT2); protein-O-mannose kinase (POMK); domain containing isoprenoid synthase (ISPD); plectin (PLEC); cholinergic receptor nicotinic epsilon subunit (CHRNE); choline O-acetyltransferase (CHAT); choline kinase β (CHKB); tail subunit similar to collagen of asymmetric acetylcholinesterase (COLQ); synapse receptor-associated protein (RAPSN); four and a half domains of LIM 1 (FHL1); β-1,4-glucuronyltransferase 1 (B4GAT1); β-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2); dystroglycan 1 (DAG1); transmembrane protein 5 (TMEM5); transmembrane protein 43 (TMEM43); SECIS 2 binding protein (SECISBP2); glucosamine (UDP-N-acetyl) -2-epimerase / N-acetylmannosamine kinase (GNE); anoctamine 5 (ANO5); containing hinge domain for structural maintenance of flexible chromosomes 1 (SMCHD1); lactate dehydrogenase A (LDHA); lactate dehydrogenase B (LHDB); calpain 3 (CAPN3); caveolin 3 (CAV3); containing tripartite motif 32 (TRIM32); CCHC-type zinc finger nucleic acid binding protein (CNBP); nebulin (NEB); skeletal muscle actin (ACTA1); cardiac muscle actin (ACTC1); nuclear protein actinin that binds to poly (A) 1 (PABPN1); protein containing LEM 3 domain (LEMD3); zinc metalloproteinase STE24 (ZMPSTE24); microsomal triglyceride transfer protein (MTTP); (CHRNA1); cholinergic receptor nicotinic subunit cholinergic receptor nicotinic subunit
[00159] [00159] In preferred embodiments, the gene products provided by the target AAV variants are selected from alpha galactosidase A (GLA), Frataxin (FXN), Dystrophin (DMD), Alpha acid glycosidase (GAA) and Glycogen phosphorylase, muscle (PYGM ). In some preferred embodiments, a target AAV variant comprises a nucleic acid segment comprising a nucleotide sequence that encodes (i) a GLA polypeptide that comprises or consists of the amino acid sequence shown as SEQ ID NO: 67, ( ii) an FXN polypeptide comprising or consisting of the amino acid sequence presented as SEQ ID NO: 69, (iii) a DMD polypeptide comprising or consisting of a functional fragment (for example, mini or micro dystrophin, preferably comprising a intact actin binding domain, at least 4 of the 24 spectrin-like repeats and the dystroglycan binding domain) of the amino acid sequence shown as SEQ ID NO: 64, (iv) a GAA polypeptide comprising or consisting of the sequence amino acid shown as SEQ ID NO: 68, (v) a PYGM polypeptide that comprises or consists of the amino acid sequence shown as SEQ ID NO: 66, (vi) or (v) a sequence amino acid content at least 80%, at least 85%, at least 90% or at least 95% identical to any of SEQ ID NOs: 64 and 66-69.
[00160] [00160] In another preferred embodiment, a target AAV variant comprises a transgene that encodes an interfering RNA, for example, an antisense RNA, a miRNA, a shRNA or a siRNA, which decreases DMPK expression. In some ways, the interfering RNA decreases the expression of DMPK encoded by a nucleic acid that has a nucleotide sequence that is presented as SEQ ID NO: 65 or a sequence of at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 65.
[00161] [00161] The genes whose gene products induce or promote apoptosis are referred to here as "pro-apoptotic genes" and the products of these genes (mRNA; protein) are referred to as "pro-apoptotic gene products". Pro-apoptotic targets include, for example, products of the Bax gene; Bid gene products; Bak gene products; Bad gene products; Bcl-2; Bcl-X1. Anti-apoptotic gene products include the X-linked apoptosis inhibitor.
[00162] [00162] The genes whose gene products induce or promote angiogenesis are referred to here as "pro-angiogenesis genes" and the products of these genes (mRNA; protein) are referred to as "pro-angiogenesis gene products". Pro-angiogenesis targets include, for example, vascular endothelial growth factor (VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factor receptor 1 (VEGFR1); vascular endothelial growth factor receptor 2 (VEGFR2); Tyrosine Kinase Related to Fms 1 (Flt1); placental growth factor (PGF); Platelet-derived growth factor (PDGF); angiopoietins; sonic hedgehog. The genes whose gene products inhibit angiogenesis are referred to here as "antiangiogenic genes" and the products of these genes (mRNA; protein) are referred to as "antiangiogenic gene products". Antiangiogenic gene products include endostatin; tumstatin; angiostatin; factor derived from pigment epithelium (PEDF) and fusion proteins or antibodies that are specific for pro-angiogenesis targets and / or their receptors, for example, the specific antibody to VEGF AvastinTM etc.
[00163] [00163] The genes whose gene products function as immunological modulators, for example, complement factors, toll-like receptors, are called "immunomodulatory genes". Examples of immunomodulatory genes include cytokines, chemokines and the fusion proteins or antibodies that are specific to them and / or their receptors, for example, the anti-IL-6 fusion protein RilonaceptTM, the antibody specific for Factor H Complement lampamizumab etc. The genes whose gene products function as protective factors for muscles, for example, insulin growth factor 1 (IGF-1); transforming growth factor β (TGFβ); fibroblast growth factor (FGF).
[00164] [00164] In some cases, a gene product of interest is a site-specific endonuclease that causes gene function-specific knock-down, for example, in which the endonuclease knocks out an allele associated with a muscle disease. For example, when a dominant allele encodes a defective copy of a gene that, when in the wild type, is a structural muscle protein and / or causes normal muscle function, a site-specific endonuclease can target the defective allele and knock out the allele defective.
[00165] [00165] In addition to knocking out a defective allele, a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, for example, a target rAAV virion can be used to deliver a site-specific endonuclease that knocks out a defective allele and can be used to provide a functional copy of the defective allele, resulting in the repair of the defective allele, thereby causing production of a functional muscle protein (for example, functional A / C lamina, functional fibrillin, functional type VI collagen, etc.). In some embodiments, an rAAV virion disclosed herein comprises a heterologous nucleotide sequence that encodes a site-specific endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional muscle protein. Functional muscle proteins include, for example, lamina A / C, fibrillin 1, COL6A1, COL6A2, COL6A3 and the like.
[00166] [00166] Site-specific endonucleases that are suitable for use include, for example, meganucleases; zinc finger nucleases (ZFNs); effector nucleases similar to the transcription activator (TALENs); and Short palindromic repetitions interspersed regularly in clusters / associated with CRISPR (Cas), in which such site-specific endonucleases do not occur naturally and are modified to target a specific gene. Such site-specific nucleases can be engineered to cut specific locations within a genome and the attachment of non-homologous ends can then repair the break while inserting or deleting several nucleotides. Such site-specific endonucleases (also referred to as "INDELs") then throw the protein out of the picture and efficiently knock out the gene. See, for example, U.S. Patent Publication No. 2011/0301073.
[00167] [00167] In some embodiments of the variant rAAV vector disclosed here, a nucleotide sequence encoding a gene product of interest is operably linked to a constitutive promoter. Suitable constitutive promoters include, for example, cytomegalovirus (CMV) promoter (Stinski et al. (1985) Journal of Virology 55 (2): 431-441), initial CMV enhancer / chicken β-actin promoter (CBA) / rabbit β-globin (CAG) intron (Miyazaki et al. (1989) Gene 79 (2): 269-277, CBSB (Jacobson et al. (2006) Molecular Therapy 13 (6): 1074-1084), promoter 1α of human elongation factor (EF1α) (Kim et al. (1990) Gene 91 (2): 217-223), promoter of human phosphoglycerate kinase (PGK) (Singer-Sam et al. (1984) Gene 32 ( 3): 409-417, promoter of the mitochondrial heavy filament (Loderio et al. (2012) PNAS 109 (17): 6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105) In other embodiments, a nucleotide sequence that encodes a gene product of interest is operably linked to an inducible promoter In some cases, a nucleotide sequence that encodes a gene product of interest is operably linked to a element regulator specific to tissue specific to cell type. For example, in some cases, a nucleotide sequence that encodes a gene product of interest is operationally linked to a muscle-specific regulatory element (e.g., a specific cardiac promoter or a specific promoter to skeletal muscle), for example, a regulatory element that confers selective expression of the gene operatively linked in a muscle cell.
[00168] [00168] For the purposes of the invention, the disclosure presented here provides an isolated nucleic acid that comprises a nucleotide sequence that encodes a variant AAV capsid protein that is described above. An isolated nucleic acid can be an AAV vector, for example, a recombinant AAV vector.
[00169] [00169] The disclosure presented here also provides a method of treating a muscle disease, the method comprising administering to an individual in need of an efficient amount of a variant rAAV virion comprising a transgene of interest which is described above and released here. A person of ordinary skill in the art would be able to easily determine an efficient amount of the target rAAV virion and that the disease was treated by testing for a change in one or more functional or anatomical parameters, for example, muscle biopsy followed by immunohistochemistry , serum sampling followed by ELISA or enzyme activity assays, walking test, maximal oxygen consumption, analysis with biomarkers, left ventricular ejection fraction, left ventricular end systolic volume, handshake dynamometry, maximum weight lifting , Timed Function Tests, the Hammersmith Motor Ability Score, timed rise from floor or 9 Hole Peg Test.
[00170] [00170] Non-limiting methods for assessing muscle function and its changes include assessment of walking test, maximum oxygen consumption, analysis with biomarkers, left ventricular ejection fraction, left ventricular end systolic volume, Vignos Scale, Timed Function, Hammersmith Motor Ability Score, timed rise from floor, Motor Function Measure Scale, North Star Ambulatory Assessment, 9 Hole Peg Test or Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders.
[00171] [00171] In some embodiments, an efficient amount of the target rAAV virion results in a reduction in the rate of loss of muscle function, anatomical muscle integrity or muscle mass, for example, a reduction of 2 times, 3 times, 4 times or 5 times or more in the rate of loss and consequently the progression of the disease, for example, a reduction of 10 times or more in the rate of loss and consequently the progression of the disease. In some modalities, the efficient amount of the target rAAV virion results in a gain in muscle function, gain in muscle strength, gain in muscle mass and / or an anatomical muscle integrity or biomarkers, for example, a 2-fold improvement, 3 times, 4 times or 5 times or more in muscle function, muscle strength, muscle mass and / or an improvement in anatomical muscle integrity or biomarkers, for example, an improvement of 10 times or more in muscle function, muscle strength , muscle mass and / or an improvement in anatomical muscle integrity or biomarkers. As will be easily considered by the ordinary person skilled in the art, the dose required to achieve the desired treatment effect will typically be in the range of 1 x 108 to approximately 1 x 1016 recombinant virions, typically referred to by the ordinary person in the art as 1 x 108 to approximately 1 x 1016 "vector genomes" and preferably will be in the range of approximately 1 x 1011 to approximately 1 x 1015 recombinant virions.
[00172] [00172] A target rAAV virion can be delivered to the skeletal muscle through intravascular administration (intravascular or intraarterial), intraperitoneal administration, limb perfusion and / or direct intramuscular injection or through another way or route of administration that will result in the delivery of the rAAV virion to the skeletal muscle. The rAAV virion can be delivered to the heart muscle through intravascular (intravenous or intraarterial) administration, direct cardiac injection (in the left atrium, right atrium, right ventricle and / or septum), anterograde or retrograde infusion into the coronary artery (through the left anterior descending or left circumflex coronary arteries), recirculation, intrapericardial injection, transendocardial injection or through any convenient mode or route of administration that will result in the delivery of the rAAV virion to the heart muscle. In a preferred embodiment, a target rAAV virion is supplied to the skeletal and / or cardiac muscle through systemic administration. When administered via intravenous injection, the target rAAV virion is able to move along the circulatory system and transduce muscle cells more efficiently, compared to the capacity of a wild-type AAV virion or an AAV virion comprising the corresponding parental AAV capsid protein.
[00173] [00173] A variant capsid protein disclosed here is isolated, for example, purified. In some embodiments, a variant capsid protein disclosed here is included in an AAV vector or a recombinant AAV virion (rAAV). In other embodiments, these AAV variant vectors and / or AAV variant virions are used in an in vivo or ex vivo method of treating a muscle disease in primate heart or skeletal muscle.
[00174] [00174] The disclosure presented here further provides host cells such as, without limitation, isolated (genetically modified) host cells that comprise an objective nucleic acid.
[00175] [00175] The disclosure presented here further provides a pharmaceutical composition comprising: a) the variant rAAV virion, which is described previously and disclosed here; and b) a pharmaceutically acceptable carrier, diluent, excipient or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient or buffer is suitable for use in a human or non-human patient. Such excipients, carriers, diluents and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail here. Pharmaceutically acceptable excipients have been described widely in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., Eds., 7th ed., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. In some aspects of the present invention, the present invention provides a pharmaceutical composition comprising approximately 1 x 108 to approximately 1 x 1016 recombinant viruses or 1 x 108 to approximately 1 x 1016 vector genomes, wherein each said recombinant virus comprises a genome which encodes one or more gene products.
[00176] [00176] Some modalities of the invention are exemplified in items 1 to 54 below:
[00177] [00177] The following examples are published in order to provide ordinary experts in the art with full disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor is it intended to represent that the following experiments are all or just the experiments performed. Efforts have been made to ensure accuracy in relation to the numbers used (for example, quantities, temperature, etc.), but some experimental errors and standard deviations must be considered. Unless otherwise stated, the parts are parts by weight, the molecular weight is average molecular weight, the temperature is in degrees Centigrade and the pressure is or is close to atmospheric.
[00178] [00178] General methods in molecular and cellular biochemistry can be found in standard textbooks such as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. Eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. Eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), whose disclosures are hereby incorporated by reference. The reagents, cloning vectors and kits for genetic manipulation referred to in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech.
[00179] [00179] Example 1
[00180] [00180] Intravenous Injection and Tissue Collection. A single male cinomolgo monkey (macaca fascicularis) aged 3-10 years old and weighing at least 3 kg received doses through intravenous injection via the saphenous vein for each round of selection. The animal was anesthetized and 1-5 mL of the library (in the first round, the library consists of variants generated using all the mutagenesis techniques described in Figure 1A; in each subsequent round, the isolated variants of the previous round), in some cases pre -incubated with human IVIG for 30 minutes at 37 ° C, were administered.
[00181] [00181] Euthanasia was performed by a team of trained veterinarians using 100 mg / kg intravenous sodium pentobarbital injection on day 14 ± 3 or 21 ± 3, depending on the selection. The cardiac and / or skeletal muscle tissue of the quadriceps was removed and the DNA was isolated from the tissue. In some cases, the cardiac tissue has been divided into several regions: the atrium, the ventricular septum, the left papillary muscle, the right papillary muscle, the left ventricle and the right ventricle.
[00182] [00182] Directed Evolution. The directed evolution process is shown in Figure 1A-1E. Briefly, a viral capsid library is created that comprises 20+ proprietary combinations of the DNA mutation technique and cap genes (Figure 1A). The viruses are then packaged (Figure 1B) - so that each particle is made up of a mutant capsid surrounding the cap gene that encodes this capsid - and purified. The capsid library is placed under selective pressure in vivo. The tissue or cell material of interest is collected to isolate the AAV variants that have successfully infected the target and successful viruses are collected. Successful clones are enriched through repeated selection (Stage I - Figure 1D). The selected cap genes then undergo property diversification and are enriched through additional selection steps to iteratively increase viral performance (Stage 2 - Figure 1D). The variants identified during Stages 1 and 2 of Vector Selection demonstrate the ability to transduce primate muscle cells (Figure 1E).
[00183] [00183] Successful Recovery of AAV Capsid Genomes. The capsids recovered from each round of selection were used to package the injected library to start the subsequent round of selection. The recovery of the capsid genes from the tissue represents the successful internalization of library vectors in the tissue of interest. The recovery of viral genomes from cardiac and skeletal muscle tissue from a representative round of selection is shown in Figure 2. The bands inside the boxes represent the successful recovery of viral genomes.
[00184] [00184] Analysis of the Sequencing. During rounds 3-4 of selections including the selective pressure of intravenous delivery to cardiac tissue or skeletal muscle tissue and rounds 1-2 of a selection including the selective pressure of intravenous delivery in the presence of neutralizing antibodies to cardiac tissue, sequencing of individual clones within the library was performed to determine the frequency of variants within the population. The variants were evaluated for the presence of motifs within the sequencing data. The variants were grouped into motifs based on the presence of a unifying variation (for example, a specific point mutation or a peptide insertion sequence at a consistent location within the capsid) that occurred in a number of sequences. The reasons that represent at least 5% of the population sequenced in two or more rounds of the selection or at least 10% of the population sequenced in one or more rounds of the selection are represented in Figure 3A (Round 4 sequencing analysis for selective supply pressure intravenous to cardiac tissue), Figure 3B (Round 2 sequencing analysis for selective pressure of intravenous delivery in the presence of neutralizing antibodies to cardiac tissue) and Figure 3C (provides Round 3 sequencing analysis for selective pressure of intravenous delivery skeletal muscle tissue).
[00185] [00185] Several representative clones that have been identified as conferring greater infectivity of cardiac and / or skeletal muscle cells are listed in Table 1 below (each clone contains the
[00186] [00186] The following chimeras have also been identified as capsids that confer greater infectivity of cardiac muscle cells and greater resistance to neutralization by anti-AAV antibodies:
[00187] [00187] A chimera with (i) amino acids 1-129 of AAV6 and (ii) amino acids 130-725 of AAV5 and which has the following amino acid substitutions for AAV5: V229I + A490T + A581T (the sequence of SEQ ID NO: 62).
[00188] [00188] A chimera with (i) amino acids 1-61 of AAV2, (ii) amino acids 62-129 of AAV6 and (iii) amino acids 130-725 of AAV5 and which has the following amino acid substitutions for AAV5: V229I + A490T + A581T (the sequence of SEQ ID NO: 63).
[00189] [00189] A chimera with (i) amino acids 1-129 of AAV6 and (ii) amino acids 130-725 of AAV5 and which has the following amino acid substitutions for AAV5: V229I + A490T + A581T + Y585S
[00190] [00190] A chimera with (i) amino acids 1-129 of AAV6 and (ii) amino acids 130-725 of AAV5 and which has the following amino acid substitutions in relation to AAV5: V229I + A447F + A490T + A581T
[00191] [00191] The variant AAV virions disclosed here may incorporate parameters, characteristics, modifications, advantages and variations of reasonable rational design that are readily apparent to those skilled in the art in the field of AAV viral vector engineering.
[00192] [00192] Example 2
[00193] [00193] Cellular tropism of recombinant AAV virions comprising the new AAV variants LANKIQRTDA + V708I (SEQ ID NO: 43), LANKTTNKDA + V708I (SEQ ID NO: 48) and LATNKIGVTA + V708I (SEQ ID NO: 46) for cardiomyocytes it was evaluated in vitro using cardiomyocytes generated from human embryonic stem cells (ESC).
[00194] [00194] Recombinant AAV virions comprising an AAV1 capsid, an AAV2 capsid, an AAV9 capsid, the new variant capsid LANKIQRTDA + V708I, the new variant capsid LANKTTNKDA + V708I or the new variant capsid LATNKIGVTA + V708I and genome comprising a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (AAV1.CAG.EGFP, AAV2.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA + V708I.CAG.EGFP, LANKTTNKDA + V708I .CAG.EGFP and LATNKIGVTA + V708I.CAG.GFP, respectively) were produced using standardized methods. Cardiomyocytes were generated from a human embryonic stem cell line, ESI-017, through the modulation of Wnt signaling using small molecules. After 14 days of cardiac mesoderm induction, cultures were further enriched in relation to cardiomyocytes by glucose deprivation. After approximately 24 days of differentiation, most cells expressed the cardiac myocyte marker, cardiac troponin T (cTnT) and a specific ventricular marker, MLC-2V. The generated cardiomyocytes were evaluated for expression of the space-binding protein (gap) Conexina 43, fluctuation of membrane potential, calcium handling and the contractile function to ensure that the generated cardiomyocytes reached a state of maturity before characterizing the vector .
[00195] [00195] Regarding AAV1, AAV2 and AAV9, the variants LANKIQRTDA + V708I, LANKTTNKDA + V708I and LATNKIGVTA + V708I produced a significantly higher transduction and transgene expression efficiency in human cardiomyocyte cultures six days after infection which are determined by immunofluorescence (Figure 6A), flow cytometry (Figure 6B) and Western blot analysis (Figures 6C-D). In addition, regarding AAV1, AAV2 and AAV9, LANKIQRTDA + V708I, LANKTTNKDA + V708I and LATNKIGVTA + V708I provided the fastest onset of gene expression in human cardiomyocyte cultures, which is determined by immunofluorescence (Figure 6E). Regarding AAV8 and AAV9, which exhibit tropism by cardiac and skeletal muscle cells, the number of infectious units per viral genome administered was several orders of magnitude higher for LANKIQRTDA + V708I and LANKTTNKDA + V708I (Figure 10A). This study illustrates the superior ability of AAV capsid variants that comprise NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and TNKIGVT (SEQ ID NO: 15) to supply genes to cardiac cells.
[00196] [00196] Example 3
[00197] [00197] The cell tropism of recombinant AAV virions comprising the new AAV6 / AAV5 chimera variant of AAV by cardiomyocytes was evaluated in vitro using cardiomyocytes generated from human embryonic stem cells (ESC).
[00198] [00198] Recombinant AAV virions comprising an AAV1 capsid, an AAV8 capsid, an AAV9 capsid or the new variant capsid AAV6 / AAV5 chimera (SEQ ID NO: 62) and a genome comprising a transgene of green fluorescent protein (EGFP) operably linked to a CAG promoter (AAV1.CAG.EGFP, AAV8.CAG.EGFP, AAV9.CAG.EGFP, AAV6 / AAV5.CAG.EGFP chimera, respectively) were produced using standard methods . Cardiomyocytes were generated from a human embryonic stem cell line, ESI-017, through the modulation of Wnt signaling using small molecules. After 14 days of cardiac mesoderm induction, cultures were further enriched in relation to cardiomyocytes by glucose deprivation. After approximately 24 days of differentiation, most cells expressed the cardiac myocyte marker, cardiac troponin T (cTnT) and a specific ventricular marker, MLC-2V. The generated cardiomyocytes were evaluated in relation to the expression of gap junction protein (Conex) 43, the fluctuation of membrane potential, calcium handling and the contractile function to ensure that the generated cardiomyocytes reached a state of maturity before characterization of the vector .
[00199] [00199] In relation to AAV1, AAV8 and AAV9, the AAV6 / AAV5 chimera produced significantly greater transduction and transgene expression efficiency in human cardiomyocyte cultures six days after infection which are determined by immunofluorescence (Figure 7A), cytometry flow (Figure 7B) and Western blot analysis (Figures 7C-D). In addition, in relation to AAV8, the AAV6 / AAV5 chimera provided faster onset of gene expression in cultures of human cardiomyocytes, which is determined by immunofluorescence (Figure 7E). In relation to AAV8 and AAV9, the number of infectious units per viral genome administered was several orders of magnitude greater for the AAV6 / AAV5 chimera (Figure 10A). This study illustrates the superior ability of the capsid variants comprising SEQ ID NO: 62 to supply genes to cardiac cells.
[00200] [00200] Example 4
[00201] [00201] Cellular tropism of recombinant AAV virions comprising the new AAV variants LANKIQRTDA + V708I, LANKTTNKDA + V708I and the AAV6 / AAV5 chimera by skeletal myofibers was evaluated in vitro using skeletal myofibers generated from primary human myoblasts.
[00202] [00202] Recombinant AAV virions comprising an AAV8 capsid, an AAV9 capsid, the new variant capsid LANKIQRTDA + V708I, the new variant capsid LANKTTNKDA + V708I or the new AAV6 / AAV5 chimera of the variant capsid and a genome comprising a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (AAV8.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA + V708I.CAG.EGFP, LANKTTNKDA + V708I.CAG.EGFP and AAV6 / chimera AAV5.CAG.GFP, respectively) were produced using standard methods. Skeletal myofibers were generated from primary human skeletal myoblasts obtained from a healthy 51-year-old man (Cook Myositis). Myoblasts were differentiated for 30 days to form mature multinucleate skeletal muscle fibers. The skeletal myofibers generated were evaluated for expression of the Heavy Myosin Chain (MHC) and Dystrophin to ensure that the majority of the skeletal myofibers generated reached a state of maturity before characterization of the vector.
[00203] [00203] Regarding AAV8 and AAV9, LANKIQRTDA + V708I, LANKTTNKDA + V708I and the AAV6 / AAV5 chimera produced a significantly higher transduction and transgene expression efficiency in human skeletal myofiber cultures seven days after infection which are determined by immunofluorescence (Figure 8A) and flow cytometry (Figure 8B). In addition, in relation to AAV8 and AAV9, LANKIQRTDA + V708I and LANKTTNKDA + V708I provided a faster onset of gene expression in human skeletal myofiber cultures, which is determined by immunofluorescence (Figure 8C). In relation to AAV8 and AAV9, the number of infectious units per viral genome administered was several orders of magnitude higher for LANKIQRTDA + V708I, LANKTTNKDA + V708I and the AAV6 / 5 chimera (Figure 10B). This study illustrates the superior ability of variants comprising NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and SEQ ID NO: 62 to provide genes for skeletal myofibers.
[00204] [00204] Example 5
[00205] [00205] Cellular tropism of recombinant AAV virions comprising the new AAV variants LANKIQRTDA + V708I, LANKTTNKDA + V708I and the AAV6 / AAV5 chimera by skeletal muscle progenitor cells was evaluated in vitro using skeletal muscle progenitor cells generated from skeletal muscle progenitor cells generated human induced pluripotent stem derived from fibroblasts (FB-iPSC) or human embryonic stem cells (ESC).
[00206] [00206] Recombinant AAV virions comprising an AAV9 capsid, the new variant capsid LANKIQRTDA + V708I, the new variant capsid LANKTTNKDA + V708I or the new chimera AAV6 / AAV5 variant capsule and a genome comprising a fluorescent protein transgene green (EGFP) operably linked to a CAG promoter (AAV8.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA + V708I.CAG.EGFP, LANKTTNKDA + V708I.CAG.EGFP and AAV6 / AAV5.CAG.GFP chimera , respectively) were produced using standardized methods. Skeletal muscle progenitor cells were generated from a human embryonic stem cell line, ESI-017 (ESI-BIO) following the differentiation strategy described in Shelton et al. Methods, 2016 with few modifications. After approximately 40 days of differentiation, the restriction of lineage to skeletal muscle progenitor cells was confirmed through the expression of PAX7 and MioD in most cells before using cultures to characterize the vector.
[00207] [00207] Regarding AAV9, LANKIQRTDA + V708I, LANKTTNKDA + V708I and the AAV6 / AAV5 chimera produced a significantly higher transduction and transgene expression efficiency in human skeletal muscle progenitor cell cultures six days after infection which are determined by immunofluorescence (Figure 9A) and flow cytometry (Figure 9B). This study illustrates the superior ability of AAV capsid variants comprising NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and SEQ ID NO: 62 to provide genes for skeletal muscle progenitor cells.
[00208] [00208] Example 6
[00209] [00209] Targeted evolution was employed to discover new variants of the adeno-associated virus (AAV) with superior gene delivery to cardiac and skeletal muscle cells after intravenous (IV) administration, an administration route with significant advantages over other methods gene supply to the human cardiac and skeletal muscles (Example 1). Cellular tropism after intracellular administration of recombinant AAV virions comprising the new variant of AAV comprising a V708I substitution and the LANKIQRTDA peptide (SEQ ID NO: 27) inserted between amino acids 587 and 588 (LANKIQRTDA + V708I; SEQ ID NO : 43) was evaluated in vivo in mice as a representative example of the ability of rAAV virions that comprise AAV capsid variants that comprise NKIQRTD (SEQ ID NO: 13) to transduce muscle cells.
[00210] [00210] Recombinant AAV virions comprising the new variant capsid LANKIQRTDA + V708I and a genome comprising a luciferase transgene operably linked to a CAG promoter (LANKIQRTDA + V708I.CAG.luciferase) were produced using standard methods. Albino B6 mice (C57BL / 6) received injections through an intravenous injection in the caudal vein of 2x1012 vg and transduction was assessed during life by obtaining images of luciferase and post-mortem through the activity of luciferase in tissues. Obtaining living images of luciferase on day 14 (left) and day 28 (right) after administration demonstrates that the capsid of the new variant LANKIQRTDA + V708I of AAV can transduce mouse cells in vivo (Figure 11A). Luciferase activity in the heart, diaphragm and quadriceps 56 days after administration demonstrates that the capsid of the new variant LANKIQRTDA + V708I of AAV can transduce mouse cardiac and skeletal muscle in vivo (Figure 11B).
[00211] [00211] This study illustrates gene supply by the variant comprising NKIQRTD (SEQ ID NO: 13) after one of several clinically acceptable routes of administration. Similar efficacy can be achieved with other variants that comprise this peptide insertion motif. Similarly, similar efficacy can be achieved with other variants disclosed here that have been identified using the same directed evolution approach.
[00212] [00212] Example 7
[00213] [00213] Targeted evolution was employed to discover new variants of the adeno-associated virus (AAV) with superior gene delivery to cardiac and skeletal muscle cells after intravenous (IV) administration, an administration route with significant advantages over other methods of gene supply to human cardiac and skeletal muscles (Example 1). Cellular tropism after intracellular administration of recombinant AAV virions comprising the new variant of AAV comprising a V708I substitution and the LANKIQRTDA peptide (SEQ ID NO: 27) inserted between amino acids 587 and 588 (LANKIQRTDA + V708I; SEQ ID NO : 43) has been evaluated in vivo in non-human primates (NHP) as a representative example of the ability of rAAV variants comprising AAV capsid variants containing NKIQRTD (SEQ ID NO: 13) to transduce muscle cells.
[00214] [00214] Recombinant AAV virions comprising the new variant capsid LANKIQRTDA + V708I and a genome comprising a green fluorescent protein (GFP) transgene operably linked to a CAG promoter (LANKIQRTDA + V708I.CAG.GFP) have been produced using standardized methods. Cynomolgus monkeys received injections through intramuscular injection of three doses of vector in the sites on the vast side of 1x1011 vg and the transduction of skeletal muscle cells was evaluated post-mortem by obtaining images by immunofluorescence. Representative images of staining with hematoxylin and eosin (H&E) and anti-GFP antibody cross sections of the proximal biopsy site in 2x, 4x and 20x magnification demonstrate that the capsid of the new variant LANKIQRTDA + V708I from AAV can transduce muscle cells skeletal structure of primates in vivo (Figure 12A). Representative images of staining with hematoxylin and eosin (H&E) and anti-GFP antibody from longitudinal sections of the distal biopsy site at 2x, 4x and 20x magnification demonstrate that the capsid of the new variant LANKIQRTDA + V708I from AAV can transduce muscle cells skeletal structure of primates in vivo (Figure 12B).
[00215] [00215] This study illustrates gene supply by the variant comprising NKIQRTD (SEQ ID NO: 13) after one of several clinically acceptable routes of administration. Similar efficacy can be achieved with other variants that comprise this peptide insertion motif. Similarly, similar efficacy can be achieved with other variants disclosed here that have been identified using the same directed evolution approach.
[00216] [00216] What has been described above merely illustrates the principles of the invention. It will be considered that those skilled in the art will be able to design various arrangements that, although not described or shown explicitly here, incorporate the principles of the invention and are included within its spirit and scope. In addition, it is intended that all examples and conditional language cited here help the reader to understand the principles of the invention and the concepts contributed by the inventors to favor the technique and should not be interpreted as limiting the examples and conditions specifically cited.
[00217] [00217] Furthermore, it is intended that all statements contained herein that cite the principles, aspects and modalities of the invention as well as their specific examples, cover both their structural and functional equivalents. In addition, it is intended that these equivalents include both equivalents known today and equivalents developed in the future, that is, any developed elements that perform the same function, regardless of structure. Therefore, it is not intended that the scope of the present invention be limited to the examples of embodiments shown and described here.
More specifically, the scope and spirit of the present invention are incorporated by the appended claims.

权利要求:
Claims (39)
[1]
1. Adeno-Associated Virus Variant Capsid Protein (AAV), characterized in that it is selected from the group consisting of: (i) an AAV capsid protein that comprises a peptide insert in relation to a protein in the corresponding parent AAV capsid, where the peptide insertion comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or NKTTNKD (SEQ ID NO: 14), where the insertion site is located between two adjacent amino acids at a position between the AAV2 VP1 amino acids 570 and 611 or the corresponding position in the capsid protein of another AAV serotype and (ii) an AAV capsid protein comprising (a) an amino acid sequence at least 85% identical, at least 90 % identical, at least 95% identical or 100% identical to the total length of the amino acid sequence shown in SEQ ID NO: 62 and (b) the following amino acid substitutions for the AAV5 capsid (SEQ ID NO: 6): V229I + A490T + A581T, where the protein AAV capsid variant gives an infectious recombinant AAV virion (rAAV) a greater infectivity of a muscle cell compared to muscle cell infectivity by an AAV virion comprising a wild-type AAV capsid protein.
[2]
2. Adeno-Associated Virus Variant Capsid Protein (AAV), characterized in that it comprises a peptide insertion in relation to a parental AAV capsid protein corresponding to two adjacent amino acids at a position between amino acids 570 and 611 of VP1 AAV2, where the insert comprises the amino acid sequence Y1Y2X1X2X3X4X5X6X7Y3 and where each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro; X1 is selected from Ala, Asn, Thr, Gly, Ser, Ala, Gln and Asp; X2 is selected from Lys, Asn, Thr, Ser, Ala and Gln; X3 is selected from Ile, Thr, Lys, Leu, Val, Asn, Asp and Arg; X4 is selected from Gln, Thr, Ile, Lys, Val, Ser and Tyr; X5 is selected from Arg, Asn, Gly, Lys, Leu, Thr, Ala, Ser and Gln; X6 is selected from Thr, Lys, Val, Gly, Ser, Ala, Arg and Pro; and X7 is selected from Asp, Thr, Asn, Ile, Ala and Ser.
[3]
3. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 2, characterized in that X1 is selected from Thr and Asn; X2 is selected from Asn and Lys; X3 is selected from Lys, Ile and Thr; X4 is selected from Ile, Gln and Thr; X5 is selected from Gly, Arg and Asn; X6 is selected from Val, Thr and Lys; and X7 is selected from Thr and Asp.
[4]
4. Adeno-Associated Virus Variant Capsid Protein (AAV) according to any one of Claims 1 to 3, characterized in that the capsid protein comprises a peptide insert, wherein the insertion site is located between the amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in the capsid protein of another serotype of AAV.
[5]
5. Adeno-Associated Virus Variant Capsid Protein (AAV) according to any one of Claims 1 to 4, characterized in that the AAV capsid protein comprises a peptide insert and the peptide insert comprises the sequence amino acid NKIQRTD (SEQ ID NO: 13) or LANKIQRTDA (SEQ ID NO: 26)
[6]
6. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 5, characterized in that the AAV capsid protein comprises an amino acid substitution V708I in relation to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position in the capsid protein of another AAV serotype.
[7]
7. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 6, characterized in that the AAV capsid protein further comprises one or more of an S109T amino acid substitution, an R588M amino acid substitution and a substitution of amino acid A593 in relation to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position (s) in the capsid protein of another AAV serotype.
[8]
8. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 6, characterized in that the AAV capsid protein further comprises an A35P amino acid substitution for AAV2 VP1 (SEQ ID NO : 2) or the corresponding position (s) in the capsid protein of another AAV serotype.
[9]
9. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 6, characterized in that the AAV capsid protein comprises an amino acid sequence of at least 85% identical, at least 90% identical, at least 95% identical or 100% identical to the total length of the amino acid sequence shown in SEQ ID NO: 43.
[10]
10. Adeno-Associated Virus Variant Capsid Protein (AAV) according to any one of Claims 1 to 4, characterized in that the AAV capsid protein comprises a peptide insert and wherein the peptide insert comprises the amino acid sequence NKTTNKD (SEQ ID NO: 14) or LANKTTNKDA (SEQ ID NO: 27).
[11]
11. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 10, characterized in that the AAV capsid protein comprises an amino acid substitution V708I in relation to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position in the capsid protein of another AAV serotype.
[12]
12. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 11, characterized in that the AAV capsid protein further comprises one or more of an S109T amino acid substitution, a W606C amino acid substitution. and a substitution of amino acid W694C in relation to VP1 of AAV2 (SEQ ID NO: 2) or the corresponding position (s) in the capsid protein of another AAV serotype.
[13]
13. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 11, characterized in that the AAV capsid protein further comprises an amino acid substitution I698V with respect to AAV2 VP1 (SEQ ID NO : 2) or the corresponding position (s) in the capsid protein of another AAV serotype.
[14]
14. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 11, characterized in that the AAV capsid protein comprises an amino acid sequence of at least 85% identical, at least 90% identical, at least 95% identical or 100% identical to the total length of the amino acid sequence shown as SEQ ID NO: 48.
[15]
15. Adeno-Associated Virus Variant Capsid Protein (AAV) according to any one of Claims 1 to 14, characterized in that the capsid protein confers an infectious rAAV virion at least 2 times or at least 5 times more infectivity of a muscle cell compared to infectivity of the muscle cell by an AAV virion comprising a wild-type AAV capsid protein.
[16]
16. Adeno-Associated Virus Variant Capsid Protein (AAV) according to any one of Claims 1 to 15, characterized in that the capsid protein further gives an infectious rAAV virion greater resistance, preferably greater resistance. resistance at least 2 times or at least 5 times greater to neutralization by a neutralizing antibody compared to an AAV comprising the corresponding parental AAV capsid protein.
[17]
17. Isolated Nucleic Acid, characterized in that it comprises a nucleotide sequence that encodes a variant AAV capsid protein as defined in any one of Claims 1 to
16.
[18]
18. Recombinant AAV Virion (rAAV) Infectious, characterized in that it comprises a variant AAV capsid protein as defined in any one of Claims 1 to 16.
[19]
19. Infectious Recombinant AAV (rAAV) Virion according to Claim 18, characterized in that it further comprises a heterologous nucleic acid, preferably a heterologous nucleic acid which comprises a nucleotide sequence encoding a gene product.
[20]
20. Method of Providing Gene Product to Individual Muscle Cell, characterized in that it comprises administering to the individual an rAAV virion as defined in Claim 19, preferably wherein the rAAV virion is administered intravenously or is administered via intramuscular injection .
[21]
21. Method of Providing Gene Product to Individual Muscle Cell according to Claim 20, characterized in that the muscle cell is a cardiac and / or skeletal muscle cell.
[22]
22. Method of Providing Gene Product to Individual Muscle Cell in accordance with Claim 20, characterized in that the gene product is a protein, a small interfering RNA, an antisense RNA, a microRNA, a short hair clip RNA or a small interfering RNA.
[23]
23. Method of Providing Gene Product to Individual Muscle Cell according to Claim 20, characterized in that the gene product is a protein selected from alpha galactosidase A (GLA), frataxin (FXN), dystrophin (DMD) or functional fragment thereof, alpha acid glycosidase (GAA) and glycogen phosphorylase, muscle (PYGM), preferably where the protein comprises or consists of an amino acid sequence that is presented in any of SEQ ID NOs: 64 and 66-69 or one amino acid sequence at least 80% identical to the amino acid sequence shown in any of SEQ ID NOs: 64 and 66-
69.
[24]
24. Method of Providing Gene Product to Muscle Cell in Individual, according to Claim 20, characterized in that the individual has a disease selected from Fabry disease, Friedreich's ataxia, Duchenne muscular dystrophy, Becker muscular dystrophy, disease de Pompe, myophosphorylase deficiency, facio-scapulohumeral muscular dystrophy, waist muscular dystrophy and myotonic dystrophy.
[25]
25. Pharmaceutical Composition, characterized in that it comprises an rAAV virion as defined in Claim 19 and a pharmaceutically acceptable excipient.
[26]
26. Adeno-Associated Virus Variant Capsid Protein (AAV) according to Claim 15, characterized in that the chimeric or variant capsid protein confers infectious recombinant AAV virion (rAAV) at least 50 times greater than a cardiac muscle cell and / or infectivity at least 2 times greater than that of a skeletal muscle cell compared to the infectivity of the cardiac muscle cell or skeletal muscle cell by an AAV virion comprising a wild-type AAV9 capsid protein.
[27]
27. Recombinant AAV Virion (rAAV) Infectious, characterized in that it comprises a variant AAV capsid protein that has an amino acid sequence of at least 95%, preferably at least 99% identical to the total length of the amino acid sequence shown in SEQ ID NO: 43 and which further comprises a nucleotide sequence encoding alpha galactosidase A (GLA) operably linked to a CAG promoter.
[28]
28. Infectious Recombinant AAV Virion (rAAV) according to Claim 27, characterized in that it comprises a variant AAV capsid protein that comprises the amino acid sequence shown in SEQ ID NO: 43.
[29]
29. Mammalian GLA-Encoding Nucleotide Sequence Method, characterized in that it comprises administering an rAAV as defined in Claim 27 or 28 to the mammal under conditions that result in the expression of GLA at a level that provides a therapeutic effect on said mammal.
[30]
30. Mammalian GLA-Encoding Nucleotide Sequence Method according to Claim 29, characterized in that the rAAV is administered intravenously to the mammal and / or is administered via intramuscular injection.
[31]
31. Mammalian GLA-Encoding Nucleotide Sequence Method according to Claim 29 or 30,
characterized by that the mammal is a human with Fabry disease.
[32]
32. Method according to any one of Claims 29 to 31, characterized in that the nucleotide sequence encodes a GLA polypeptide that comprises or consists of the amino acid sequence shown as SEQ ID NO: 67 or a sequence of at least 80% identical to the same.
[33]
33. Recombinant AAV Virion (rAAV) Infectious, characterized in that it comprises a variant AAV capsid protein that has an amino acid sequence at least 95% identical to the total length of the amino acid sequence shown in SEQ ID NO: 43 and which comprises a nucleotide sequence that encodes muscle glycogen phosphorylase (PYGM) operationally linked to a CAG promoter.
[34]
34. Infectious Recombinant AAV Virion (rAAV) according to Claim 33, characterized in that it comprises a variant AAV capsid protein that comprises the amino acid sequence shown in SEQ ID NO: 43.
[35]
35. Method of Providing Nucleotide Sequence Encoding Mammalian PYGM, characterized in that it comprises administering a rAAV as defined in Claim 33 or 34 to the mammal under conditions that result in the expression of PYGM at a level that provides a therapeutic effect on said mammal.
[36]
36. Method of Providing Nucleotide Sequence Encoding PYGM for Mammal according to Claim 35, characterized in that the rAAV is administered intravenously to the mammal and / or is administered via intramuscular injection.
[37]
37. Nucleotide Sequence Provision Method that
It encodes PYGM for Mammal according to Claim 35 or 36, characterized in that the mammal is a human with myophosphorylase deficiency (McArdle's disease).
[38]
38. Method of Providing Mammalian Nucleotide Sequence Encoding PYGM according to any one of Claims 35 to 37, characterized in that the nucleotide sequence encodes a PYGM polypeptide that comprises or consists of the amino acid sequence shown as SEQ ID NO: 66 or a sequence at least 80% identical to the same.
[39]
39. Pharmaceutical composition, characterized in that it comprises an rAAV virion as defined in any one of Claims 27, 28, 33 and 34 and a pharmaceutically acceptable excipient.
FIGURE 1
Petition 870200036291, of 03/18/2020, p. 325/353 4DMT Property Libraries
In Vivo Vector Selection: Stage 1 In Vivo Vector Selection: Stage 2
Identified Vector Results Identified Vector Results
AAV Capsid Variants
FIGURE 2
Skeletal Heart
Muscle
FIGURE 3A
Petition 870200036291, of 03/18/2020, p. 327/353 FIGURE 3B FIGURE 3C
Reason Reason Reason
Reason Reason Reason Reason
Chimera Reason Another Reason
FIGURE 4B
FIGURE 4C FIGURE 4A
FIGURE 5
FIGURE 5 (CONTINUED)
FIGURE 5 (CONTINUED)
FIGURE 5 (CONTINUED)
FIGURE 5 (CONTINUED)
Time: 6 days FIGURE 6A
FIGURE 6B
Two-tailed T tests Medium + SD
% EGFP + / cTnT + Cells
39-second exposure FIGURE 6C
FIGURE 6D
Petition 870200036291, of 03/18/2020, p. 337/353 Quantification of Band Intensity
9-second exposure
FIGURE 6E
Day
FIGURE 7A
AAV6 / AAV5 chimera
Time: 6 days
FIGURE 7B
Petition 870200036291, of 03/18/2020, p. 340/353 Chimera of AAV6 / AAV5
% EGFP + cTnT + Average Cells + SD Two-tailed T-tests
FIGURE 7C FIGURE 7D of AAV6 / AAV5
Quantification of the Chimera Band Intensity
Α-Tubulin vehicle
AAV6 / AAV5 Vehicle Chimera
FIGURE 7E
Chimera Day of AAV6 / AAV5
FIGURE 8A
Myosin Heavy Chain
Petition 870200036291, of 03/18/2020, p. 342/353 Chimera of AAV6 / AAV5
Time: 7 days
FIGURE 8B
Petition 870200036291, of 03/18/2020, p. 343/353 Chimera of AAV6 / AAV5
% EGFP-MHC + / MHC + Medium Cells + SD Two-tailed T-tests
FIGURE 8C
Petition 870200036291, of 03/18/2020, p. 344/353 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Day 6 Day 7 Day 6 Day 7
AAV6 / AAV5 chimera
Time: 6 days FIGURE 9A
FIGURE 9B% EGFP-PAX7 + / PAX7 + Cells
AAV6 / AAV5 chimera
Average + SD Two-tailed T tests
AAV6 / AAV5 Chimera of AAV6 / AAV5 Chimera of FIGURE 10A
Amount of Increase vs.
Natural Serotype of AAV6 / AAV5 Chimera of AAV6 / AAV5 Chimera FIGURE 10B
Amount of Increase vs.
Natura serotype
FIGURE 11A
Luciferase Expression In Life
Petition 870200036291, of 03/18/2020, p. 349/353 14 Days After Administration 28 Days After Administration
Radiance
Color Scale
FIGURE 11B Luciferase Expression in the Individual's Tissue
Petition 870200036291, of 03/18/2020, p. 350/353 ng Luciferase / ng Protein Heart Quadriceps Diaphragm
Proximal Biopsy - Cross Section FIGURE 12A
Distal Biopsy - Longitudinal Section FIGURE 12B

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法律状态:
2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-11-30| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2022-03-03| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

优先权:

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