Adeno-associated virus variant capsids and methods of use thereof

Abstract

The present invention relates to adeno-associated virus variant capsids and methods of use thereof. Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more amino acid sequence modifications relative to a parent AAV capsid protein, which, when present in an AAV virion, are more effective than when the AAV virion comprising the unmodified parent AAV capsid protein is infectious to muscle cells. The variant AAV capsid proteins confer increased infectivity to one or more types of muscle cells. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising the variant AAV capsid proteins as described herein; methods of making these rAAV capsid proteins and virions; and methods of using these rAAV capsid proteins and virions in research and clinical practice, such as in, for example, delivery of nucleic acid sequences to one or more muscle cells to treat muscle disorders and diseases.

Description

[0001] This application is a divisional application. The application date of the original application is September 19, 2018, the application number is 201880071752.3 (PCT / US2018 / 051812), and the name of the invention is “Adeno-associated virus variant capsid and its use method”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 560,901, filed September 20, 2017, the entire disclosure of which is incorporated herein by reference. Technical Field

[0004] The invention disclosed herein generally relates to the field of adeno-associated virus (AAV) virions comprising variant capsid proteins and the generation of such variant capsids using directed evolution techniques. Background Art

[0005] Muscle is associated with a variety of serious genetic disorders. Muscle is the target tissue in many gene therapies for muscular dystrophy and can also be used as a biofactory to produce secreted factors to treat systemic diseases. Delivery of therapeutic genes to human muscle tissue can be considered the most urgent unmet need for the treatment of muscle-related diseases.

[0006] One method of achieving muscle-directed gene delivery is a gene-based adeno-associated virus (AAV)-mediated therapy, in which recombinant adeno-associated virus (rAAV) is used to deliver genes to one or more muscle cells to, for example, replace missing genes, correct dominant defective genes, or provide a template for continuous protein therapy. Although clinical gene therapy based on AAV has been increasingly successful, there are still shortcomings in terms of viral vector properties, including, for example, targeting desired muscle cells with high efficiency. Therefore, there is a need in the art for novel AAV variants with excellent transduction ability, which will provide more effective gene-based delivery to muscle cells to treat diseases. The art needs such AAV variants, which present an enhanced muscle transduction profile-compared to wild-type AAV and AAV variants known in the art, in some cases, the AAV variants are widely used in certain muscle cell types, and in other cases, the AAV variants are preferentially used in certain muscle cell types.

[0007] 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 (Rep78, Rep68, Rep52, and Rep40) essential for genome replication, the second gene, cap, expresses three structural proteins (VP1-3) that assemble to form the viral capsid, and the third gene expresses the assembly-activating protein (AAP) that is essential for capsid assembly. AAV depends on the presence of a helper virus (such as adenovirus or herpes virus) for active replication. In the absence of a helper virus, AAV forms a latent state in which its genome is maintained episomally or integrated into the host chromosome in the AAVS1 locus.

[0008] In vitro and in vivo directed evolution techniques can be used to select AAV variants that provide improvements that are superior to current AAV-based gene delivery vectors. Such directed evolution techniques are known in the art and are described, for example, in PCT Publication WO2014 / 194132 and Kotterman & Schaffer (Nature Review Genetics, AOP, published online on May 20, 2014 doi: 10.1038 / nrg3742), both of which are incorporated herein by reference in their entirety. Directed evolution is a capsid engineering method that simulates natural evolution through multiple rounds of iterative genetic diversification and selection processes, thereby being able to accumulate beneficial mutations that gradually improve the function of biomolecules (such as AAV-based virions). In this method, wild-type AAV cap genes are diversified to produce a larger gene library that is packaged to produce a library of viral particles, and selective pressure is applied to isolate unique variants with superior phenotypes that can overcome gene delivery barriers.

[0009] AAV variants have been disclosed in, for example, U.S. Patent Nos. 9,193,9569; 186; 419; 8,632,764; 8,663,624; 8,927,514; 8,628,966; 8,263,396; 8,734,809; 8,889,641; 8,632,764; 8,691,948; 8,299,295; 8,802,440; 8,445,267; 8,906,307; 8,574,583; 8,067,015; 7,588,7 72; 7,867,484; 8,163,543; 8,283,151; 8,999,678; 7,892,809; 7,906,111; 7,259,151; 7,629,322; 7,220,577; 8,802,080; 7,198,951; 8,318,480; 8,962,332; 7,790,449; 7,282,199; 8,906,675; 8,524,446; 7,712,893; 6,491,907; 8,637 ,255; 7,186,522; 7,105,345; 6,759,237; 6,984,517; 6,962,815; 7,749,492; 7,259,151 and 6,156,303; U.S. Publication Nos. 2013 / 0295614; 2015 / 0065562; 2014 / 0364338; 2013 / 0323226; 2014 / 0359799; 2013 / 0059732; 2014 / 0037585; 2014 / 0056854; 2013 / 0296409; 2014 / 03350542013 / 0195801; 2012 / 0070899; 2011 / 0275529; 2011 / 0171262; 2009 / 0215879; 2010 / 0297177; 2010 / 0203083; 2009 / 0317417; 2009 / 0202490; 2012 / 0220492; 2006 / 0292117 and 2004 / 0002159 European Publication No. 2692731A1; 2383346 B1;2359865 B1;2359866 B1;2359867 B1 and 2357010 B1;1791858 B1;1668143B1;1660678 B1;1664314 B1;1496944 B1;1456383 B1;2341068 B1;2338900 B1;1456419B1;1310571 B1;1456383 B1;1633772 B1 and 1135468 B1;and International (PCT) Publication Nos. WO 2014 / 124282; WO 2013 / 170078; WO 2014 / 160092; WO 2014 / 103957; WO 2014 / 052789; WO 2013 / 174760; WO 2013 / 123503; WO 2011 / 038187 and WO 2008 / 124015; WO 2003 / 054197; however, these references do not disclose the embodiments and / or features and / or compositions of matter structures of the AAV variants disclosed herein. ;

[0010] All documents and references in the patent literature cited and referenced herein are hereby incorporated by reference. Summary of the invention

[0011] Provided herein are variant adeno-associated virus (AAV) capsid proteins having one or more amino acid sequence modifications relative to a parent AAV capsid protein, which, when present in an AAV virion, imparts increased infectivity to one or more types of muscle cells compared to the infectivity of the AAV virion comprising the unmodified parent AAV capsid protein to muscle cells. Also provided are recombinant AAV virions and pharmaceutical compositions thereof comprising the variant AAV capsid proteins as described herein; methods for preparing variant rAAV capsid proteins and virions; and methods for using these rAAV capsid proteins and virions in research and clinical practice, such as delivering nucleic acid sequences to one or more muscle cells to treat conditions and diseases.

[0012] In some aspects of the present disclosure, variant adeno-associated virus (AAV) capsid proteins are provided, which have one or more amino acid sequence modifications relative to the parent AAV capsid, and when present in AAV virions, the variant AAV capsid proteins confer increased infectivity to one or more types of muscle cells (e.g., skeletal muscle cells and / or cardiac muscle cells) compared to the infectivity of AAV virions comprising the parent AAV capsid protein that does not include the amino acid sequence modifications to muscle cells. In related aspects of the present disclosure, when present in AAV virions, the variant AAV capsid proteins also confer enhanced resistance to neutralization by anti-AAV antibodies.

[0013] In some aspects of the present disclosure, recombinant AAV (rAAV) virions are provided, which include variant capsid proteins as described herein, wherein the rAAV virions exhibit increased infectivity to one or more types of muscle cells (e.g., skeletal muscle cells and / or cardiomyocytes) relative to the infectivity of AAV virions including corresponding unmodified parental AAV capsid proteins to muscle cells. In some embodiments, relative to AAV virions including parental AAV capsid proteins, the rAAV virions exhibit increased infectivity to all muscle cells. In other embodiments, the rAAV virions exhibit increased infectivity to certain muscle cell types rather than other cell types relative to AAV virions including parental AAV capsid proteins. In other words, rAAV virions exhibit increased infectivity to certain muscle cell types rather than other types, for example, rAAV exhibits increased infectivity to 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 have not been shown to have increased infectivity to all cell types.

[0014] In some embodiments, the rAAV virion comprises a heterologous nucleic acid. In some such embodiments, the heterologous nucleic acid encodes an RNA encoding a polypeptide. In other such embodiments, the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, such as a heterologous nucleic acid sequence that is a guide RNA for an RNA interfering agent, a nuclease, etc.

[0015] Also provided herein are pharmaceutical compositions comprising infectious rAAV virions of the invention and a pharmaceutically acceptable carrier.

[0016] Also provided is the use of rAAV virions comprising variant capsid proteins as described herein in a method of delivering heterologous nucleic acids to target cells (such as cardiomyocytes) by contacting the target cells with rAAV virions. In some embodiments, the target cells are in vivo, such as in the heart of an individual in need of treatment for a cardiovascular disorder. In other embodiments, the target cells are in vitro.

[0017] Also provided are methods of treating and / or preventing a disease (e.g., a cardiac or skeletal muscle disorder) by administering to an individual in need of such treatment an effective amount of rAAV virions comprising a variant capsid protein as described herein, or a pharmaceutical composition comprising an effective amount of rAAV virions.

[0018] Also provided is an isolated nucleic acid comprising a sequence encoding a variant AAV capsid protein as described herein and a host cell comprising the isolated nucleic acid. In yet other embodiments, the isolated nucleic acid and / or isolated host cell comprises rAAV.

[0019] In some aspects, the variant AAV capsid protein comprises an insertion of about 5 amino acids to about 20 amino acids (a "heterologous peptide" or "peptide insertion") into the GH-loop of the capsid protein relative to the corresponding parental AAV capsid protein, wherein when present in an AAV virion, the variant capsid protein confers increased infectivity to muscle cells compared to the infectivity of AAV virions comprising the corresponding parental AAV capsid protein to muscle cells. In some embodiments, the peptide comprises 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: 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), NKISAKD (SEQ ID NO: 31), NQDYTKT (SEQ ID NO: 32), QADTTKN (SEQ ID NO: 33), TNRTSPD (SEQ ID NO: 34), SNTTQKT (SEQ ID NO: 35), ASDSTKA (SEQ ID NO: 36), LANKIQRTDA (SEQ ID NO: 37), LANKTTNKDA (SEQ ID NO: 38), LATNKIGVTA (SEQ ID NO: 39), LAGNLTKGNA (SEQ ID NO: 40), 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 comprises or is mainly composed 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).

[0020] In some aspects, the variant AAV capsid protein comprises one or more amino acid substitutions relative to the corresponding parental AAV capsid protein, wherein when present in an AAV virion, the variant capsid protein confers increased infectivity to muscle cells compared to the infectivity of the AAV virion comprising the corresponding parental AAV capsid protein to muscle cells.

[0021] In some embodiments, a variant AAV capsid protein is disclosed that comprises a P363L substitution relative to AAV2 and, optionally, further comprises an E347K and / or V708I substitution relative to AAV2.

[0022] In a related aspect, the variant AAV capsid protein comprises a peptide insertion and one or more amino acid substitutions relative to a corresponding parental AAV capsid protein, wherein when present in an AAV virion, the variant capsid protein confers increased infectivity to muscle cells compared to the infectivity of AAV virions comprising the corresponding parental AAV capsid protein to muscle cells.In several embodiments, variant AAV capsid proteins are provided relative to AAV2 comprising a peptide insertion and a V708I substitution, wherein the peptide insertion 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), NO: 39), LATNRTSPDA (SEQ ID NO: 40), LASNTTQKTA (SEQ ID NO: 41), and LAASDSTKAA (SEQ ID NO: 42), preferably 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: 16), NKTTNKD (SEQ ID NO: 17), LANKISAKDA (SEQ ID NO: 18), LANQDYTKTA (SEQ ID NO: 19), LATNKIGVTS (SEQ ID NO: 20), LATNKIGVTA (SEQ ID NO: 21), LAQADTTKNA (SEQ ID NO: 22), LATNRTSPDA (SEQ ID NO: 23), LASNTTQKTA (SEQ ID NO: 24), and LAASDSTKAA (SEQ ID NO: 25), preferably 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: 16), LANKISAKDA (SEQ ID NO: 17), LANQDYTKTA (SEQ ID NO: 18), LATNKIGVTS (SEQ ID NO: 19), NO:27), LANKTTNKDA (SEQ ID NO:28), LATNKIGVTA (SEQ ID NO:29) and LATNKIGVTS (SEQ ID NO:37).In several embodiments, a variant AAV capsid protein is provided relative to AAV2P comprising a peptide insertion and a 363L substitution, wherein the peptide insertion 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).

[0023] In some embodiments, a variant AAV capsid protein is disclosed that includes a heterologous peptide LANKIQRTDA (SEQ ID NO: 27) and includes a V708I substitution relative to AAV2 and, optionally, further includes an A593E and / or S109T and / or T330A and / or R588M substitution relative to AAV2. In other embodiments, a variant AAV capsid protein is disclosed that includes a heterologous peptide LANKIQRTDA (SEQ ID NO: 27) and includes an A35P substitution relative to AAV2. In other embodiments, a variant AAV capsid protein is disclosed that includes a heterologous peptide LANKIQRTDA (SEQ ID NO: 27) and includes amino acid substitutions N312K, N449D, N551S, I698V and L735Q relative to AAV2 and, optionally, further includes a V708I substitution relative to AAV2.

[0024] In some embodiments, a variant AAV capsid protein is disclosed that includes a heterologous peptide LANKTTNKDA (SEQ ID NO: 28) and includes a V708I substitution relative to AAV2 and, optionally, further includes S109T and / or W694C and / or W606C substitutions relative to AAV2. In other embodiments, a variant AAV capsid protein is disclosed that includes a heterologous peptide LANKTTNKDA (SEQ ID NO: 28) and includes an I698V substitution relative to AAV2. In other embodiments, a variant AAV capsid protein is disclosed that includes a heterologous peptide LANKTTNKDA (SEQ ID NO: 28) and includes amino acid substitutions N312K, N449D, N551S, I698V and L735Q relative to AAV2 and, optionally, further includes a V708I substitution relative to AAV2.

[0025] In some embodiments, a variant AAV capsid protein is disclosed, comprising a heterologous peptide LATNKIGVTA (SEQ ID NO: 29) and comprising a V708I substitution relative to AAV2 and, optionally, further comprising a N449K and / or G222S substitution relative to AAV2. In other embodiments, a variant AAV capsid protein is disclosed, comprising a heterologous peptide LATNKIGVTA (SEQ ID NO: 29) and comprising amino acid substitutions N312K, N449D, N551S, I698V and L735Q relative to AAV2 and, optionally, further comprising a V708I substitution relative to AAV2.

[0026] In some embodiments, disclosed is a variant AAV capsid protein comprising a heterologous peptide as described herein and comprising a P363L substitution relative to AAV2.

[0027] Also disclosed herein are methods for making and / or delivering rAAV comprising variant AAV capsids as disclosed herein. In addition, provided herein are kits comprising rAAV comprising variant AAV capsids as disclosed herein and uses for the methods described herein.

[0028] In other embodiments, the AAV virus particles comprising the variant capsid proteins in the preceding paragraphs may be incorporated into any of the preceding or subsequently disclosed embodiments. In fact, it should be understood that certain features of the invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, the various features of the invention described in the context of a single embodiment for brevity may also be provided separately or in any suitable sub-combination. The present invention specifically encompasses all combinations of embodiments related to the present invention and is disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, the present invention also specifically encompasses all sub-combinations of various embodiments and elements thereof and is disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein.

[0029] This summary is not intended to define the claims, nor is it intended to limit the scope of the invention in any way.

[0030] Other features and advantages of the invention disclosed herein will be apparent from the following drawings, detailed description, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application document contains at least one drawing in color. Copies of this patent or patent application publication with one or more color drawings will be provided by the Patent Office upon request and payment of the necessary fee. It should be emphasized that, in accordance with common practice, the various features of the drawings are not drawn to scale. Instead, the dimensions of the various features have been arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0032] Figure 1 Describe the embodiment of the directed evolution method.Step (a) describes the generation of a viral capsid library including a combination of DNA mutation technology and cap genes.Step (b) describes the packaging of the virus so that each viral particle is composed of a mutant capsid around the cap gene encoding that capsid and purification.The capsid library is then placed under in vitro or in vivo selection pressure.In this aspect of the directed evolution technique, relevant tissues or cell materials are harvested to separate the AAV variants that have successfully infected that target, and the successful virus is recovered.Step (c) describes the stage 1 enrichment of successful cloning by repeated selection.Step (d) describes the stage 2 enrichment of the selected cap gene, which undergoes re-diversification and further selection steps to iteratively increase viral fitness.Step (e) describes the variants identified as hits during the vector selection stages 1 and 2, which will be manufactured as recombinant AAV vectors and characterize the transduction levels of various cell types and tissue targets.According to the nature of the AAV directed evolution process, the variants disclosed herein have demonstrated the ability to transduce muscle cells and deliver genomes (genomes encoding variant cap genes) during the selection process.

[0033] Figure 2 PCR amplification of viral genomes from heart and skeletal muscle tissues from representative selection rounds is shown. The bands within the red boxes indicate successful amplification of viral genomes.

[0034] Figures 3A-3C Displays the frequency of motifs within the sequencing analysis. Figure 3A Round 4 sequencing analysis of selective pressures for intravenous delivery to myocardial tissue is provided. Figure 3B Round 2 sequencing analysis of selective pressures delivered intravenously to myocardial tissue in the presence of neutralizing antibodies is provided. Figure 3C Round 3 sequencing analysis of selective pressures for intravenous delivery to skeletal muscle tissue is provided. Figure 3A 57.40% LANKIQRTDA motif, 16.96% LANKTTNKDA motif, 7.32% A593E motif, 7.32% others, 4.88% V708I motif, and 4.88% LASNTVKAIA motif were displayed. Figure 3B21.14% others, 20.33% LAQADTTKNA motifs, 15.45% LANKTTNKDA motifs, 15.45% LAASNITKAA motifs, 15.45% AAV6 / AAV5 chimera motifs, and 12.20% LANTVKLSTA motifs were displayed. Figure 3C 43.21% A593E motif, 41.98% P363L motif and 14.81% others were displayed.

[0035] Figures 4A-4C Figure 4A is a representative three-dimensional model of AAV2 containing a random heptamer after amino acid 587 and V708I substitutions. Figure 4B is a representative three-dimensional model of an AAV6 / AAV5 chimera containing V229I, A490T and A581T substitutions (corresponding to the amino acid sequence shown as SEQ ID NO: 62). Figure 4C A representative 3D model of AAV2 containing the P363L substitution.

[0036] Figure 5 An alignment of wild-type AAV SEQ ID NOs: 1-11 is provided, showing the amino acid positions between and across wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

[0037] Figures 6A-6E Data are provided on in vitro transduction of human cardiomyocytes by recombinant AAV viruses comprising a novel AAV variant LANKIQRTDA+V708I capsid, a novel AAV variant LANKTTNKDA+V708I capsid, and a novel LATNKIGVTA+V708I variant capsid, each expressing a GFP transgene under the control of a CAG promoter. Fig. 6A : Cells differentiated into cardiomyocytes from human pluripotent stem cell lines were infected with novel AAV variants LANKIQRTDA+V708I.CAG.GFP, novel AAV variants LANKTTNKDA+V708I.CAG.GFP, novel AAV variants LATNKIGVTA+V708I.CAG.GFP, or wild-type controls AAV1.CAG.GFP, AAV2.CAG.GFP, and AAV9.CAG.GFP at MOIs of 20, 100, 500, and 2500. Immunofluorescence imaging of cell cultures 6 days post-infection at all MOIs demonstrated that the novel AAV variant capsids were better able to transduce cardiomyocytes than wild-type AAV1, AAV2, or AAV9 capsids. Figure 6B: Quantification of the percentage of GFP-positive cardiomyocytes in each culture by flow cytometry revealed that the novel AAV variant capsids provided significant, dose-dependent improvements in the number of cells transduced by wild-type AAV1, AAV2, or AAV9 capsids. *p<0.05 Figure 6C-6D : Quantification of the amount of GFP in each culture by Western blot revealed that the novel AAV variants provide significant improvement in the expression of the transgene over wild-type AAV1, AAV2 or AAV9 capsids. NT = not transduced. Fig. 6E : Cells differentiated into cardiomyocytes from human pluripotent stem cell lines were infected with novel AAV variants LANKIQRTDA+V708I.CAG.GFP, novel AAV variants LANKTTNKDA+V708I.CAG.GFP, novel AAV variants LATNKIGVTA+V708I.CAG.GFP, or wild-type controls AAV 1.CAG.GFP, AAV2.CAG.GFP, and AAV9.CAG.GFP. Immunofluorescence imaging of cell cultures at days 1, 2, 3, and 5 post-infection at an MOI of 500 showed that novel AAV variant capsids were better able to transduce cardiomyocytes and began to express the GFP transgene earlier than wild-type AAV1, AAV2, or AAV9 capsids.

[0038] Fig. 7A -E provides data on in vitro transduction of human cardiomyocytes by recombinant AAV virus comprising the novel AAV variant AAV6 / AAV5 chimeric capsid of SEQ ID NO: 62 expressing a GFP transgene under the control of the CAG promoter. Fig. 7A : Cells differentiated into cardiomyocytes from human pluripotent stem cell lines were infected with novel AAV variant AAV6 / AAV5 chimera capsids or wild-type controls AAV1.CAG.GFP, AAV8.CAG.GFP, and AAV9.CAG.GFP at MOIs of 100, 500, and 2500. Immunofluorescence imaging of cell cultures 6 days after infection at all MOIs showed that the novel AAV variant capsids were better able to transduce cardiomyocytes than wild-type AAV1, AAV8, or AAV9 capsids. Figure 7B : Quantification of the percentage of GFP-positive cardiomyocytes in each culture by flow cytometry revealed that the novel AAV variant capsids provided significant, dose-dependent improvements in the number of cells transduced by wild-type AAV1, AAV8, or AAV9 capsids. *p<0.05 Figure 7C-7D: Quantification of the amount of GFP in each culture by Western blot revealed that the novel AAV variants provided significant improvement in the expression of transgenes over wild-type AAV1, AAV8 or AAV9 capsids. Vehicle = no transduction. Fig. 7E : Cells differentiated into cardiomyocytes from a human pluripotent stem cell line were infected with novel AAV variant AAV6 / AAV5 chimera capsids or wild-type control AAV8.CAG.GFP. Immunofluorescence imaging of cell cultures at days 3, 4, 5, and 6 post-infection at an MOI of 2500 showed that novel AAV variant capsids better transduced cardiomyocytes and began to express the GFP transgene earlier than wild-type AAV8 capsids.

[0039] Fig. 8A -C provides data on in vitro transduction of human skeletal muscle fibers by recombinant AAV viruses comprising a novel AAV variant LANKIQRTDA+V708I capsid, a novel AAV variant LANKTTNKDA+V708I capsid, and a novel variant AAV6 / AAV5 chimeric capsid, each expressing a GFP transgene under the control of the CAG promoter. Fig. 8A : Cells differentiated into skeletal muscle fibers from human primary myoblasts were infected with novel AAV variant LANKIQRTDA+V708I.CAG.GFP, novel AAV variant LANKTTNKDA+V708I.CAG.GFP, novel AAV variant AAV6 / AAV5 chimera.CAG.GFP, or wild-type controls AAV8.CAG.GFP and AAV9.CAG.GFP at MOIs of 100, 500, and 2500. Immunofluorescence imaging of cell cultures 7 days post-infection at all MOIs showed that novel AAV variant capsids were better able to transduce skeletal muscle fibers than wild-type AAV8 or AAV9 capsids. Figure 8B : Quantification of the percentage of GFP-positive skeletal muscle fibers in each culture by flow cytometry revealed that the novel AAV variant capsids provided significant, dose-dependent improvements in the number of cells transduced by wild-type AAV8 or AAV9 capsids. *p<0.05 Figure 8C: Cells differentiated into skeletal muscle fibers from human primary myoblasts were infected with the novel AAV variant LANKIQRTDA+V708I.CAG.GFP, the novel AAV variant AAV6 / AAV5 chimera.CAG.GFP, or the wild-type controls AAV8.CAG.GFP and AAV9.CAG.GFP. Immunofluorescence imaging of cell cultures at days 2-7 post-infection at an MOI of 2500 showed that the novel AAV variant capsids better transduced skeletal muscle fibers and began to express the GFP transgene earlier than wild-type AAV8 or AAV9 capsids.

[0040] Fig.9A -B provides data on in vitro transduction of human muscle progenitor cells by recombinant AAV viruses comprising a novel AAV variant LANKIQRTDA+V708I capsid, a novel AAV variant LANKTTNKDA+V708I capsid, and a novel AAV variant AAV6 / AAV5 chimeric capsid, each expressing a GFP transgene under the control of a CAG promoter. Fig.9A : Cells differentiated into muscle progenitor cells from a human pluripotent stem cell line were infected with the novel AAV variant LANKIQRTDA+V708I.CAG.GFP, the novel AAV variant LANKTTNKDA+V708I.CAG.GFP, the novel AAV variant AAV6 / AAV5 chimera.CAG.GFP, or the wild-type control AAV9.CAG.GFP at an MOI of 500. Immunofluorescence imaging of cell cultures 6 days post-infection at all MOIs demonstrated that the novel AAV variant capsids were better able to transduce muscle progenitor cells than wild-type AAV9. Fig. 9B : Quantification of the percentage of GFP-positive muscle progenitor cells in each culture by flow cytometry revealed that the novel AAV variant capsids provided significant improvement in the number of cells transduced by wild-type AAV9. *p<0.05

[0041] Fig. 10A -B provides data on in vitro transduction of human cardiomyocytes and human skeletal muscle fibers by recombinant AAV viruses comprising a novel AAV variant LANKIQRTDA+V708I capsid, a novel AAV variant LANKTTNKDA+V708I capsid, and a novel variant AAV6 / AAV5 chimeric capsid, each expressing a GFP transgene under the control of a CAG promoter. Fig. 10A : Novel AAV capsid variants show fold-increase in transduction of human cardiomyocytes compared with wild-type AAV8 and AAV9, the serotypes most widely used in clinical applications for muscle diseases. Fig. 10B: Fold increase in transduction of human skeletal muscle fibers by novel AAV capsid variants compared with wild-type AAV8 and AAV9.

[0042] Fig.11A -B presents data on in vivo transduction of mouse tissues by recombinant AAV viruses comprising a novel AAV variant LANKIQRTDA+V708I capsid expressing a luciferase transgene under the control of the CAG promoter. Mice were injected intravenously via the tail vein with a single dose of 2 × 10 11 A viral genome. Fig.11A : Vital imaging of luciferase at day 14 (left) and day 28 (right) after administration demonstrates that the novel AAV variant LANKIQRTDA+V708I capsid can transduce mouse cells in vivo. Fig. 11B : Luciferase activity in the heart, diaphragm, and quadriceps muscle 56 days after administration indicated that the novel AAV variant LANKIQRTDA+V708I capsid could transduce mouse cardiac and skeletal muscle in vivo.

[0043] Fig. 12A -B presents data on in vivo transduction of non-human primate skeletal muscle by recombinant AAV virus comprising a novel AAV variant LANKIQRTDA+V708I capsid expressing a GFP transgene under the control of the CAG promoter. Non-human primates were injected intramuscularly three times with 10 11 The virus genomes were delivered to the left vastus lateralis muscle, and muscle tissue was analyzed 4 weeks after administration. Fig. 12A : Representative images of cross sections of proximal biopsy sites stained with hematoxylin and eosin (H&E) and anti-GFP antibodies at 2x, 4x, and 20x magnification demonstrate that the novel AAV variant LANKIQRTDA+V708II capsid can transduce primate skeletal muscle in vivo. Fig. 12B : Representative images of longitudinal sections of distal biopsy sites stained with hematoxylin and eosin (H&E) and anti-GFP antibodies at 2x, 4x, and 20x magnification demonstrate that the novel AAV variant LANKIQRTDA+V708I capsid can transduce primate skeletal muscle cells in vivo. DETAILED DESCRIPTION

[0044] Before describing the methods and compositions of the present invention, it should be understood that the present invention is not limited to the specific methods or compositions described, and therefore may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be restrictive, as the scope of the present invention is limited only by the appended claims.

[0045] The invention disclosed herein is illustrated in the drawings and the specification. However, although specific embodiments are illustrated in the drawings, it is not intended to limit the invention to the specific embodiments or the illustrated and / or disclosed embodiments. On the contrary, the invention disclosed herein is intended to cover all modifications, alternative constructions, and equivalents that fall within the spirit and scope of the invention. Therefore, the drawings are intended to be illustrative rather than restrictive.

[0046] Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed, up to one tenth of the lower limit unit (unless the context clearly specifies otherwise). Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in that stated range is encompassed in the present invention. The upper and lower limits of these smaller ranges may be independently included in the described range or excluded therefrom, and each range (where one, none, or both of the two limits are included in the described smaller range) is also encompassed in the present invention, subject to any specifically excluded limits in the described range. When the stated range includes one or two limits, the range excluding any or both of those included limits is also encompassed in the present invention.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the 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 disclosures mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials related to the listed disclosures. It should be understood that in the event of a conflict, the present disclosure replaces any disclosure incorporated into the disclosure.

[0048] As will be apparent to those skilled in the art after reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method may be performed in the order of the recited events or in any other order that is logically possible.

[0049] It should be noted that as used herein and in the appended claims, the singular forms "a / an", "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "recombinant AAV virions" includes a plurality of such virions and reference to "myocytes" includes reference to one or more myocytes and equivalents thereof known to those of ordinary skill in the art, etc. It should be further noted that the claims may be written to exclude any optional elements. Therefore, such statements are intended to serve as antecedent basis for the use of exclusive terminology such as "solely", "only", etc., in connection with the recitation of claim elements, or the use of a "negative" limitation.

[0050] The disclosure discussed herein is provided only for the disclosure content before the filing date of the present application. Any content of this article should not be interpreted as admitting that the present invention has no right to precede such disclosure by virtue of prior invention. In addition, the publication date provided may be different from the actual publication date, and the actual publication date may need to be determined independently.

[0051] definition

[0052] Adeno-associated virus is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that are the origin of viral replication and packaging signals. The rep ORF encodes four nonstructural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The cap ORF encodes three structural proteins (VP1-3) that assemble to form a 60-mer viral capsid. Finally, the ORF that exists as an alternative reading frame within the cap gene produces assembly activation protein (AAP), a viral protein that localizes the AAV capsid protein to the nucleus and plays a role in the capsid assembly process.

[0053] There are several naturally occurring ("wild-type") serotypes and over 100 known AAV variants, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid protein and thus in its gene delivery properties. AAV has not yet been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0054] For the purposes of this disclosure, the term "AAV" is an abbreviation for adeno-associated virus, including but not limited to the virus itself and its derivatives. Unless otherwise specified, the term refers to all subtypes or serotypes and both replication-competent and recombinant forms. The term "AAV" includes, but is not limited to, 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), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine 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, and so on.

[0055] The genomic sequences of various serotypes of AAV, as well as the natural terminal repeat (TR) sequences, Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank Accession Nos. 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_00615 2.1 (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 by reference for teaching AAV nucleic acid and amino acid sequences. See also, e.g., 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 U.S. Pat. No. 6,156,303.

[0056] The sequences of naturally occurring cap (capsid) proteins associated with AAV serotypes are known in the art and include those disclosed herein: such 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 term "variant AAV capsid protein" or "AAV variant" refers to an AAV capsid protein comprising an amino acid sequence comprising at least one modification or substitution (including deletions, insertions, point mutations, etc.) relative to a naturally occurring or "wild-type" AAV capsid protein sequence, such as shown in SEQ ID NOs: 1-12 herein. The variant AAV capsid protein may have about 80% identity or greater to the amino acid sequence of the wild-type capsid protein, such as 85% identity or greater, 90% identity or greater, or 95% identity or greater, such as 98% or 99% identity to the wild-type capsid protein. The variant AAV capsid protein may not be a wild-type capsid protein.

[0057] For the purposes of this disclosure, "AAV virion" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0058] For the purposes of this disclosure, the term "rAAV" refers to the abbreviation for recombinant adeno-associated virus. "Recombinant" as applied to polynucleotides means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other processes that produce constructs that are different from polynucleotides found in nature. Recombinant viruses are viral particles that include recombinant polynucleotides. The terms include copies of the original polynucleotide construct and the progeny of the original viral construct, respectively.

[0059] The term "rAAV vector" encompasses rAAV virions (i.e., rAAV virus particles) (e.g., infectious rAAV virions), which are defined to include rAAV polynucleotides; and also encompasses polynucleotides encoding rAAV (e.g., single-stranded polynucleotides encoding rAAV (ss-rAAV); double-stranded polynucleotides encoding rAAV (ds-rAAV), such as plasmids encoding rAAV; etc.).

[0060] If an AAV virion includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a target cell, an RNAi agent or a CRISPR agent to be delivered to a target cell, etc.), it is often referred to as a "recombinant AAV (rAAV) virion" or "rAAV viral particle." Typically, the heterologous polynucleotide is flanked by at least one and usually two AAV inverted terminal repeats (ITRs).

[0061] The term "packaging" refers to the series of intracellular events that produce the assembly and encapsidation of AAV particles. The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0062] The term "helper virus" of AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses, and poxviruses such as vaccinia viruses. Adenoviruses cover many different subgroups, but the most commonly used is adenovirus type 5 in subgroup C. Many adenoviruses of human, non-human mammalian, and avian origin are known and can be obtained from depositories such as ATCC. The viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (Epstein-Barr viruse; EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (pseudorabies viruse; PRV); they can also be obtained from depositories such as ATCC.

[0063] The term "one or more helper virus functions" refers to one or more functions encoded in the helper virus genome that allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus functions" can be provided in a variety of ways, including by providing a helper virus or providing a polynucleotide sequence encoding one or more essential functions to a trans-producing cell. For example, a plasmid or other expression vector comprising a nucleotide sequence encoding one or more adenoviral proteins is transfected into a producing cell together with a rAAV vector.

[0064] The term "infectious" virus or virus particle is a term that includes an effectively assembled viral capsid and is capable of delivering a polynucleotide component to a cell in which the viral species is tropical. The term does not necessarily mean any replication ability of the virus. An assay for counting infectious virus particles is described in the present disclosure and the art. Viral infectivity can be expressed as a ratio of infectious virus particles to total virus particles. Methods for determining the ratio of infectious virus particles to total virus particles are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11: S337 (describing TCID50 infectivity titer analysis); and Zolotukhin et al. (1999) Gene Ther. 6: 973. See also Examples.

[0065] As used herein, the term "tropism" refers to a virus (e.g., AAV) preferentially targeting cells of a particular host species or a specific cell type within a host species. For example, relative to viruses that may only infect lungs and myocytes, viruses that may infect heart, lungs, liver, and myocytes have a wider (i.e., increased) tropism. Tropism can also include the dependence of viruses on cell surface molecules of a particular type of host. For example, some viruses may only infect cells with surface glycosaminoglycans, while other viruses may only infect cells with sialic acid (this dependence can be tested using various cell lines lacking specific class molecules as potential host cells for viral infection). In some cases, the tropism of a virus describes the relative preference of the virus. For example, a first virus may be able to infect all cell types, but is more successful in infecting those cells with surface glycosaminoglycans. If a second virus also prefers the same features (e.g., a second virus is also more successful in infecting those cells with surface glycosaminoglycans), it can be considered that the second virus has a similar (or identical) tropism to the first virus, even if the absolute transduction efficiency is not similar. For example, a second virus may be more effective than a first virus at infecting each given cell type tested, but if the relative preferences are similar (or the same), the second virus can still be considered to have a tropism similar (or the same) as the first virus. In some embodiments, the tropism of the virions comprising the variant AAV capsid proteins of the invention is not altered relative to naturally occurring virions. In some embodiments, the tropism of the virions comprising the variant AAV capsid proteins of the invention is expanded (i.e., widened) relative to naturally occurring virions. In some embodiments, the tropism of the virions comprising the variant AAV capsid proteins of the invention is reduced relative to naturally occurring virions.

[0066] The term "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and also capable of replicating in infected cells (i.e., in the presence of a helper virus or helper virus function). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. Typically, rAAV vectors as described herein are not replicable in mammalian cells (especially in human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences so as to minimize the possibility of producing replication-competent AAV by recombination between AAV packaging genes and incoming rAAV vectors. In many embodiments, rAAV vector preparations as described herein are those containing very little (if any) replication-competent AAV (rcAAV, also referred to as RCA) (e.g., 10 2 Each rAAV particle is smaller than about 1 rcAAV particle. 4 Each rAAV particle is smaller than about 1 rcAAV, each 10 rAAV particles are smaller than about 1 rcAAV, each 10 12 less than about 1 rcAAV particle, or no rcAAV).

[0067] The term "polynucleotide" refers to nucleotides in a polymeric form of any length, including deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interspersed with non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of polynucleotides herein encompasses each of the double-stranded form and the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0068] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, which means that when compared, the percentage of bases or amino acids is the same when compared with the two sequences. Sequence similarity can be determined in a variety of different ways. In order to determine sequence identity, the sequences can be compared using methods and computer programs including BLAST, which can be obtained through the World Wide Web ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, which can be obtained from the Genetics Computing Group (GCG) package in Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group. Other techniques for alignment are described in Methods in Enzymology, Volume 266: Computer Methods for Macromolecular Sequence Analysis (1996), edited by Doolittle, Academic Press, a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow gaps in sequences. Smith-Waterman is a type of algorithm that allows gaps in sequence alignment. 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 sequences. See J. Mol. Biol. 48: 443-453 (1970).

[0069] The term "gene" refers to a polynucleotide that performs a certain function in a cell. For example, a gene may contain an open reading frame that can encode a gene product. An example of a gene product is a protein transcribed and translated from a gene. Another example of a gene product is an RNA that is transcribed but not translated, such as a functional RNA product, such as an aptamer of a nuclease, an interfering RNA, a ribosomal ribonucleic acid (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA, etc.

[0070] The term "gene expression product" or "gene product" is a molecule produced by the expression of a specific gene as defined above. Gene expression products include, for example, polypeptides, aptamers, interfering RNAs, messenger ribonucleic acid (mRNA), rRNA, tRNA, non-coding RNA (ncRNA), etc.

[0071] The term "siRNA agent" ("small interfering" or "short interfering RNA" (or siRNA)) is an RNA duplex of nucleotides targeting a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule that form a double-stranded RNA region (dsRNA). siRNA is "targeted" to a gene because the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the duplex of the siRNA is less than 30 nucleotides. In some embodiments, the length of the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides. In some embodiments, the length of the duplex is 19-25 nucleotides. In some embodiments, siRNA-mediated gene targeting is achieved by using DNA-guided RNA interference (ddRNAi), which is a gene silencing technique that utilizes DNA constructs to activate endogenous RNA interference (RNAi) pathways of animal cells. Such DNA constructs are designed to express a self-complementary double-stranded RNA that causes one or more target genes to be silenced after treatment, typically short hairpin RNA (shRNA). Any RNA comprising endogenous mRNA or viral RNA can be silenced by designing constructs to express double-stranded RNA complementary to the desired mRNA target. Therefore, the RNA duplex portion of the siRNA agent can be a part of a short hairpin structure referred to as shRNA. In addition to the duplex portion, the hairpin structure can contain a loop portion between two sequences forming a duplex. The length of the loop can vary. In some embodiments, the length of the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides. The hairpin structure can also contain a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang with a length of 0, 1, 2, 3, 4 or 5 nucleotides. Typically, the level of the expression product (e.g., mRNA, polypeptide, etc.) of the target gene is reduced by an siRNA agent (e.g., siRNA, shRNA, etc.) containing a specific double-stranded nucleotide sequence, which is complementary to a target gene transcript of at least 19-25 nucleotides (e.g., 20-21 nucleotide sequences) in length, including a 5' untranslated (UT) region, an ORF, or a 3'UT region. In some embodiments, the length of the short interfering RNA is about 19-25 nt.See, for example, PCT applications WO 00 / 44895, WO 99 / 32619, WO 01 / 75164, WO 01 / 92513, WO 01 / 29058, WO 01 / 89304, WO 02 / 16620 and WO 02 / 29858; and U.S. Patent Publication No. 2004 / 0023390 for describing siRNA technology. siRNA and / or shRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter. The nucleic acid sequence can also include a polyadenylation signal. In certain embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[0072] The term "antisense RNA" encompasses RNA that is complementary to a gene expression product. For example, an antisense RNA targeting a specific mRNA is an RNA-based agent (or may be a modified RNA) that is complementary to the mRNA, wherein hybridization of the antisense RNA to the mRNA changes the expression of the mRNA (e.g., by changing the stability of the RNA, changing the translation of the RNA, etc.). "Antisense RNA" also includes nucleic acids encoding the antisense RNA.

[0073] About "CRISPR / Cas9 agent", the term "CRISPR" covers aggregated regular interspersed short palindromic repeats / CRISPR-related (Cas) systems, which are evolved by using CRISPR RNA (crRNA) to guide the silencing of invading nucleic acids to provide adaptive immunity to bacteria and archaea against viruses and plasmids. Cas9 protein (or its functional equivalent and / or variant, i.e., Cas9-like protein) naturally contains DNA endonuclease activity, which depends on the association of protein with two naturally occurring or synthetic RNA molecules referred to as crRNA and tracrRNA (also referred to as guide RNA). In some cases, two molecules are covalently linked to form a single molecule (also referred to as a single guide RNA ("sgRNA")). Therefore, Cas9 or Cas9-like protein is associated with DNA targeting RNA (the term covers both double-molecule guide RNA configuration and single-molecule guide RNA configuration), and the DNA targeting RNA activates Cas9 or Cas9-like protein and guides the protein to the target nucleic acid sequence.

[0074] If Cas9 or a Cas9-like protein retains its native enzymatic function, it will cleave the target DNA to produce a double-strand break, which can result in genomic changes (i.e., editing: deletion, insertion (when a donor polynucleotide is present), substitution, etc.), thereby altering gene expression. Some variants of Cas9 (variants of which are covered by the term Cas9-like) have been altered so that they have reduced DNA cleavage activity (in some cases, they cleave a single strand instead of both strands of the target DNA, and in other cases, they are severely reduced to no DNA cleavage activity). Cas9-like proteins with reduced DNA cleavage activity (or even no DNA cleavage activity) can still be guided to the target DNA to block RNA polymerase activity. Alternatively, Cas9 or a Cas9-like protein can be modified by fusing the VP64 transcriptional activation domain to the Cas9 protein and co-delivering it with an MS2-P65-HSF1 auxiliary protein and a single guide RNA encoding an MS2 RNA aptamer on the tetraloop to form a synergistic activation mediator (Cas9-SAM) complex in cells that activate transcription. Therefore, the enzymatically inactivated Cas9-like protein can be targeted to a specific position in the target DNA by the DNA-targeting RNA to block or activate the transcription of the target DNA. As used herein, the term "CRISPR / Cas9 agent" encompasses all forms of CRISPR / Cas9 as described above or known in the art.

[0075] Detailed information about CRISPR agents can be found in the following literature; for example, (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) U.S. Patent Application No. 13 / 842,859 and PCT Application No. PCT / US13 / 32589; all of which are hereby incorporated by reference in their entirety. Thus, the term "CRISPR agent" as used herein encompasses any agent (or nucleic acid encoding such an agent) including naturally occurring and / or synthetic sequences that can be used in a Cas9-based system (e.g., Cas9 or Cas9-like proteins; any component of a DNA-targeting RNA, such as crRNA-like RNA, tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; etc.).

[0076] "Zinc finger nuclease" (ZFN) refers to an artificial DNA endonuclease produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target a desired DNA sequence, and this enables the zinc finger nuclease to cleave a unique target sequence. When introduced into a cell, ZFNs can be used to edit target DNA in a cell (e.g., the genome of the cell) by inducing double-strand breaks. For more information on the use of ZFNs, see, e.g., 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):87575-85; Takasu et al., Insect Biochem Mol. Biol. 2010 Oct;40(10):759-65; Ekker et al., Zebrafish Summer 2008;5(2):121-3; Young et al., PNAS 2011 Apr 26;108(17):7052-7; Goldberg et al., Cell. 2010 May 5;140(5):678-91; Geurts et al., Science 2009 Jul 24;325(5939):433; Flisikowska et al., PLoS One One) 2011;6(6):e21045.doi:10.1371 / joumal.pone.0021045. Epub 2011 Jun 13; Hauschild et al., Proc Natl Acad Sci U S A 2011 Jul 19;108(29):12013-7; and Yu et al., Cell Res. 2011 Nov;21(11):1638-40; all of which are incorporated herein by reference for their teachings related to ZFNs. The term "ZFN agent" encompasses zinc finger nucleases and / or polynucleotides comprising nucleotide sequences encoding zinc finger nucleases.

[0077] The term "transcription activator-like effector nuclease" or "TALEN" agent refers to a transcription activator-like effector nuclease (TALEN). TALEN is an artificial DNA endonuclease produced by fusing a TAL (transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALEN can be rapidly engineered to bind to virtually any desired DNA sequence, and when introduced into a cell, TALEN can edit a target DNA in a cell (e.g., the genome of a cell) by inducing double-strand breaks. For more information on the use of TALENs, see, e.g., 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):69%6; and Huang et al., Nat Biotechnol. 2011 Aug 5;29(8):699-700; all of which are incorporated herein by reference for their teachings on TALENs. The term "TALEN agent" encompasses TALENs and / or polynucleotides comprising a nucleotide sequence encoding a TALEN.

[0078] The term "control element" or "control sequence" refers to a nucleotide sequence that participates in molecular interactions, which contributes to the functional regulation of a polynucleotide, including replication, repetition, transcription, splicing, translation or degradation of a polynucleotide. The regulation may affect the frequency, speed or specificity of the process, and may be enhancing or inhibiting in nature. Control elements known in the art include, for example, transcriptional regulatory sequences, such as promoters and enhancers. A promoter is a DNA region that is able to bind to RNA polymerase under certain conditions and initiate transcription of a coding region that is usually located downstream (3' direction) of the promoter. Promoters can be broadly active, i.e., active in many cell types (e.g., CAG or CMV promoters); or tissue or cell specific, for example, a promoter can be tissue specific for expression in cardiomyocytes.

[0079] The term "operatively linked" or "operably linked" refers to the juxtaposition of genetic elements, wherein the elements are in a relationship that allows them to operate in the intended manner. For example, a promoter is operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0080] The term "expression vector" encompasses a vector that includes a polynucleotide region encoding a polypeptide of interest, and is used to achieve expression of the protein in the intended target cell. The expression vector may also include a control element operably linked to the coding region to promote expression of the protein in the target. The combination of a control element and a gene or genes to which it is operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art, or can be readily constructed from components available in the art.

[0081] The term "heterologous" refers to an entity that is derived from a genotype different from the rest of the entity to which it is compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence not found naturally linked is a heterologous promoter. Thus, for example, an rAAV comprising a heterologous nucleic acid sequence encoding a heterologous gene product is an rAAV comprising a polynucleotide that is not normally contained in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by a naturally occurring wild-type AAV.

[0082] The terms "genetic alteration" and "genetic modification" (and grammatical variations thereof) are used interchangeably herein to refer to a process in which a genetic element (e.g., a polynucleotide) is introduced into a cell other than mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration can be achieved, for example, by transfecting the cell with a recombinant plasmid or other polynucleotide by any method known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic alteration can also be achieved, for example, by transduction or infection with a DNA or RNA virus or viral vector. Typically, genetic elements are introduced into a chromosome or mini-chromosome in a cell; however, any alteration that changes the phenotype and / or genotype of the cell and its progeny is included in this term.

[0083] With respect to cell modification, the term "genetic modification" or "transformation" or "transfection" or "transduction" by exogenous DNA (e.g., by a recombinant virus) refers to when such DNA has been introduced into a cell. The presence of exogenous DNA results in permanent or transient genetic changes. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. A "clone" is a population of cells derived from a single cell or a 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.

[0084] As used herein, a cell is said to be "stably" altered, transduced, genetically modified, or transformed with a gene sequence if the sequence can be used to exert its function during prolonged cell culture in vitro and / or for an extended period of time in vivo. Typically, such cells are "heritably" altered (genetically modified) because a genetic change is introduced that is also heritable by the progeny of the altered cell.

[0085] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation or conjugation with a marker component. When discussed in the context of delivering gene products to mammalian individuals, polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like and compositions thereof refer to the corresponding complete polypeptides, or any fragments or genetically engineered derivatives thereof, that retain the biochemical function required for the complete protein. Similarly, reference to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for delivering gene products to mammalian individuals (which may be referred to as "transgenes" to be delivered to recipient cells) include polynucleotides encoding complete polypeptides or any fragments or genetically engineered derivatives having the desired biochemical function.

[0086] As used herein, an "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, protein or other substance refers to a preparation of a substance lacking at least some of the other components, which may also be present in a naturally occurring or originally prepared substance or similar substance. Thus, for example, the isolated substance can be prepared by enriching the isolated substance from a source mixture using a purification technique. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or can be measured relative to a second potential interfering substance present in the source mixture. The increased enrichment of the embodiments of the present disclosure is increasingly isolated. In some embodiments, the isolated plasmid, nucleic acid, vector, virus, host cell or other substance is purified, for example, about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure or greater.

[0087] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or side effects attributable to a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease (and / or symptoms caused by a disease) that may occur in an individual who may be susceptible to the disease or at risk of the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease (and / or symptoms caused by the disease), i.e., preventing its development; and (c) alleviating the disease (and / or symptoms caused by the disease), i.e., causing the disease (and / or symptoms caused by the disease) to subside, i.e., improving the disease and / or one or more symptoms of the disease. For example, the compositions and methods of the present invention may be directed to the treatment of muscle diseases. Non-limiting methods for assessing muscle disease and its treatment include measuring therapeutic protein production (e.g., muscle biopsy followed by immunohistochemistry or serum sampling followed by ELISA or "or" logistic and activity analysis), measuring symptoms of heart failure (e.g., New York Heart Association Functional Classification or the Minnesota Living With Heart Failure Questionnaire), functional myocardial status (e.g., 6-minute walk test or peak maximum oxygen consumption), biomarker analysis (e.g., N-terminal prohormone brain natriuretic peptide), left ventricular function / remodeling (e.g., left ventricular ejection fraction or left ventricular end-systolic volume), muscle strength (e.g., Duchenne muscular dystrophy), and left ventricular function / remodeling (e.g., left ventricular ejection fraction or left ventricular end-systolic volume). Dystrophy), handheld dynamometer, Medical Research Council Scale for Maximum Lift Clinical Research), muscle function (e.g., Vignos Scale, Timed Function Test, Hammersmith Motor Ability Score, Timed Rise from Floor, Walk Test, Motor Function Measure Scale, North Star Ambulatory Assessment, 9 Hole Peg Test, or Children's Hospital of Philadelphia Neuromuscular Disorders Infant Test), muscle disease symptoms (e.g., Neuromuscular Symptom Score or Clinical Global Impression), mitochondria (e.g., 31 P magnetic resonance spectroscopy), questionnaire-based quality of life assessment, patient-reported outcomes, or daily activities.

[0088] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to: humans; non-human primates, including monkeys; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0089] In some embodiments, the individual is a person who has previously been naturally exposed to AAV and therefore carries anti-AAV antibodies (i.e., AAV neutralizing antibodies). In some embodiments, the individual is a person who has previously been administered an AAV vector (and therefore may carry anti-AAV antibodies) and needs to be administered the vector again to treat a different condition or further treat the same condition. Based on the positive results in clinical trials that deliver AAV genes to all tissues affected by neutralizing antibodies to this vector, such as liver, muscle, and retina, there are many such therapeutic applications / disease targets.

[0090] As used herein, the term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. The effective amount can be given in one or more administrations. For the purposes of this disclosure, the effective amount of a compound (e.g., an infectious rAAV virion) is an amount sufficient to alleviate, improve, stabilize, reverse, prevent, slow down or delay (and / or symptoms associated therewith) the progress of a specific disease state (e.g., muscle disease). Therefore, the effective amount of an infectious rAAV virion is the amount of infectious rAAV virions that can effectively deliver heterologous nucleic acids to individual target cells (or multiple target cells). The effective amount can be determined preclinically by, for example, detecting gene products (RNA, proteins) encoded by heterologous nucleic acid sequences in cells or tissues using techniques such as RT-PCR, Western blotting, ELISA, fluorescence or other reporter readings known in the art. The effective amount can be determined clinically by, for example, detecting changes in the onset or progression of the disease using methods known in the art, such as 6-minute walking tests, left ventricular ejection fractions, handheld dynamometers, Vignos scales, etc. as described herein and as known in the art.

[0091] The term "myoblast" or "muscle tissue" herein refers to a cell or cell population derived from any type of muscle, including but not limited to skeletal muscle, cardiac muscle, smooth muscle (e.g., from the digestive tract, bladder, and blood vessels), and diaphragm muscle. Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and myocardioblasts. Since muscle tissue is easily accessible to the circulatory system, proteins produced and secreted by muscle cells and tissues in vivo will logically enter the blood to produce systemic benefits, thereby providing sustained therapeutic levels of protein secretion from muscle.

[0092] The term "directed evolution" refers to an in vitro and / or in vivo shell engineering method that mimics natural evolution through multiple rounds of iterative genetic diversification and selection processes, thereby accumulating beneficial mutations that gradually improve the function of the biomolecule. Directed evolution often involves an in vivo method known as "biopanning," which is used to select AAV variants from a library that have more effective levels of infectivity in relevant cell or tissue types.

[0094] Adeno-associated virus (AAV) is a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within a non-enveloped capsid. The viral genome of naturally occurring AAV has two inverted terminal repeats (ITRs) - which function as viral sources of replication and packaging signals - flanked by two major open reading frames (ORFs): rep (encoding proteins that function in viral replication, transcriptional regulation, site-specific integration, and virion assembly) and cap. The cap ORF encodes three structural proteins that assemble to form a 60-mer viral capsid. Many naturally occurring AAV variants and serotypes have been isolated, and none have been associated with human disease.

[0095] Recombinant versions of AAV can be used as gene delivery vectors, where relevant markers or therapeutic genes are inserted between the ITRs replacing rep and cap. These vectors have been shown to transduce both dividing and non-dividing cells in vitro and in vivo, and can produce stable transgene expression in post-mitotic tissues for many years. See, for example, Knipe DM, Howley PM, Fields' Virology, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, USA, 2007; Gao GP, Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM, A novel adeno-associated virus from rhesus monkeys as a vector for human gene therapy. PNAS (2002);99:11854-9; Atchison RW, Casto BC, Hammon WM, Defective virus particles associated with adenovirus, 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, PNAS (1966);55:1467-74; Blacklow NR, Hoggan MD, Rowe WP, Isolation of adenovirus-associated viruses from humans, PNAS (1967);58:1410-5; Bantel-Schaal U, zur Hausen H, Characterization of defective human parvovirus DNA isolated from the genital area, Virology (1984);134:52-63; Mayor HD, Melnick J JL, Small DNA-containing viruses (picodnavirus group), Nature, 1966;210:331-2; Mori S, Wang L, Takeuchi T, Kanda T, Two adeno-associated viruses from cynomolgus macaques: pseudotype characterization of capsid proteins, Virology, 2004;330:375-83; Flotte TR, Progress and prospects in gene therapy: recombinant adeno-associated virus (rAAV) vectors, Gene Ther, 2004;11:805-10.

[0096] Recombinant AAV (referred to herein as "AAV") produces promising results in an increasing number of clinical trials. However, obstacles to gene delivery may limit the utility of AAV, such as anti-capsid immune response, low transduction of certain tissues, inability to target delivery to specific cell types, and relatively low carrying capacity. In many cases, there is not enough mechanistic knowledge to effectively empower rational design with the ability to improve AAV. As an alternative, directed evolution has become a strategy for establishing novel AAV variants that meet specific biomedical needs. Directed evolution strategies utilize genetic diversification and selection processes to accumulate beneficial mutations that can gradually improve the function of biomolecules. In this process, wild-type AAVcap genes are diversified by several methods to produce a larger gene library that is packaged to produce a virion library, and selective pressure is subsequently applied to separate novel variants that can overcome gene delivery obstacles. Importantly, the mechanistic basis of the gene delivery problem does not need to be known by the directed evolution of function, which can therefore accelerate the development of enhanced vectors.

[0097] Typically, the variants disclosed herein are generated using an AAV library and / or multiple AAV libraries. Such an AAV library or multiple AAV libraries are generated by mutating the cap gene, which is a gene encoding an AAV capsid structural protein, through a series of directed evolution techniques known and readily available to those skilled in the art of viral genome engineering. See, for example, Bartel et al., AM. Soc. Gene Cell Ther., 15th Annu. Meet. 20, S140 (2012); Bowles, D. et al., Journal of Virology (J. Virol.) 77, 423-432 (2003); Gray et al., Molecular Therapy 18, 570-578 (2010); Grimm, D. et al., Journal of Virology 82, 5887-5911; Koerber, JT et al., Molecular Therapy 16, 1703-1709 (2008); Li W. et al., Molecular Therapy 16, 1252-1260 (2008); Koerber, JT et al., Methods in Molecular Biology 434, 161-170 (2008); Koerber, JT et al., Human Gene Therapy 18, 367-378 (2007); and Koerber, JT et al., Molecular Therapy 17, 2088-2095 (2009). Such techniques are as follows, without limitation: i) error-prone PCR for introducing random point mutations into the AAV cap open reading frame (ORF) at a predetermined, modifiable rate; ii) in vitro or in vivo viral recombination or "DNA shuffling" to generate random chimeras of the AAV cap gene to obtain a gene library with multiple AAV serotypes; iii) random peptide insertion at defined sites of the capsid by ligating degenerate oligonucleotides in the cap ORF; iv) defined insertion of peptide coding sequences into random positions of the AAV cap ORF using transposon mutagenesis; v) replacement of surface loops of the AAV capsid with a library of peptide sequences bioinformatically designed based on the conservation level of each amino acid position in natural AAV serotypes and variants to generate a "loop swap" library; vi) random amino acid substitutions at degenerate positions between AAV serotypes to generate a library of ancestral variants (Santiago-Ortiz et al., 2015); and combinations of such techniques.

[0098] DNA shuffling produces chimeras that combine the properties of their parents in a unique and often beneficial way; however, some may not be able to be packaged, which can actually reduce the diversity of the library. The diversity concentration of the library in one or more specific regions of the capsid is achieved by peptide insertion techniques such as, but not limited to, iii-iv) above. In techniques such as v) above, the diversity of the library is also concentrated into one or more specific regions of the capsid, and such concentration is directed to multiple hypervariable regions located on the surface-exposed loops of the AAV capsid. Although many techniques produce variant capsids with only a small portion of capsid mutations, these techniques can be paired with additional mutagenesis strategies to modify the entire capsid.

[0099] Once an AAV library or multiple AAV libraries are generated, the virus is then packaged so that each AAV particle is composed of a mutant capsid surrounding the cap gene encoding that capsid and purified. The variants of the library are then subjected to in vitro and / or in vivo selective pressure techniques known and readily available to those skilled in the art of AAV. See, for example, Maheshri, N. et al., Nature Biotechnology 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., Molecular Therapy 13, 77-87 (2006); and Bell, P. et al., Hum. Gene. Ther. 22, 985-997 (2011). For example, but not by way of limitation, AAV variants may be selected using: i) affinity columns, where elution of different fractions yields variants with altered binding properties; ii) primary cells - isolated from tissue samples or immortalized cell lines that mimic the behavior of human cells - which produce AAV variants with improved efficiency and / or tissue specificity; iii) animal models - which mimic the clinical gene therapy environment - which produce AAV variants that have successfully infected target tissues; iv) human xenograft models, which produce AAV variants that have infected transplanted human cells; and / or combinations of selection techniques thereof.

[0100] Once the viruses are selected, they can be recovered by known techniques, such as but not limited to adenovirus-mediated replication, PCR amplification, next generation sequencing and cloning, etc. Viral clones are then enriched by repeated rounds of selection techniques, and AAV DNA is isolated to recover the selected related variant cap gene. Such selected variants can be subjected to further modification or mutation, and thus serve as a new starting point for further selection steps to iteratively increase AAV virus fitness. However, in some cases, successful capsids have been produced without additional mutations.

[0101] The AAV variants disclosed herein are generated at least in part by using an in vivo directed evolution method, such as the techniques described above, which involves screening using primate myocardial and skeletal muscles after intravenous administration. Therefore, the AAV variant capsid disclosed herein includes one or more modifications in the amino acid sequence that confer more effective primate myocyte transduction than the corresponding parent AAV capsid protein. As used herein, "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same wild-type or variant AAV serotype as the variant AAV capsid protein of the present invention, but does not include one or more amino acid sequence modifications of the variant AAV capsid protein of the present invention. In a specific embodiment, an AAV including a variant AAV capsid protein as described herein has a systemic tropism toward myocardial and / or multiple skeletal muscle groups throughout the body after systemic or tissue-targeted administration.

[0102] In some embodiments, the variant AAV capsid protein of the present invention comprises a heterologous peptide of about 5 amino acids to about 20 amino acids inserted into the GH loop or loop IV of the AAV capsid protein by covalent linkage relative to the corresponding parent AAV capsid protein. The "GH loop" or loop IV of the AAV capsid protein refers to the solvent-accessible portion of the GH loop or loop IV of the AAV capsid protein referred to in the art. For the GH loop / loop IV of the AAV capsid, see, e.g., van Vliet et al., (2006) Mol. Therap. 14:809; Padron et al., (2005) J. Virol. 79:5047; and Shen et al., (2007) Molecular Therapy 15:1955. Thus, for example, the insertion site can be within amino acids about 411-650 of the AAV VP1 capsid protein. For example, the insertion site can be within amino acids 571-612 of AAV1 VP1, within amino acids 570-611 of AAV2 VP1, within amino acids 571-612 of AAV3A VP1, within amino acids 571-612 of AAV3B VP1, within amino acids 569-610 of AAV4 VP1, within amino acids 560-601 of AAV5 VP1, within amino acids 571 to 612 of AAV6 VP1, within amino acids 572 to 613 of AAV7 VP1, within amino acids 573 to 614 of AAV8 VP1, within amino acids 571 to 612 of AAV9 VP1, or within amino acids 573 to 614 of AAV10 VP1, or the corresponding amino acids of any variants thereof. Based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, one of ordinary skill in the art will know that the insertion site of "amino acids corresponding to AAV2" will be in the capsid protein of any given AAV serotype. See also Figure 6 for an alignment of wild-type AAV SEQ ID NOs: 1-11, which provides amino acid positions between and across wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

[0103] In certain embodiments, the insertion site is a single insertion site between two adjacent amino acids between amino acids 570-614 of VP1 of any wild-type AAV serotype or AAV variant, e.g., 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 of VP1 of any AAV serotype or variant. For example, the insertion site can be between amino acids 580 and 581, between amino acids 581 and 582, between amino acids 583 and 584, between amino acids 584 and 585, between amino acids 585 and 586, between amino acids 586 and 587, between amino acids 587 and 588, between amino acids 588 and 589, or between amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, between amino acids 576 and 577, between amino acids 577 and 578, between amino acids 578 and 579, or between amino acids 579 and 580. The insertion site can be between amino acids 590 and 591, between amino acids 591 and 592, between amino acids 592 and 593, between amino acids 593 and 594, between amino acids 594 and 595, between amino acids 595 and 596, between amino acids 596 and 597, between amino acids 597 and 598, between amino acids 598 and 599, or between amino acids 599 and 600. For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 588 and 589 of AAV3A, between amino acids 588 and 589 of AAV3B, between amino acids 584 and 585 of AAV4, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

[0104] In some embodiments, the length of the peptide insertion disclosed herein is 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, the peptide insertion disclosed herein includes 1 to 4 spacer amino acids at the amino terminus (N-terminus) and / or carboxyl terminus (C-terminus) of any of the peptide insertions disclosed herein. Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In certain embodiments, the peptide insertion includes 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insertion includes 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus.

[0105] The peptide insertions disclosed herein have not been previously described and / or inserted into the AAV capsid. Without wishing to be bound by theory, the presence of any of the disclosed peptide insertions may function to reduce the affinity of the variant capsid for heparin sulfate, which may alter extracellular or intracellular steps in the viral transduction pathway. In addition, the peptide insertion motifs disclosed herein may confer enhanced transduction to muscle cells (e.g., cardiomyocytes) by adding cell surface receptor binding domains.

[0106] In some preferred embodiments, the inserted peptide comprises an amino acid sequence of any one of the following formulae.

[0107] In some aspects, the inserted peptide can be a peptide of Formula 1a having a length of 7 to 10 amino acids:

[0108] Y1Y2X1X2X3X4X5X6X7Y3

[0109] wherein each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

[0110] X1 is selected from the group consisting of Ala, Asn, Thr, Gly, Ser, Ala, Gln and Asp

[0111] X2 is selected from Lys, Asn, Thr, Ser, Ala and Gln

[0112] X3 is selected from the group consisting of Ile, Thr, Lys, Leu, Val, Asn, Asp and Arg

[0113] X4 is selected from the group consisting of Gln, Thr, Ile, Lys, Val, Ser and Tyr

[0114] X5 is selected from Arg, Asn, Gly, Lys, Leu, Thr, Ala, Ser and Gln

[0115] X6 is selected from Thr, Lys, Val, Gly, Ser, Ala, Arg and Pro

[0116] X7 is selected from the group consisting of Asp, Thr, Asn, He, Ala and Ser.

[0117] In certain embodiments, the insertion peptide of Formula 1a 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: 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 insertion peptide of Formula 1a does not include an amino acid sequence selected from the group consisting of NKTTNKD (SEQ ID NO: 14), QADTTKN (SEQ ID NO: 23), TNRTSPD (SEQ ID NO: 24), and NQDYTKT (SEQ ID NO: 22).

[0118] In other aspects, the inserted peptide can be a peptide of Formula 1b having a length of 7 to 10 amino acids:

[0119] Y1Y2X1X2X3X4X5X6X7Y3

[0120] wherein each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

[0121] X1 is selected from Thr and Asn

[0122] X2 is selected from Asn and Lys

[0123] X3 is selected from Lys, Ile and Thr

[0124] X4 is selected from Ile, Gln and Thr

[0125] X5 is selected from Gly, Arg and Asn

[0126] X6 is selected from Val, Thr and Lys

[0127] X7 is selected from Thr and Asp

[0128] In certain embodiments, the insertion peptide of Formula 1b comprises an amino acid sequence selected from the group consisting of: NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), and TNKIGVT (SEQ ID NO: 15). In other embodiments, the insertion peptide of Formula 1a does not comprise the amino acid sequence NKTTNKD (SEQ ID NO: 14).

[0129] In other aspects, the inserted peptide can be a peptide of Formula 1c having a length of 7 to 10 amino acids:

[0130] Y1Y2X1X2X3X4X5X6X7Y3

[0131] wherein each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

[0132] X1 is selected from Thr and Asn

[0133] X2 is selected from Asn and Lys

[0134] X3 is selected from Lys and Ile

[0135] X4 is selected from Ile and Gln

[0136] X5 is selected from Gly and Arg

[0137] X6 is selected from Val and Thr

[0138] X7 is selected from Thr and Asp

[0139] In certain embodiments, the insertion peptide of Formula 1c comprises an amino acid sequence selected from the group consisting of: NKIQRTD (SEQ ID NO: 13) and TNKIGVT (SEQ ID NO: 15).

[0140] In other aspects, the inserted peptide can be a peptide of 7 to 10 amino acids in length of Formula 1d:

[0141] Y1Y2X1X2X3X4X5X6X7Y3

[0142] wherein each of Y1-Y3, if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

[0143] X1 is selected from Asn and Thr

[0144] X2 is selected from Asn and Lys

[0145] X3 is selected from Lys and Thr

[0146] X4 is selected from Ile and Thr

[0147] X5 is selected from Gly, Lys and Thr

[0148] X6 is selected from Lys, Arg and Val

[0149] X7 is selected from Asp, Thr and Asn

[0150] In certain embodiments, the insertion peptide of Formula 1d comprises the amino acid sequence TNKIGVT (SEQ ID NO: 15).

[0151] In other embodiments, the insertion peptide comprises an amino acid sequence selected from the group consisting of: 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 the group consisting of: NKIQRTD (SEQ ID NO: 13) and TNKIGVT (SEQ ID NO: 15).

[0152] In some embodiments, the insertion peptide 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: 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).

[0153] In other preferred embodiments, the inserted peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl terminus of 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: 20), NKISAKD (SEQ ID NO: 21), NQDYTKT (SEQ ID NO: 22), QADTTKN (SEQ ID NO: 23), TNNRTSPD (SEQ ID NO: 24), SNTTQKT (SEQ ID NO: 25), and ASDSTKA (SEQ ID NO: 26). In certain such embodiments, 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).

[0154] In some embodiments, the variant AAV capsid proteins of the invention do not comprise any other amino acid sequence modifications other than a peptide insertion of about 5 amino acids to about 20 amino acids in the GH loop or loop IV. For example, in some embodiments, the variant AAV capsid protein of the invention comprises a peptide insertion comprising 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: 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), NKISAKD (SEQ ID NO: 30), NQDYTKT (SEQ ID NO: 31), QADTTKN (SEQ ID NO: 32), TNRTSPD (SEQ ID NO: 33), SNTTQKT (SEQ ID NO: 34), ASDSTKA (SEQ ID NO: 35), LANKIQRTDA (SEQ ID NO: 36), LANKTTNKDA (SEQ ID NO: 37), LATNKIGVTA (SEQ ID NO: 38), 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), and the variant AAV capsid does not contain any other amino acid substitutions, insertions or deletions (i.e., the variant AAV capsid protein includes the insertion and is otherwise identical to the corresponding AAV capsid protein). In other words, the variant AAV capsid protein comprising the insertion is otherwise identical to the parent AAV capsid protein into which the peptide has been inserted.As another example, the variant AAV capsid protein of the present invention comprises a peptide insertion, wherein the peptide insertion 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: 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), 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), LADNTWTRSA (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: 35) NO:42), wherein the peptide insertion is located between amino acids 587 and 588 of VP1 of AAV2 capsid; between amino acids 588 and 589 of VP1 of AAV3A, AAV3B, AAV9 or AAV10; between amino acids 589 and 590 of VP1 of AAV7; between amino acids 590 and 591 of VP1 of AAV1, AAV6 or AAV8; between amino acids 584 to 585 of VP1 of AAV4 or between amino acids 575 and 576 of AAV5, wherein the variant AAV capsid protein sequence is otherwise identical to the corresponding parental AAV capsid protein sequence, such as any one of SEQ ID NOs:1-12.

[0155] In other embodiments, the variant AAV capsid proteins of the invention include about 1 to about 100 amino acid substitutions or deletions, such as 1 to about 5, about 2 to about 4, about 2 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25-50, about 50-100 amino acid substitutions or deletions, compared to the parent AAV capsid protein, in addition to including, for example, a peptide insertion in the GH loop as disclosed herein or as known in the art. Thus, in some embodiments, the variant capsid proteins of the invention include a sequence identity with a corresponding parent AAV capsid having 85% or more, 90% or more, 95% or more, or 98% or more, for example, or 99% identity, such as the wild-type capsid proteins shown in SEQ ID NOs: 1-12.

[0156] In another embodiment, the one or more amino acid substitutions are at one or more amino acid residues 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 as numbered before peptide insertion, or at the corresponding one or more amino acid residues of another AAV capsid protein. In some such embodiments, the one or more amino acid substitutions are selected from the group consisting of 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, K649E, R651H, W694C, I698V, V708I, and L735Q of the AAV2 VP1 capsid protein as numbered prior to peptide insertion, or the corresponding one or more amino acid residues of another AAV capsid protein.

[0157] In a preferred embodiment, a variant AAV capsid protein is provided, comprising a) an inserted peptide in the GH-loop of the capsid protein, wherein the peptide insertion comprises an amino acid sequence selected from the group consisting of NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and TNKIGVT (SEQ ID NO: 15), and b) an amino acid sequence identical to that of AAV2 (SEQ ID NO: 16). NO: 2), or a corresponding substitution of another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids do not naturally occur at the corresponding position: 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, K649E, R651H, W694C, I698V, V708I, L735Q, and combinations thereof. In some embodiments, the 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, N312K+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, AAV3B, AAV9 or AAV10 capsid; between amino acids 589 and 590 of the AAV7 capsid; between amino acids 590 to 591 of the AAV1, AAV6 or AAV8 capsid; between amino acids 584 and 585 of the AAV4 capsid or between amino acids 575 and 576 of the AAV5 capsid.

[0158] In particularly preferred embodiments, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence NKIQRTD (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting 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 V708I amino acid substitution at amino acid sequence residue 708 relative to the AAV2 capsid (SEQ ID NO: 2), and optionally, further comprises an A593E and / or S109T and / or T330A and / or R588M substitution relative to AAV2 or the corresponding substitution in another AAV parental serotype, wherein the substituted amino acid or amino acids do not naturally occur at the corresponding position. In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence NKIQRTD (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting 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 an A35P amino acid substitution at amino acid sequence residue 35 relative to the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parental AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or is 100% identical to the following amino acid sequence:

[0159]

[0160]

[0161] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence NKIQRTD (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKIQRTDA (SEQ ID NO: 27) between amino acids 587 and 588 of the AAV2 capsid protein or at corresponding positions in the capsid protein of another AAV serotype, and comprises a peptide insertion that is identical to the amino acid sequence NKIQRTD (SEQ ID NO: 13) of the AAV2 capsid protein or at corresponding positions in the capsid protein of another AAV serotype. NO:2) or the corresponding substitution of another AAV parent serotype, and optionally further comprises (i) N449D, N551S, I698V and L735Q or (ii) N449D, N551S, I698V, L735Q and V708I amino acid substitutions compared to the amino acid sequence of AAV2 capsid or the corresponding substitution of another AAV parent serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98% or more amino acid sequence identity to the entire length of the amino acid sequence shown in SEQ ID NO:2. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence or is 100% identical to the following amino acid sequence:

[0162]

[0163]

[0164] In another embodiment, a variant AAV capsid protein is provided, comprising a) a peptide insertion 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, wherein the peptide insertion comprises a peptide selected from the group consisting of NKIQRTD (SEQ ID NO: 13) and LANKIQRTDA (SEQ ID NO: 14). NO:27), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally is otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence NKIQRTD (SEQ ID NO: 13) or the amino acid sequence LANKIQRTDA (SEQ ID NO: 27) comprising between amino acids 587 and 588 of the AAV2 capsid, and b) a valine to isoleucine amino acid substitution at amino acid 708 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.

[0165] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising the amino acid sequence NKIQRTD (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKIQRTDA (SEQ ID NO: 27) between amino acids 587 and 588 of the AAV2 capsid, and b) a valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, and is otherwise identical to the amino acid sequence SEQ ID NO: 2.

[0166] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising the amino acid sequence NKIQRTD (SEQ ID NO: 13) or comprising, consisting essentially of, or consisting 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 SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or is 100% identical to the following amino acid sequence:

[0167]

[0168] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence TNKIGVT (SEQ ID NO: 15), or comprising, consisting essentially of, or consisting 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 at corresponding positions in the capsid protein of another AAV serotype, and comprising a V708I amino acid substitution, or a corresponding substitution in another AAV parent serotype, compared to the amino acid sequence of AAV2, and optionally further comprising a N449K and / or G222S substitution, or a corresponding substitution in the capsid protein of another AAV parent serotype, relative to AAV2, wherein the substituted amino acid does not naturally occur at the corresponding position. In another preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence TNKIGVT (SEQ ID NO: 15) or the amino acid sequence LATNKIGVTA (SEQ ID NO: 29) or LATNKIGVTS (SEQ ID NO: 37) comprising between amino acids 587 and 588 of the AAV2 capsid or the corresponding position in the capsid protein of another AAV serotype, and comprises N312K, N449D, N551S, I698V and L735Q and optionally V708I amino acid substitutions compared to the amino acid sequence of AAV2 or one or more corresponding substitutions in another AAV parent serotype, wherein the substituted one or more amino acids do not naturally occur at the corresponding position. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98% or more amino acid sequence identity to the entire length of the amino acid sequence shown in SEQ ID NO: 2. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or is 100% identical to the following amino acid sequence:

[0169]

[0170] In another embodiment, a variant AAV capsid protein is provided, comprising a) a peptide insertion 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, wherein the peptide insertion comprises TNKIGVT (SEQ ID NO: 15), LATNKIGVTA (SEQ ID NO: 29), and LATNKIGVTS (SEQ ID NO: 30). NO:37), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5. In preferred embodiments, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence TNKIGVT (SEQ ID NO: 15) or comprising, consisting essentially of, or consisting 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 comprising a valine to isoleucine amino acid substitution 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 about 85%, at least about 90%, at least about 95%, at least about 98% or more amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO: 2.

[0171] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence TNKIGVT (SEQ ID NO: 15) or comprising, consisting essentially of, or consisting 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 valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, and is otherwise identical to the amino acid sequence SEQ ID NO: 2.

[0172] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion comprising the amino acid sequence TNKIGVT (SEQ ID NO: 15) or comprising, consisting essentially of, or consisting 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 SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or is 100% identical to the following amino acid sequence:

[0173]

[0174] In another preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising 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 comprising a V708I amino acid substitution at residue 708 relative to the AAV2 capsid amino acid sequence (SEQ ID NO: 2), or a corresponding substitution of another AAV parent serotype, and optionally, further comprising an S109T and / or W694C and / or W606C amino acid substitution or a corresponding substitution of another AAV parent serotype compared to the amino acid sequence of AAV2, wherein one or more of the substituted amino acids does not naturally occur at the corresponding position. In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO: 28) between amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, and further comprising an I698V amino acid substitution of residue 698 or the corresponding residue of another AAV capsid relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2). The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98% or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2 or the corresponding parental AAV capsid. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or is 100% identical to the following amino acid sequence:

[0175]

[0176] In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid protein or at corresponding positions in the capsid protein of another AAV serotype, and comprises an N312K amino acid substitution compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or a corresponding substitution of another AAV parent serotype, and optionally further comprises N449D, N551S, I698V and L735Q and optionally V708I amino acid substitutions compared to the amino acid sequence of the AAV2 capsid or a corresponding substitution in another AAV parent serotype. The variant AAV capsid can have at least about 85%, at least about 90%, at least about 95%, at least about 98% or more amino acid sequence identity to the entire length of the amino acid sequence set forth in SEQ ID NO: 2. In particularly preferred embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or is 100% identical to the following amino acid sequence:

[0177]

[0178] In another embodiment, a variant AAV capsid protein is provided, comprising a) a peptide insertion 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, wherein the peptide insertion comprises a peptide selected from the group consisting of NKTTNKD (SEQ ID NO: 14) and LANKTTNKDA (SEQ ID NO: 15). NO:28), and b) a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5, and optionally is otherwise identical to any one of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the variant capsid protein comprises a) a peptide insertion comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid, and b) a valine to isoleucine amino acid substitution at amino acid 708 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.

[0179] In yet another embodiment, the variant capsid protein comprises a) a peptide insertion of the amino acid sequence NKTTNKD (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTINKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid, and b) a valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, and is otherwise identical to the amino acid sequence SEQ ID NO: 2.

[0180] In another embodiment, the variant capsid comprises a peptide insertion comprising the amino acid sequence NKTTNKD (SEQ ID NO: 14) or comprising, consisting essentially of, or consisting of the amino acid sequence LANKTINKDA (SEQ ID NO: 28) between amino acids 587 and 588 of the AAV2 capsid, and is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0181]

[0182] In other embodiments, a variant AAV capsid protein is provided, comprising a) a peptide insertion in the GH loop of the capsid protein, wherein the peptide insertion comprises an amino acid sequence selected from the group consisting of 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) an amino acid sequence that is identical to that of AAV2 (SEQ ID NO: 27). NO: 2) or a corresponding substitution in another AAV parent serotype (i.e., other than AAV2): wherein the substituted one or more amino acids do not naturally occur at the corresponding position: A35P, S109T, P195L, D213N, G222S, V229I, N312K, A319T, T330A, A333S, E347K, P363L, A427D, V447F, N449D, N449K, G453R, A490T, KS27Q, N551S, A581T, Y585S, R588M, A593E, W606C, K649E, R651H, W694C, I698V, V708I, L735Q, and combinations 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 combinations thereof. In some preferred embodiments, the one or more amino acid substitutions include at least V708I and / or P363L amino acid substitutions or corresponding substitutions in another AAV parent serotype. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or a corresponding position in the capsid protein of another AAV serotype.

[0183] In some embodiments, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting 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 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitutions in another AAV parental serotype, wherein one or more of the substituted amino acids does not naturally occur at the corresponding position: V708I, V708I+S109T, R651H, A319T+P195L, P363L, P363L+V708I. In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) comprising amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, and (ii) a V708I substitution relative to the amino acid sequence of AAV2 (SEQ ID NO: 2) or the corresponding residue of another AAV capsid and comprising 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 insertion comprising, consisting essentially of, or consisting of the amino acid sequence GNLTKGN (SEQ ID NO: 16) or the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) comprising, consisting essentially of, or consisting 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 relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid and comprising 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 NO2 or the corresponding parental AAV capsid protein sequence.In other embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or the amino acid sequence LAGNLTKGNA (SEQ ID NO: 30) comprising amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, and (ii) a R651H substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid and comprising 2 to 5, 5 to 10 or 10 to 15 amino acid substitutions or 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 insertion comprising the amino acid sequence GNLTKGN (SEQ ID NO: 16) or comprising, consisting essentially of, or consisting 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 set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or is 100% identical to the following amino acid sequence:.

[0184]

[0185]

[0186] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising the amino acid sequence NTVKLST (SEQ ID NO: 17) or the amino acid sequence LANTVKLSTA (SEQ ID NO: 31) comprising amino acids 587 and 588 of VP1 of AAV2 or the corresponding amino acids of another AAV capsid, and (ii) a V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or a corresponding substitution in another AAV parental serotype, wherein one or more of the substituted amino acids does not naturally occur at 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 a corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insertion comprising the amino acid sequence NTVKLST (SEQ ID NO: 17) or comprising, consisting essentially of, or consisting 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 set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0187]

[0188]

[0189] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence SNTVKAI (SEQ ID NO: 18) or the amino acid sequence LASNTVKAIA (SEQ ID NO: 32) comprising, consisting essentially of, or consisting of the amino acid sequence LASNTVKAIA (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 V708I amino acid substitution relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2), or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising the amino acid sequence SNTVKAI (SEQ ID NO: 18) or comprising, consisting essentially of, or consisting 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 set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0190]

[0191]

[0192] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence ASNITKA (SEQ ID NO: 19) or the amino acid sequence LAASNITKAA (SEQ ID NO: 33) comprising, consisting essentially of, or consisting of amino acids 587 and 588 of VP1 of AAV2, or the corresponding amino acids of another AAV capsid, and (ii) a V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising the amino acid sequence ASNITKA (SEQ ID NO: 19) or comprising, consisting essentially of, or consisting 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 set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0193]

[0194]

[0195] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence DNTVTRS (SEQ ID NO: 20) or the amino acid sequence LADNTVTRSA (SEQ ID NO: 34) comprising, consisting essentially of, or consisting 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 V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising, consisting essentially of, or consisting of the amino acid sequence DNTVTRS (SEQ ID NO: 20) or the amino acid sequence LADNTVTRSA (SEQ ID NO: 34) comprising, consisting essentially of, or consisting 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) an I698V amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2), or a corresponding substitution in another AAV parental serotype, wherein the substituted amino acid does not naturally occur at 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 a corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insertion comprising the amino acid sequence DNTVTRS (SEQ ID NO: 20) or comprising, consisting essentially of, or consisting 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 set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0196]

[0197] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence NKISAKD (SEQ ID NO: 21) or the amino acid sequence LANKISAKDA (SEQ ID NO: 35) comprising, consisting essentially of, or consisting 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 V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion. The peptide insertion comprises the amino acid sequence NKISAKD (SEQ ID NO: 21) or comprises, consists essentially of, 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 set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0198]

[0199] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence NQDYTKT (SEQ ID NO: 22) or the amino acid sequence LANQDYTKTA (SEQ ID NO: 36) comprising, consisting essentially of, or consisting 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 V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising, consisting essentially of, or consisting of the amino acid sequence NQDYTKT (SEQ ID NO: 22) or the amino acid sequence LANQDYTKTA (SEQ ID NO: 36) comprising, consisting essentially of, or consisting 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 I698V amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2), or a corresponding substitution in another AAV parental serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally occur at 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 a corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insertion comprising the amino acid sequence NQDYTKT (SEQ ID NO: 22) or comprising, consisting essentially of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 36) between amino acids 587 and 588 of the AAV2 capsid, and is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0200]

[0201] In some embodiments, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence QADTTKN (SEQ ID NO: 23) or comprising, consisting essentially of, or consisting 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 relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitutions in another AAV parental serotype, wherein one or more of the substituted amino acids does not naturally occur at the corresponding position: V708I, D213N, P363L, G453R. In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence QADTTKN (SEQ ID NO: 23) or the amino acid sequence LAQADTTKNA (SEQ ID NO: 39) comprising, consisting essentially of, or consisting 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 (ii) a V708I substitution relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising, consisting essentially of, or consisting of the amino acid sequence QADTTKN (SEQ ID NO: 23) or the amino acid sequence LAQADTTKNA (SEQ ID NO: 39) comprising, consisting essentially of, or consisting 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 (ii) a P363L substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding residue of another AAV capsid and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising the amino acid sequence QADTTKN (SEQ ID NO: 23) or comprising, consisting essentially of, or consisting 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 (ii) a D213N substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising the amino acid sequence QADTTKN (SEQ ID NO: 23) or comprising, consisting essentially of, or consisting 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. NO: 23) or comprising, consisting essentially of, or consisting 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 (ii) a G453R substitution relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 insertion comprising the amino acid sequence QADTIXN (SEQ ID NO: 23) or comprising 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. NO: 39), consisting essentially of or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO: 39) between amino acids 587 and 588 of the AAV2 capsid, and 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 about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or is 100% identical to the following amino acid sequence:.

[0202]

[0203] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence TNRTSPD (SEQ ID NO: 24) or the amino acid sequence LATNRTSPDA (SEQ ID NO: 40) comprising, consisting essentially of, or consisting 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 V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or the corresponding substitution in another AAV parental serotype, and comprising 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions or 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 about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or is 100% identical to the following amino acid sequence:

[0204]

[0205] In some embodiments, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence SNTTQKT (SEQ ID NO: 25) or the amino acid sequence LASNTTQKTA (SEQ ID NO: 41) comprising 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 about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or is 100% identical to the following amino acid sequence:

[0206]

[0207] In some embodiments, the variant AAV capsid comprises (i) a peptide insertion comprising, consisting essentially of, or consisting of the amino acid sequence ASDSTKA (SEQ ID NO: 26) or the amino acid sequence LAASDSTKAA (SEQ ID NO: 42) comprising, consisting essentially of, or consisting 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 V708I amino acid substitution relative to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2), or a corresponding substitution in another AAV parental serotype, wherein one or more of the substituted amino acids does not naturally occur at 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 a corresponding parental AAV capsid protein sequence. In another embodiment, the variant capsid comprises a peptide insertion comprising the amino acid sequence ASDSTKA (SEQ ID NO: 26) or comprising, consisting essentially of, or consisting of the amino acid sequence LAASDSTKAA (SEQ ID NO: 42) between amino acids 587 and 588 of the AAV2 capsid, and is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to, or is 100% identical to, the following amino acid sequence:

[0208]

[0209] In several aspects, a variant AAV capsid protein is provided that comprises one or more amino acid substitutions relative to a corresponding parental AAV capsid protein, wherein when present in an AAV virion, the variant capsid protein confers increased infectivity to muscle cells (e.g., skeletal or cardiac myocytes) compared to the infectivity of the AAV virion comprising the corresponding parental AAV capsid protein to muscle cells.

[0210] In some embodiments, the variant AAV capsid protein comprises an amino acid substitution at amino acid 363 compared to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or at a corresponding position in another AAV parent serotype (i.e., other than AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or greater amino acid sequence identity over the entire 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 AAV2 capsid (SEQ ID NO: 2). In some preferred embodiments, the variant AAV capsid protein comprises a P363L amino acid substitution compared to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2), AAV3A capsid (SEQ ID NO: 3) or AAV3B capsid (SEQ ID NO: 4); or a P364L amino acid substitution compared to the amino acid sequence of AAV1 capsid (SEQ ID NO: 1) or AAV6 capsid (SEQ ID NO: 7); or a P354L amino acid substitution compared to the amino acid sequence of AAV4 capsid (SEQ ID NO: 5) or AAV5 capsid (SEQ ID NO: 6); or a P365L amino acid substitution compared to the amino acid sequence of AAV7 capsid (SEQ ID NO: 8) or AAV9 capsid (SEQ ID NO: 10);or a P366L amino acid substitution compared to the amino acid sequence of AAV8 capsid (SEQ ID NO: 9) or AAV10 capsid (SEQ ID NO: 11). In some preferred embodiments, the variant capsid protein comprises a P363L substitution compared to the amino acid sequence of SEQ ID NO: 2, or a corresponding substitution compared to any one of SEQ ID NOs: 1 and 3-12, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO: 2 or any one of SEQ ID NOs: 1 and 3-12. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence comprising a P363L amino acid substitution compared to the amino acid sequence shown in SEQ ID NO: 2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO: 2. In related embodiments, the variant capsid protein comprises a P363L amino acid substitution compared to the amino acid sequence of SEQ ID NO: 2 or a corresponding substitution in another AAV parent serotype (i.e., other than AAV2), wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO: 2 or compared to the amino acid sequence of a capsid protein in another AAV parent serotype. In another preferred embodiment, the variant capsid comprises a P363L amino acid substitution and further comprises one or more E347K and / or V708I amino acid substitutions compared to the amino acid sequence of SEQ ID NO: 2, or a corresponding substitution in a capsid from another AAV AAV serotype (i.e., other than AAV2). In another preferred embodiment, the variant capsid comprises the P363L amino acid substitution compared to the amino acid sequence of SEQ ID NO: 2 or a corresponding substitution in a capsid from another AAV parent serotype, and further comprises a peptide insertion, preferably between amino acids 587 and 588 of VP1 of AAV2; between amino acids 588 and 589 of AAV3A, AAV3B, AAV9 or AAV10; between amino acids 589 and 590 of VP1 of AAV7; between amino acids 590 to 591 of VP1 of AAV1, AAV6 or AAV8; between amino acids 584 and 585 of VP1 of AAV4;or between amino acids 575 and 576 of AAV5, wherein the peptide insertion preferably 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: 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), LANKIQRTDA (SEQ ID NO: 30), LANKTTNKDA (SEQ ID NO: 31), LATNKIGVTA (SEQ ID NO: 32), 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), more preferably selected from GNLTKGN (SEQ ID NO: 42) NO: 16), LAGNLTKGNA (SEQ ID NO: 30), QADTTKN (SEQ ID NO: 23) and LAQADTTKNA (SEQ ID NO:39), and optionally includes 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 a corresponding parent AAV capsid protein sequence. ;

[0211] In other embodiments, the variant AAV capsid protein comprises an amino acid substitution at amino acid 593 compared to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or a corresponding position in another AAV parent serotype (i.e., other than AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 593 compared to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or greater amino acid sequence identity over the entire length of the amino acid sequence set forth in SEQ ID NO 2, or is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the variant capsid protein comprises a glycine to glutamic acid amino acid substitution at amino acid 594, at amino acid 583 of AAV5, or at amino acid 596 of AAV8 or AAV10, or an arginine to glutamic acid amino acid substitution at amino acid 594 of AAV3B, or an aspartic acid to glutamic acid amino acid substitution at amino acid 592 of AAV4, or a glutamine to glutamic acid amino acid substitution at position 595 of AAV7, compared to the amino acid sequence of AAV1, AAV3A, AAV6, or AAV9. In other embodiments, the variant capsid protein comprises an A593E amino acid substitution compared to the amino acid sequence of AAV2, and does not comprise one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2: I19V, V369A, K26R, N215D, G355S, V46A, and S196P. In related embodiments, the variant capsid protein comprises A593E and V708I amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity over the entire length of the amino acid sequence set forth in SEQ ID NO 2, or is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In related embodiments, the variant capsid protein comprises A593E and S109T amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity over the entire length of the amino acid sequence set forth in SEQ ID NO 2, or is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In related embodiments, the variant capsid protein comprises A593E, V708I, and S109T amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or is otherwise identical to SEQ ID NO: 2.In other embodiments, the variant capsid comprises A593E, V708I, and N551S amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In other embodiments, the variant capsid protein comprises A593E, V708I, and K649E amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2. In other embodiments, the variant capsid protein comprises A593E, V708I, S109T, and K527Q amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity over the entire length of the amino acid sequence set forth in SEQ ID NO: 2, or is otherwise identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0212] In other embodiments, the variant AAV capsid protein comprises an amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) or at a corresponding position in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally occur at the corresponding position. Preferably, the rAAV virion does not comprise a proline to serine substitution at amino acid 250 compared to AAV2 or at a corresponding amino acid in another AAV parent serotype. In some embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2 capsid (SEQ ID NO: 2) and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or greater amino acid sequence identity over the entire 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 includes a valine to isoleucine (V708I) substitution at amino acid 708 compared to the amino acid sequence of the AAV2 capsid and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or greater amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO: 2, and is otherwise identical to the amino acid sequence of SEQ ID NO: 2, wherein the variant capsid protein does not include the P250S amino acid substitution. In some embodiments, the variant capsid protein comprises a valine to isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid 697 of AAV5. In related embodiments, the variant capsid protein comprises a V708I amino acid substitution 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 include a P250S amino acid substitution. In other embodiments, the variant capsid protein comprises a V708I amino acid substitution and further comprises an A333S and / or S721L amino acid substitution compared to the amino acid sequence of AAV2.In other related embodiments, the variant capsid includes a V708I amino acid substitution and further includes an A333S and / or S721L amino acid substitution compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or greater amino acid sequence identity over the entire 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.

[0213] In other embodiments, the variant AAV capsid protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 98% identical to a wild-type AAV capsid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12, and further comprises i) one or more amino acid substitutions selected from the group consisting of A35P, D213N, A319T+P195L, P363L, P363L+V708I, G453R, R651H, I698V, V708I, V708I+A593, V708I+S 109T, V708I+T330A, V708I+R588M, V708I+W694C, V708I+W606C, V708I+N449K, V708I+ G222S, N312K+N449D+N551S+I698V+L735Q, N312K+N449D+N551S+I698V+V708I+L735Q,and / or (ii) a peptide insertion 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), NKISAKD (SEQ ID NO: 31), NQDYTKT (SEQ ID NO: 32), QADTTKN (SEQ ID NO: 33), TNRTSPD (SEQ ID NO: 34), SNTTQKT (SEQ ID NO: 35), ASDSTKA (SEQ ID NO: 36), LANKIQRTDA (SEQ ID NO: 37), LANKTTNKDA (SEQ ID NO: 38), LATNKIGVTA (SEQ ID NO: 39), LAGNLTKGNA (SEQ ID NO: 40), 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 embodiments, the variant AAV capsid comprises the specified one or more amino acid substitutions and / or peptide insertions and is otherwise identical to a sequence selected from the group consisting of: SEQ ID NOs: 1-12.

[0214] In some embodiments, variant AAV capsid proteins are ancestral capsid proteins comprising one or more peptide insertions and / or amino acid substitutions as described herein. An ancestral capsid protein refers to an evolutionary ancestor of a capsid protein found in nature today, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12, AAV13, which are generated in silico by random amino acid substitutions at degenerate positions between AAV capsid proteins found in nature today.

[0215] In other embodiments, the variant AAV capsid protein is a chimera comprising amino acids 130-725 of AAV5 capsid (SEQ ID NO: 6) or an amino acid sequence at least 90%, at least 95%, or at least 98% identical thereto.

[0216] In some aspects, the variant AAV capsid protein is a chimera comprising (i) amino acids 1-129 of AAV6 (SEQ ID NO: 7), or an amino acid sequence at least 90%, at least 95%, or at least 98% identical thereto, and (ii) amino acids 130-725 of AAV5 (SEQ ID NO: 6), or an amino acid sequence at least 90%, at least 95%, or at least 98% identical thereto, and further comprising V229I, A490T, and A581T and optionally V447F or Y585S amino acid substitutions relative to the sequence of AAV5 (SEQ ID NO: 6). In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or 100% identical to the following amino acid sequence:

[0217]

[0218] In other aspects, the variant AAV capsid protein is a chimera comprising (i) amino acids 1-61 of AAV2 (SEQ ID NO: 2) or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity thereto; (ii) amino acids 62-129 of AAV6 (SEQ ID NO: 7) or an amino acid sequence having at least 90%, at least 95%, or at least 98% identity thereto and (iii) amino acids 130-725 of AAV5 (SEQ ID NO: 6) and further comprising V229I, A490T, and A581T amino acid substitutions relative to the sequence of AAV5 (SEQ ID NO: 6). In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity to the following amino acid sequence, or 100% identity to the following amino acid sequence:

[0219]

[0220]

[0221] The AAV variants disclosed herein are generated by using in vivo directed evolution, which involves screening using primate cardiac and skeletal muscles after intravenous administration. In some embodiments, when present in AAV virions, the variant capsid proteins disclosed herein confer increased transduction to muscle cells compared to transduction of muscle cells by AAV virions comprising corresponding parental AAV capsid proteins or wild-type AAV. For example, in some embodiments, when present in AAV virions, the variant capsid proteins disclosed herein confer more efficient transduction to primate muscle cells than AAV virions comprising corresponding parental AAV capsid proteins or wild-type AAV capsid proteins, such as muscle cells taking up more AAV virions comprising variant AAV capsid proteins of the present invention than AAV virions comprising parental AAV capsid proteins or wild-type AAV. In some such embodiments, the AAV variant virion or variant rAAV exhibits at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold or greater than 50-fold increased transduction of muscle cells compared to wild-type AAV virions or rAAVs comprising corresponding corresponding AAV capsid proteins. In preferred embodiments, the AAV variant virion or variant rAAV exhibits at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold or greater than 1000-fold increased transduction of muscle cells compared to wild-type AAV8 or AAV9 virions. In certain such embodiments, when present in AAV virions, the variant capsid proteins disclosed herein confer broader transduction of primate muscle cells than AAV virions comprising corresponding parental AAV capsid proteins or wild-type AAV capsid proteins. In other words, the variant AAV virions transduce cell types that are not transduced by the virions including the corresponding parental AAV capsid protein, so there are more cell types in the muscle than in the corresponding parental AAV virions. In some embodiments, the AAV variant virions preferentially transduce muscle cells, for example, compared with another muscle cell or non-muscle cell, the rAAV virions of the present invention specifically infect muscle cells by 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 50 times or more than 50 times. In some embodiments, the transduced muscle cells are cardiomyocytes (e.g., cardiomyocytes, cardiac fibroblasts, or cardiac progenitor cells). In some embodiments, the muscle cells are skeletal muscle cells (e.g., myoblasts, myotubes, or satellite cells). By any number of methods for measuring gene expression in the art, it is easy to assess the increase of muscle cell transduction in vitro or in vivo, such as increased transduction efficiency, more extensive transduction, more preferential transduction, etc.For example, AAV can be packaged with a genome comprising an expression cassette comprising a reporter gene, such as a fluorescent protein, under the control of a ubiquitous or tissue-specific promoter, and the extent of transduction is assessed by detecting the fluorescent protein, such as by fluorescence microscopy. As another example, AAV can be packaged with a genome comprising a barcoded nucleic acid sequence, and the extent of transduction is assessed by detecting the nucleic acid sequence, such as by PCR. As another example, AAV can be packaged with a genome comprising an expression cassette comprising a therapeutic gene for treating a muscle disease, and the extent of transduction is assessed by detecting the treatment of the muscle disease in a treated patient administered the AAV.

[0222] Diseases that may be treated using the variant rAAV vectors or viral particles and / or the methods 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, integrin deficiency, partitionin deficiency muscular dystrophy, and Walker-Warburgh syndrome), distal (including but not limited to Gowers-Laing, Miyoshi, and Nonaka), Emery-Dreifuss, facioscapulohumeral, limb girdle, myotonia, and dystrophia; myotonia congenita and myotonia congenita; myotubular myopathy; centronuclear myopathy; myofibrillary myopathy; desmin-related disease (desmin related; anemia; Andersen-Tawil syndrome; Nemaline myopathy; Brody disease; lysosomal storage disorders, such as alpha-mannosidosis, aspartylglucosaminuria, beta-mannosidosis, cystinosis, Farber disease, fucosidosis, Gaucher disease, galactosialidosis, gangliosidose (including but not limited to AB variant, activator deficiency, beta-galactosidase deficiency, Fabry disease, Sandhoff disease, and Schindle disease), glycogen storage (including but not limited to Andersen disease, Corey disease, Danon disease, Forbes disease, disease), glucose-6-phosphate deficiency, Hers disease, lactate dehydrogenase A deficiency, Pompe disease, Tarui disease, and glycogen storage disease.gierkedisease), infantile free sialic acid storage disease, lysosomal acid lipase deficiency, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidose (including but not limited to hyaluronidase deficiency, Hunter syndrome, Hurler syndrome, Hurler-Scheiesyndrome, Maroteaux-Lamy syndrome, Morquio syndrome, Sanfilippo syndrome, Scheie syndrome, and Sly syndrome), mucolipidosis (including but not limited to sialidosis, I-cell disease, mucolipidin 1 deficiency, 1 deficiency and Psuedy-Hurler Polydystrophy), multiple sulfase deficiency, Niemann-Pick disease, neuronal ceroid (including but not limited to Batten-Spielmeyer-Vogt disease, congenital cathepsin D deficiency, German / Serbian Late Infantile, Jansky-Bielschowsky disease, Kufs disease, late infantile type, late infantile variant Northern Epilepsy, Santavuori-Haltia disease, and Turkish late infantile), pyknodysostosis, Salla disease, saposin B deficiency, Tay-Sach's disease, and Wolman's diseasedisease; metabolic disorders, such as adenosine monophosphate deaminase deficiency, alkaptonuria, carnitine deficiency, carnitine palmitate transferase deficiency, Hartnup disorder, homocystinuria, maple syrup urine disease, myophosphorylase deficiency, phosphofuctokinase deficiency, phosphoglycerate kinase deficiency, phosphoglycerate mutase deficiency, phosphorylase deficiency, and Tangier disease; Friedreich's ataxia; ataxia telangiectasia; vitamin E deficiency ataxia; periodic paralysis, such as Gamstorp disease and hypokalemic periodic paralysis. paralysis; mitochondrial diseases such as Barth syndrome, Kearns-Sayre syndrome, mitochondrial myopathy, mitochondrial encephalopathy lactic acidosis and stroke-like episodes, myoclonic epilepsy with ragged-red fiber, and Pearson syndrome; familial hypertrophic cardiomyopathy, dilated cardiomyopathy, familial congenital heart diseases such as familial aortic valve disease and congenital heart disease with noncompacted left ventricle, familial arrhythmias such as Anderson cardiodysrhythmic periodic paralysis, atrial septal defect with AV conduction defect, Brugada syndrome, cardiac conduction defects, catecholaminergicpolymorphic ventriculartachycardia and congenital heart block; familial vascular diseases such as arterial tortuosity syndrome, cerebral autosomal dominant arteriopathy with cerebral cortical lesions and cerebral white matter lesions, cerebral concave dominant arteriopathy with cerebral cortical lesions and cerebral white matter lesions, familial aortic aneurysm, Marfan syndrome, Ehlers-Danlos syndrome, Beal congenital contractual arachnodactyly, Loeys-Dietz syndrome, and pseudoxanthoma elasticum; arrhythmic right ventricular cardiomyopathy; familial arrhythmic right ventricular dysplasia; Naxos disease; left ventricular noncompaction; familial atrial fibrillation; familial ventricular tachycardia; familial Wolff-Parkinson-White syndrome. syndrome; long QT syndrome; short QT syndrome; sick sinus syndrome; lipoproteinopathies such as abetalipoproteinemia and lipoprotein lipase deficiency, alpha-1-antitrypsin deficiency, factor VIII deficiency (hemophilia A) or factor IX deficiency (hemophilia B); thalassemia; fibrodysplasia ossificans progressive; laminopathies; Huntington disease; congenital myasthenic syndromes; Hutchinson-Gilford Progeria syndrome; Noonan syndrome; congenital fibre type disproportion myopathy; congenital fibrosis of the extraocular muscles; minicore myopathy; ripple myopathy; Schwartz-Jampel syndrome syndrome; tubular aggregate myopathymyopathy); and zebra myopathy examples of complex diseases include, but are not limited to, heart / cardiovascular disease (e.g., congestive heart failure, myocardial infarction, angina, coronary artery disease, ischemic heart disease, cardiomyopathy); cancer; diabetes; and infection. Examples of traumatic injury include, but are not limited to, viral infection of muscle, muscle tear; and muscle contusion. In a preferred embodiment, the variant rAAV vectors or viral particles and / or the methods disclosed herein are used to treat Fabry disease, Friedreich's ataxia, Duchenne muscular dystrophy, Becker muscular dystrophy, Pompe disease, muscle phosphorylase deficiency, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, or myotonic dystrophy

[0223] In another embodiment, the variant capsid disclosed herein comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product, such as but not limited to an interfering RNA, a long noncoding RNA, a short noncoding RNA, an antisense RNA, an aptamer, a polypeptide, a secreted antibody, a single-chain antibody, a V HH domains, soluble receptors, affibodies, knottins, DARPins, centurins, chaperones, site-specific nucleases that provide site-specific knockdown of gene function or modified site-specific nucleases that provide gene-specific activation of transcription.

[0224] The rAAV variant virions disclosed herein include heterologous nucleic acids including nucleotide sequences encoding gene products. 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 thereof. In some embodiments, the gene product is a long noncoding RNA. In some embodiments, the gene product is a short noncoding 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 V HH 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 centurin. In some embodiments, the gene product is a chaperone. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knockdown of gene function.

[0225] The uses of gene products include but are not limited to: increasing the level of a factor in a cell; increasing the level of a factor in an adjacent or distal cell by secreting a factor; decreasing the level of a factor in a cell; or decreasing the level of a factor in an adjacent or distal cell by secreting a factor. Gene products can be designed to: supplement the level of a defective gene product; reduce the level of a defective gene product; introduce a new supporting gene product; supplement the level of a supporting gene product; reduce the level of an obstructing gene product; or both reduce the level of an obstructing gene product and introduce or supplement the level of a supporting gene product.

[0226] The gene products delivered by the AAV variants of the invention can be used to alter the level of gene products or gene product activities directly or indirectly associated with muscle diseases and trauma. Skeletal, cardiac or smooth muscle transduced with the AAV variants of the invention can also be used as a biofactory for the production and secretion of therapeutic proteins to treat trans diseases in remote organs. Gene products that are genes directly or indirectly linked to genetic diseases include, for example, genes encoding any of the following gene products: myofascin; including mini myofascin and micro myofascin (DMD; e.g., GenBank Accession No. NP_003997.1; SEQ ID NO: 64); titin (TTN); titin cap (TCAP) α-sarcoglycan (SGCA), β-sarcoglycan (SGCB), γ-sarcoglycan (SGCG) or δ-sarcoglycan (SGCD); α-1-antitrypsin (A1-AT); myosin heavy chain 6 (MYH6); myosin heavy chain 7 (MYH7); myosin heavy chain 11 (MYH11); myosin light chain 2 (ML2); myosin light chain 3 (ML3); myosin light chain kinase 2 (MYLK2); myosin binding protein C (MYBPC 3); desmin (DES); dynamin 2 (DNM2); lamin alpha 2 (LAMA2); lamin A / C (LMNA); lamin B (LMNB); lamin B receptor (LBR); dysferlin (DYSF); emerin (EMD); insulin; coagulation factors, including but not limited to factor VIII and factor IX; erythropoietin (EPO); lipoprotein lipase (LPL); sarcoplasmic reticulum Ca2 ++-ATPase (SERCA2A), S100 calcium binding protein A1 (S100A1); myotubularin (MTM); DM1 protein kinase (DMPK; e.g., GenBank Accession No. NG_009784.1; SEQ ID NO: 65); glycogenoylase L (PYGL); muscle-associated glycogenoylase (PYGM; e.g., GenBank Accession No. NP_005600.1; SEQ ID NO: 66); glycogenoyl synthase 1 (GYS1); glycogenoyl synthase 2 (GYS2); αα-galactosidase A (GLA; e.g., GenBank Accession No. NP_000160.1; SEQ ID NO: 67); α-N-acetylgalactosaminidase (NAGA); acid α-glucosidase (GAA; e.g., GenBank Accession No. NP_000143.2; SEQ ID NO: 68); NO: 68), sphingomyelinase phosphodiesterase 1 (SMPD1); lysosomal acid lipase (LIPA); type I collagen alpha 1 chain (COL1A1); type I collagen alpha 2 chain (COL1A2); type III collagen alpha 1 chain (COL3A1); type V collagen alpha 1 chain (COL5A1); type V collagen alpha 2 chain (COL5A2); type VI collagen alpha 1 chain (COL6A1); type VI collagen alpha 2 chain (COL6A2); type VI collagen alpha 3 chain (COL6A3); procollagen-lysine 2-oxoglutarate 5-dioxygenase (PLOD1); lysosomal acid lipase (LIPA); comitatin (FXN; e.g., GenBank accession number NP_000135.2; SEQ ID NO: 69); myostatin (MSTN); β-N-acetylhexosaminidase A (HEXA); β-N-acetylhexosaminidase B (HEXB); β-glucocerebrosidase (GBA); adenosine monophosphate deaminase 1 (AMPD1); β-hemoglobin (HBB); iduronidase (IDUA); iduronic acid 2-sulfate (IDS); troponin 1 (TNNI3); troponin T2 (TNNT2); troponin C (TNNCl); tropomyosin 1 (TPM1); tropomyosin 3 (TPM3); N-acetyl-α-glucosaminidase (NAGLU); N-sulfoglucosamine sulfohydrolase (SGSH); heparan-α-glucosaminidase N-acetyltransferase (HGSNAT); integrin α7 (IGTA7); integrin α9 (IGTA9); glucosamine (N-acetyl)-6-sulfatase (GNS); galactosamine (N-acetyl)-6-sulfatase (GALNS); β-galactosidase (GLB1); β-glucuronidase (GUSB); hyaluronoglucosaminidase 1 (HYAL1); acid ceramidase (ASAH1);Galactosylcermidase (GALC); cathepsin A (CTSA); cathepsin D (CTSA); cathepsin K (CTSK); GM2 ganglioside activator (GM2A); arylsulfatase A (ARSA); arylsulfatase B (ARSB); formylglycine-generating enzyme (SUMF1); neuraminidase 1 (NEU1); N-acetylglucosamine-1-phosphotransferase α (GNPTA); N-acetylglucosamine-1-phosphotransferase β (GNPTB); N-acetylglucosamine-1-phosphotransferase γ (GNPTG); mucolipin-1 (mucolipin -1)(MCOLN1); NPC intracellular transporter 1 (NPC1); NPC intracellular transporter 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); DNAJ heat shock protein family 40 member C5 (DNAJC5); major facilitator containing eight superfamily domains (MFSD8); mannosidase alpha class 2B member 1 (MAN2B1); mannosidase beta (MANBA); aspartylglucosaminidase (AGA); α-L-fucoidase Glycosidase (FUCA1); cystinosin, lysosomal cysteine ​​transporter (CTNS); sialic acid transporter (sialin); solute carrier family 2 member 10 (SLC2A10); solute carrier family 17 member 5 (SLC17A5); solute carrier family 6 member 19 (SLC6A19); solute carrier family 22 member 5 (SLC22A5); solute carrier family 37 member 4 (SLC37A4); lysosomal associated membrane protein 2 (LAMP2); sodium voltage-gated channel alpha subunit 4 (SCN4A); sodium voltage-gated channel beta subunit 4 (SCN4B); sodium voltage-gated channel alpha subunit 5 (SCN5A); sodium voltage-gated Calcium voltage-gated channel alpha subunit 4 (SCN4A); calcium voltage-gated channel subunit alpha 1c (CACNA1C); calcium voltage-gated channel subunit alpha 1s (CACNA1S); phosphoglycerate kinase 1 (PGK1); phosphoglycerate mutase 2 (PGAM2); amylo-α-1,6-glucosidase, 4-α-glucanotransferase (AGL); potassium voltage-gated channel ISK-related subfamily member 1 (KCNE1); potassium voltage-gated channel ISK-related subfamily member 2 (KCNE2); potassium voltage-gated channel subfamily J member 2 (KCNJ2); potassium voltage-gated channel subfamily J member 5 (KCNJ5); potassium voltage-gated channel subfamily H member 2 (KCNH2);Potassium voltage-gated channel KQT-like family member 1 (KCNQ1); hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4); chloride voltage-gated channel 1 (CLCN1); carnitine palmitoyltransferase 1A (CPT1A); ryanodine receptor 1 (RYR1); ryanodine receptor 2 (RYR2); bridging integrator 1 (BIN1); LARGE xylosyl- and glucuronosyltransferase 1 (LARGE1); docking protein 7 (DOK7); fukutin (FKTN); fukutin-related protein (FKRP); selenoprotein N ( N)(SELENON); protein O-mannosyltransferase 1 (POMT1); protein O-mannosyltransferase 2 (POMT2); protein O-linked mannose N-acetylglucosamine transferase 1 (POMGNT1); protein O-linked mannose N-acetylglucosamine transferase 2 (POMGNT2); protein-O-mannose kinase (POMK); isoprenoid synthase domain-containing (ISPD); plectin (PLEC); cholinergic receptor Nicotinic epsilon subunit (CHRNE); choline O-acetyltransferase (CHAT); choline kinase beta (CHKB); asymmetric acetylcholinesterase collagen tail subunit (COLQ); receptor associated protein of synapse (RAPSN); four and half LIM domains 1 (FHL1); beta-1,4-glucuronyltransferase 1 (B4GAT1); beta-1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2); dystroglycan 1 (DAG1); transmembrane protein 5 (TMEM5); transmembrane protein 43 (TMEM43); SECIS binding protein 2 (SECISBP2); glucosamine (UDP-N-acetyl)-2-epimerase / N-acetylmannosamine kinase (GNE); anoctamin 5 (ANO5); structural maintenance of chromosomes containing 1 flexible hinge domain (SMCHD1); lactate dehydrogenase A (LDHA); lactate dehydrogenase B (LHDB); calpain 3 (CAPN3); caveolin 3 (CAV3); containing 32 tripartite motifs (TRIM32); CCHC-type zinc finger nucleic acid binding protein (CNBP); nebulin (NEB); actin, alpha 1, skeletal muscle (ACTA1); actin, alpha 1, cardiac muscle (ACTC1); actinin alpha 2 (ACTN2); poly (A)-binding protein nuclear 1 (PABPN1); LEM domain-containing protein 3 (LEMD3); zinc metalloproteinase STE24 (ZMPSTE24); microsomal triglyceride transfer protein (MTTP);Cholinergic receptor nicotinic ɑ1 subunit (CHRNA1); cholinergic receptor nicotinic ɑ2 subunit (CHRNA2); cholinergic receptor nicotinic α3 subunit (CHRNA3); cholinergic receptor nicotinic α4 subunit (CHRNA4); cholinergic receptor nicotinic ɑ5 subunit (CHRNA5); cholinergic receptor nicotinic ɑ6 subunit (CHRNA6); cholinergic receptor nicotinic ɑ7 subunit (CHRNA7); cholinergic receptor nicotinic α8 subunit (CHRNA3); NA8 =; cholinergic receptor nicotinic alpha 9 subunit (CHRNA9); cholinergic receptor nicotinic alpha 10 subunit (CHRNA10); cholinergic receptor nicotinic beta 1 subunit (CHRNB1); cholinergic receptor nicotinic beta 2 subunit (CHRNB2); cholinergic receptor nicotinic beta 3 subunit (CHRNB3); cholinergic receptor nicotinic beta 4 subunit (CHRNB4); cholinergic receptor nicotinic gamma subunit (CHRNG1); cholinergic receptor nicotinic; subunit (CHRND); cholinergic receptor nicotinic epsilon subunit (CHRNE1); ATP cassette family A member 1 (ABCA1); ATP cassette family C member 6 (ABCC6); ATP cassette family C member 9 (ABCC9); ATP cassette family D member 1 (ABCD1); ATPase sarcoplasmic / endoplasmic reticulum Ca2+ transporter 1 (ATP2A1); ATM serine / threonine kinase (ATM); TTPA protein; kinesin family member 21A (KIF21A); paired homeobox 2a-like (PHOX2A); heparan sulfate proteoglycan 2 (HSPG2); stroma interacting molecule 1 (STIM1); notch 1 (NOTCH1); notch 3 (NOTCH3); dystrobrevin alpha (DTNA); protein kinase AMP-activated, non-catalytic gamma 2 (PRKAG2); cysteine- and glycine-rich protein 3 (CSRP3); viniculin (VCL); myozenin 2 (MyoZ2); myopalladin (MYPN); junctophilin 2 (JPH2); phospholamban (PLN); calreticulin 3 (CALR3); microtubule junction protein F-actin-binding protein (NEXN); LIM domain binding 3 (LDB3); eyelet absent 4 (EYA4); huntingtin (HTT); androgen receptor (AR); protein tyrosine phosphatase nonreceptor type 11 (PTPN11); junction discoidin (JN) plakoglobin (JUP); desmoplakin (DSP); desmoplakin 2 (PKP2); desmoglein 2 (DSG2); desmoglein 2 (DSC2); catenin alpha 3 (CTNNA3); NK2 homeobox 5 (NKX2-5); A-kinase anchor protein 9 (AKAP9); A-kinase anchor protein 10 (AKAP10); guanine nucleotide-binding protein alpha-inhibitory activity polypeptide 2 (GNAI2); ankyrin 2 (ANK2); synaptic protein alpha-1 (SNTA1); calmodulin 1 (CALM1); calmodulin 2 (CALM2); HTRA serine peptidase 1 (HTRA1); fibrillin 1 (FBN1); fibrillin 2 (FBN2); xylosyltransferase 1 (xylosyltransferase 1)(XYLT1); xylosyltransferase 2 (XYLT2); tafazzin (TAZ); homogentisate 1,2-dioxygenase (HGD); glucose-6-phosphatase catalytic (G6PC);1,4-α-Glucanase 1 (GBE1); Phosphofructokinase, muscle (PFKM); Phosphokinase kinase regulatory subunit α1 (PHKA1); Phosphokinase kinase regulatory subunit α2 (PHKA2); Phosphokinase kinase regulatory subunit β (PHKB); Phosphokinase kinase catalytic subunit γ2 (PHKG2); Phosphoglycerate mutase 2 (PGAM2); Cystathionine-β-synthase (CBS); Methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase)(MTHFR); 5-methylenetetrahydrofolate-homocysteine ​​methyltransferase (MTR); 5-methylenetetrahydrofolate-homocysteine ​​methyltransferase reductase (MTRR); methylmalonic aciduria and homocystinuria, cblD type (MMADHC); mitochondrial DNA, including but not limited to mitochondrial encoding NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1); mitochondrial encoding NADH:ubiquinone oxidoreductase core subunit 5 (MT-ND5); mitochondrial encoding tRNA glutamate (MT-TE); mitochondrial encoding tRNA histidine (MT-TH); mitochondrial encoding tRNA leucine 1 (MT-TL1); mitochondrial encoding tRNA lysine (MT-TK); mitochondrial encoding tRNA serine 1 (MT-TS1); mitochondrial encoding tRNA valine (MT-TV); mitogen-activated protein MAP2K1; B-Raf proto-oncogene, serine / threonine kinase (BRAF); raf-1 proto-oncogene, serine / threonine kinase (RAF1); growth factors, including but not limited to insulin growth factor 1 (IGF-1); transforming growth factor beta 3 (TGFβ3); transforming growth factor beta receptor, type I (TGFβR1); transforming growth factor beta receptor, type II (TGFβR2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 4 (FGF4), vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B); vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 1 (VEGFR1) and vascular endothelial growth factor receptor 2 (VEGFR2); interleukins; immunoadhesins; cytokines; and antibodies. ;

[0227] In preferred embodiments, the gene product delivered by the AAV variants of the invention is selected from alpha galactosidase A (GLA), frataxin (FXN), myofascin (DMD), acid alpha glucosidase (GAA), and muscle glycogenoylphosphorylase (PYGM). In some preferred embodiments, the AAV variants of the present invention include a nucleic acid segment that encodes (i) a GLA polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:67; (ii) a FXN polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:69; (iii) a DMD polypeptide comprising or consisting of a functional fragment (e.g., micromyosin, preferably comprising an entire actin-binding domain, at least 4 of the 24 spectrin-like repeats and a dystrobin-binding domain) of the amino acid sequence set forth in SEQ ID NO:64; (iv) a GAA polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:68; (v) a PYGM polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:66; or (vi) an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of SEQ ID NOs:64 and 66-69.

[0228] In another preferred embodiment, the AAV variants of the invention include a transgene encoding an interfering RNA, such as an antisense RNA, miRNA, shRNA or siRNA, which reduces the expression of DMPK. In some aspects, the interfering RNA reduces the expression of DMPK encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 65 or a sequence having at least 80%, at least 85%, at least 90% or at least 95% identity to SEQ ID NO: 65.

[0229] Genes whose gene products induce or promote apoptosis are referred to herein as "pro-apoptotic genes," and the products of those genes (mRNA; protein) are referred to as "pro-apoptotic gene products." Pro-apoptotic targets include, for example, Bax gene products; Bid gene products; Bak gene products; Bad gene products; Bcl-2; Bcl-X1. Anti-apoptotic gene products include X-linked inhibitors of apoptosis.

[0230] Genes whose gene products induce or promote angiogenesis are referred to herein as "pro-angiogenic genes", and the products of those genes (mRNA; protein) are referred to as "pro-angiogenic gene products". Pro-angiogenic targets include, for example, vascular endothelial growth factors (VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factor receptor 1 (VEGFR1); vascular endothelial growth factor receptor 2 (VEGFR2); Fms-related tyrosine kinase 1 (Flt1); placental growth factor (PGF); platelet-derived growth factor (PDGF); angiopoietin; sonic hedgehog. Genes whose gene products inhibit angiogenesis are referred to herein as "anti-angiogenic genes", and the products of those genes (mRNA; protein) are referred to as "anti-angiogenic gene products". Anti-angiogenic gene products include endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF), and fusion proteins or antibodies specific for pro-angiogenic targets and / or their receptors, such as the VEGF-specific antibody Avastin. TM wait.

[0231] Genes whose gene products act as immunomodulators, such as complement factors, toll-like receptors, are referred to as "immunomodulatory genes". Exemplary immunomodulatory genes include cytokines, chemokines, and fusion proteins or antibodies specific to them and / or their receptors, such as the anti-IL-6 fusion protein Rilonacept. TM , complement factor H-specific antibody lampazopanib (1ampamizumab), etc. Genes whose gene products act as muscle protective factors, such as insulin growth factor 1 (IGF-1); transforming growth factor β (TGFβ); fibroblast growth factor (FGF).

[0232] In some cases, the gene product of interest is a site-specific endonuclease that provides site-specific knockdown of gene function, e.g., where the endonuclease knocks out an allele associated with a muscle disease. For example, in the case where a dominant allele encodes a defective copy of a gene that is a muscle structural protein and / or provides normal muscle function when wild type, the site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.

[0233] In addition to knocking out defective alleles, site-specific nucleases can also be used to stimulate homologous recombination with donor DNA, and the donor DNA encodes a functional copy of a protein encoded by a defective allele. Therefore, for example, the rAAV virion of the present invention can be used to deliver the site-specific endonuclease that knocks out defective alleles, and can be used to deliver a functional copy of a defective allele, thereby causing the repair of defective alleles, thereby providing for the production of functional muscle protein (e.g., functional lamin A / C, functional myofibrillar protein, functional collagen VI type, etc.). In certain embodiments, rAAV virions disclosed herein include heterologous nucleotide sequences encoding site-specific endonucleases; and heterologous nucleotide sequences encoding functional copies of defective alleles, wherein functional copies encode functional muscle protein. Functional muscle protein includes, for example, lamin A / C, myofibrillar protein 1, COL6A1, COL6A2, COL6A3, etc.

[0234] Site-specific endonucleases suitable for use include, for example, meganucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and regularly aggregated interspaced short palindromic repeats / CRISPR-related (Cas), wherein such site-specific endonucleases are non-naturally occurring and modified to target specific genes. Such site-specific nucleases can be engineered to cut specific positions within the genome, and then non-homologous end joining can repair breaks when several nucleotides are inserted or deleted. Such site-specific endonucleases (also referred to as "INDELs") then throw proteins out of the framework and effectively knock out genes. See, for example, U.S. Patent Publication No. 2011 / 0301073.

[0235] In some embodiments of the variant rAAV vectors disclosed herein, the nucleotide sequence encoding the gene product of interest is operably linked to a constitutive promoter. Suitable constitutive promoters include, for example, the cytomegalovirus promoter (CMV) (Stinski et al., (1985) Journal of Virology 55(2): 431-441), the CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al., (1989) Gene 79(2): 269-277, CB SB(Jacobson et al., (2006) Mol Therapy 13(6): 1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al., (1990) Gene 91(2): 217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al., (1984) Gene 32(3): 409-417), mitochondrial heavy chain promoter (Loderio et al., (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al., (1990) FEBS Letters 261: 101-105). In other embodiments, the nucleotide sequence encoding the gene product of interest is operably linked to an inducible promoter. In some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a tissue-specific or cell-type-specific regulatory element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a muscle-specific regulatory element (e.g., a cardiac muscle-specific promoter or a skeletal muscle-specific promoter), such as a regulatory element that confers selective expression of the operably linked gene in muscle cells. Suitable muscle-specific regulatory elements include, for example, the skeletal muscle α-actin promoter (Muscat and Kedes (1987) Mol. Cell. Biol. 7: 4089-4099); the cardiac muscle α-actin promoter (Minty and Kedes (1986) Mol. Cell. Biol.6:2125-2136); smooth muscle α-actin promoter (Nakano et al., (1991) Gene 99:285-289); vascular smooth muscle α-actin promoter (Keogh et al., (1999) Mol Therapy 6(4):616-628); muscle creatine kinase promoter (Bartlett et al., (1996) Cell Transplantation 5(3):411-419); myosin light chain 1 and myosin light chain 3 promoters (Seidel and Arnold (1989) J Biol Chem 264(27):16109-16117); myosin light chain 2v (MLC2v) promoter (Su et al., (2004) PNAS 101(46):16280-16285); myogenic factor 5 (Myf5) promoter (Fujimaki et al., (2004) Journal of Biochemical Methods 289(11):7399-7412); myogenic differentiation 1 (Myodl) promoter (Zingg et al., (1994) Nucleic Acids Res. 22(12):2234-2241); myogenin (Myog) promoter (Salminen et al., (1991) Journal of Cell Biology 115(4):905-917); paired box gene 7 (Pax7) promoter (Murmann et al., (2000) Biol. Chem. 381(4):331-335); paired-like homeodomain 3 (Pitx3) promoter (Coulon et al. (2007) Journal of Biochemical Methods 282:33192-33200); MHCK7 promoter (Salva et al. (2007) Mol Therapy 15(2):320-329); MCK / SV40 promoter (Takeshita et al. (2007) Int J Molecular Pharmacol 19:309-315); C5-12 Promoter (Li et al., (1999) Nature Biotechnology 17:241-245); secondary and tertiary tandem MCK enhancer / promoter (Wang et al., (2008) Gene Therapy 15:1489-1499); myosin heavy chain 7 (MYH7) promoter; (Iwaki et al., (2104) PLoS ONE 9(4):e88610); myosin heavy chain 6 (MYH6) promoter (Pacak et al., (2008) Genet. Vaccines Ther. 6:13); cardiac troponin T (TNNT2) promoter (Farza et al., (1998) J. Mol. Cell Cardiol.30(6):1247-53); α-tropomyosin promoter (Helfman et al. (1986) Molecular and Cellular Biology 6(11):3582-3595); cardiac troponin C (TNNC1) promoter (Scheier et al. (1990) J. Biochem. Methods 34(5):21247-21253); cardiac myosin-binding protein C promoter (Lin et al. (2013) PLoS ONE 8(7):e69671); cardiac troponin I (TNNI3) promoter (Bhavsar et al. (1996) Genomics 35(1):11-23); desmin promoter (Li et al. (1991) J. Biochem. Methods 10(5):6562-6570); sodium-calcium exchanger (NCX1) promoter (Scheller et al. (1997) J. Biochem. Methods 273(13):7643-7649); atrial natriuretic factor promoter (Durocher et al. (1996) Mol. Cell. Biol. 16(9):4648-4655); and SM22α promoter (Kemp et al. (1995) J. Biochem. 310(3):1037-1043.

[0236] For the purposes of the present invention, the disclosure herein provides an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as described above. The isolated nucleic acid can be an AAV vector, such as a recombinant AAV vector.

[0237] The disclosure herein also provides a method of treating a muscle disease, comprising administering to an individual in need thereof an effective amount of rAAV variant virions comprising a transgene of interest as described above and disclosed herein. One of ordinary skill in the art will readily be able to determine the effective amount of the rAAV virions of the invention, and the disease has been treated by testing for changes in one or more functional or anatomical parameters, such as muscle biopsy followed by immunohistochemistry, serum sampling followed by ELISA or enzyme activity analysis, walking test, peak maximum oxygen consumption, biomarker analysis of left ventricular ejection fraction, left ventricular end-systolic volume, handheld dynamometer, maximum weight lift, timed functional test, Hammersmith motor ability score, timed climb from the floor, or 9-hole peg test.

[0238] Non-limiting methods for assessing muscle function and changes therein include assessment of walking test, peak maximal oxygen consumption, biomarker analysis, left ventricular ejection fraction, left ventricular end-systolic volume, Vignos scale, timed function test, Hammersmith motor ability score, timed rise from the floor, motor function measurement scale, North Star ambulatory assessment, 9-hole peg test, or Children's Hospital of Philadelphia Neuromuscular Disorders Infant Test.

[0239] In some embodiments, an effective amount of the rAAV virions of the invention results in a reduction in the rate of loss of muscle function, anatomical integrity, or muscle mass, such as a 2-fold, 3-fold, 4-fold, or 5-fold or more reduction in the rate of loss and, therefore, the progression of the disease, such as a 10-fold or more reduction in the rate of loss and, therefore, the progression of the disease. In some embodiments, an effective amount of the rAAV virions of the invention results in an increase in muscle function, an increase in muscle strength, an increase in muscle mass, and / or an improvement in anatomical muscle integrity or a biomarker, such as a 2-fold, 3-fold, 4-fold, or 5-fold improvement or more in muscle function, muscle strength, muscle mass, and / or an improvement in anatomical muscle integrity or a biomarker, such as a 10-fold or more improvement in muscle function, muscle strength, muscle mass, and / or an improvement in anatomical muscle integrity or a biomarker. As will be readily appreciated by those of ordinary skill in the art, the dose required to achieve the desired therapeutic effect will generally be in the range of 1×10 8 To about 1×10 16 The range of recombinant virus particles is usually referred to as 1×10 by those skilled in the art. 8 To about 1×10 16 "vector genomes", and preferably will be approximately 1 × 10 11 To about 1×10 15 recombinant virus particles.

[0240] The rAAV virions of the present invention can be delivered to skeletal muscle by intravascular (intravenous or intraarterial) administration, intraperitoneal administration, limb perfusion and / or direct intramuscular injection or by any other convenient mode of administration or route that will result in the delivery of rAAV virions to skeletal muscle. The rAAV virions can be delivered to the myocardium by intravascular (intravenous or intraarterial) administration, direct myocardial (to the left atrium, right atrium, right ventricle and / or septum), antegrade or retrograde infusion into the coronary artery (via the left anterior descending or left circumflex coronary artery), recirculation, intrapericardial injection, endocardial injection or by any other convenient mode of administration or route that will result in the delivery of rAAV virions to the myocardium. In a preferred embodiment, the rAAV virions of the present invention are delivered to the skeleton and / or myocardium by systemic intravenous administration. When administered by intravenous injection, the rAAV virions of the present invention are able to move through the circulatory system and transduce muscle cells more effectively than the ability of wild-type AAV virions or AAV virions comprising the corresponding parental AAV capsid protein.

[0241] The variant capsid proteins disclosed herein are isolated, e.g., purified. In some embodiments, the variant capsid proteins disclosed herein are contained in an AAV vector or recombinant AAV (rAAV) virion. In other embodiments, such AAV variant vectors and / or AAV variant virions are used in an in vivo or in vitro method for treating muscle diseases in primate cardiac or skeletal muscle.

[0242] The disclosure herein further provides host cells, such as but not limited to isolated (genetically modified) host cells including nucleic acids of the present invention. According to the host cells of the present invention disclosed herein, it can be isolated cells, such as cells from in vitro cell culture. As described herein, such host cells are used to produce rAAV variant virions of the present invention. In one embodiment, such host cells are stably genetically modified with nucleic acids. In other embodiments, host cells are transiently genetically modified with nucleic acids. Such nucleic acids are stably or transiently introduced into host cells using established techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. For stable transformation, nucleic acids generally further include selectable markers, for example, any of several well-known selectable markers, such as neomycin resistance, etc. Such host cells, such as mammalian cells, are produced by introducing nucleic acids into any of a variety of cells, including, for example, mouse cells and primate cells (e.g., human cells). Exemplary mammalian cells include, but are not limited to, primary cells and cell lines, wherein exemplary cell lines include, but are not limited to, HEK293 cells, HEK293T cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and the like. Exemplary host cells include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK293) cells (ATCC No. CRL1573), HLHepG2 cells, etc. Host cells can also be prepared using baculovirus to infect insect cells (such as Sf9 cells) that produce AAV (see, e.g., U.S. Pat. No. 7,271,002; U.S. Patent Application No. 12 / 297,958). In some embodiments, in addition to nucleic acids comprising nucleotide sequences encoding variant AAV capsid proteins, genetically modified host cells comprise nucleic acids as described above, comprising nucleotide sequences encoding one or more AAV rep proteins. In other embodiments, the host cell further comprises a rAAV variant vector. Such host cells can be used to produce rAAV variant virions.Methods of producing rAAV virions are described, for example, in U.S. Patent Publication Nos. 2005 / 0053922 and 2009 / 0202490.

[0243] The disclosure herein further provides a pharmaceutical composition comprising: a) rAAV variant virus particles as described above and disclosed herein; b) a pharmaceutically acceptable carrier, diluent, excipient or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient or buffer is suitable for human or non-human patients. Such excipients, carriers, diluents and buffers include any medicament that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts may be included therein, such as inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids such as acetates, propionates, malonates, benzoates, etc. In addition, auxiliary substances such as wetting agents or emulsifiers, surfactants, pH buffer substances, etc. may be present in such vehicles. A variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are well described 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) HC Ansell et al., ed., 7th edition, Lippincott, Williams, & Wilkins; and "Handbook of Pharmaceutical Excipients" (2000) AK Kibbe et al., ed., 3rd edition, Amer. Pharmaceutical Assoc. In some aspects of the present invention, the present invention provides a pharmaceutical composition comprising about 1×10 8 To about 1×10 16 recombinant virus or 1×10 8 To about 1×10 16 The invention relates to a plurality of vector genomes, wherein each of the recombinant viruses comprises a genome encoding one or more gene products.

[0244] Some embodiments of the present invention are illustrated in the following items 1 to 54:

[0245] 1. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion in the GH loop of the capsid protein, wherein the insertion is at an AAV2 or corresponding position in the capsid portion of a wild-type AAV serotype other than AAV2 or an AAV variant, and wherein the peptide insertion is 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), 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), LADNTWTRSA (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).

[0246] 2. The variant AAV according to item 1, wherein the capsid protein comprises one or more point mutations relative to AAV2 or one or more corresponding point mutations relative to other wild-type AAV serotypes or AAV variants.

[0247] 3. The variant AAV according to item 2, wherein the one or more point mutations are selected from the group consisting of: 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, K649E, R651H, W694C, I698V, V708I and L735Q, and is preferably selected from the group consisting of V708I, V708I+A593E, V708I+S109T, V708I+T 330A, A35P, V708I+R588M, V708I+W606C, V708I+W694C, I698V, N312K+N449D+N551S+I698V+L735Q, N312K+N449D+N551S+I698V+V708I+L735Q, V708I+N449K and V708I+G222S.

[0248] 4. The variant AAV according to item 1, wherein the peptide insertion is inserted after any of the amino acids in positions 570-671 in VP1 of AAV2 or at the corresponding position in another wild-type AAV serotype or AAV variant.

[0249] 5. The variant AAV according to item 4, wherein the peptide is inserted after amino acid 587 in VP1 of AAV2 or a corresponding position in another AAV serotype.

[0250] 6. An infectious recombinant adeno-associated virus (rAAV) virion comprising: (a) a variant AAV capsid protein according to any one of items 1 to 5 and a heterologous nucleic acid.

[0251] 7. The rAAV according to item 6, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding an RNA interfering agent or a polypeptide.

[0252] 8. A method for delivering a heterologous nucleic acid to a target cell, comprising contacting the target cell with the rAAV virus particle according to item 7.

[0253] 9. The method according to item 8, wherein the target cells are cardiac and / or skeletal muscle cells.

[0254] 10. The method according to item 8, wherein the target cell is in vitro.

[0255] 11. The method according to item 8, wherein the target cell is in vivo.

[0256] 12. An isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus (AAV) capsid protein, the variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion in the GH-loop of the capsid protein, wherein the insertion is at the AAV2 or corresponding position in the capsid portion of a wild-type AAV serotype other than AAV2 or an AAV variant, and wherein the peptide insertion is 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), 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 IDNO: 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).

[0257] 13. An isolated host cell comprising the nucleic acid according to item 12.

[0258] 14. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion relative to a parent AAV capsid protein, the peptide insertion corresponding to two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2, wherein the insertion comprises the amino acid sequence Y1Y2X1X2X3X4X5X6X7Y3, and wherein X1 is selected from T and N; X2 is selected from N and K; X3 is selected from K, I and T; X4 is selected from I, Q and T; X5 is selected from G, R and N; X6 is selected from V, T and K; and X7 is selected from T and D. .

[0259] 15. The variant AAV according to item 14, wherein the peptide insertion is selected from the group consisting of: NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14) and TNKIGVT (SEQ ID NO: 15).

[0260] 16. The variant AAV according to item 15, wherein the peptide insertion is flanked by N-terminal amino acids LA and C-terminal amino acids A.

[0261] 17. The variant AAV according to item 15, wherein the peptide insertion is between amino acids 587 and 588 of VP1 of AAV2 or the corresponding position in another wild-type AAV serotype or AAV variant.

[0262] 18. An infectious recombinant adeno-associated virus (rAAV) virion comprising: (a) a variant AAV capsid protein according to any one of items 14 to 17 and a heterologous nucleic acid.

[0263] 19. The rAAV according to item 18, wherein the heterologous nucleic acid comprises an RNA interfering agent or a nucleotide sequence encoding a polypeptide.

[0264] 20. A method of delivering a heterologous nucleic acid to a target cell, comprising contacting the target cell with the rAAV virion according to item 18.

[0265] 21. The method according to item 20, wherein the target cells are cardiac muscle and / or bone cells.

[0266] 22. The method according to item 21, wherein the target cell is in vitro or in vivo.

[0267] 23. A variant adeno-associated virus (AAV) capsid protein comprising i) an AAV amino acid sequence having at least 90% identity to a wild-type AAV selected from the group consisting of SEQ ID NOs: 1-10 and 11; and ii) one or more amino acid substitutions selected from the group consisting of: P363L, P363L+V708I, P363L+E347K, V708I+A593E, V708I+A333S, V708I+S721L, V708I+A593E+N551S, V708I+A593E+K649E, V708I+A593E+S109T, V708I+A593E+S109T+K527Q, A593E+S109T, wherein the one or more substitutions are relative to AAV2 or the one or more corresponding substitutions are relative to other AAV serotypes.

[0268] 24. The variant AAV according to item 23, wherein the capsid protein comprises a peptide insertion.

[0269] 25. The variant AAV according to item 24, wherein the peptide insertion is 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), 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).

[0270] 26. The variant AAV according to item 23, wherein the AAV amino acid sequence is at least 95% identical to the wild-type AAV.

[0271] 27. The variant AAV according to item 23, wherein the AAV amino acid sequence is at least 99% identical to the wild-type AAV.

[0272] 28. The variant AAV according to item 23, wherein the capsid protein is a chimeric capsid protein or an ancestral capsid protein.

[0273] 29. An infectious recombinant adeno-associated virus (rAAV) virion comprising: (a) a variant AAV capsid protein according to any one of items 23 to 28 and a heterologous nucleic acid.

[0274] 30. The rAAV according to item 29, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding an RNA interfering agent or a polypeptide.

[0275] 31. A method of delivering a heterologous nucleic acid to a target cell, comprising contacting the target cell with the rAAV virion according to item 29.

[0276] 32. The method according to item 31, wherein the target cells are cardiac and / or skeletal muscle cells.

[0277] 33. The method according to item 32, wherein the cardiomyocytes are selected from the group consisting of cardiomyocytes, cardiomyocytes, cardiac fibroblasts and cardiac progenitor cells.

[0278] 34. The method according to item 31, wherein the target cell is in vitro.

[0279] 35. The method according to item 31, wherein the target cell is in vivo.

[0280] 36. An isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus (AAV) capsid protein, wherein the variant adeno-associated virus (AAV) capsid protein comprises an amino acid sequence that is at least 90% identical to a wild-type AAV or an AAV variant selected from the group consisting of SEQ ID NOs: 1-12; and ii) one or more amino acid substitutions selected from the group consisting of: P363L, P363L+V708I, P363L+E347K, V708I+A593E, V708I+A333S, V708I+S721L, V708I+A593E+N551S, V708I+A593E+K649E, V708I+A593E+S109T, V708I+A593E+S109T+K527Q, A593E+S109T.

[0281] 37. An isolated host cell comprising the nucleic acid according to item 36.

[0282] 38. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion in the GH-loop of the capsid protein and optionally comprising one or more point mutations, wherein the peptide insertion is selected from the group consisting of NKIQRTD (SEQ ID NO: 13) and LANKIQRTDA (SEQ ID NO: 26).

[0283] 39. The variant AAV capsid protein according to item 38, comprising a V708I amino acid substitution.

[0284] 40. The variant AAV capsid protein according to item 39, comprising V708I+A593E, V708I+S109T, V708I+T330A, V708I+R588M or V708I+N312K+N449D+N551S+I698V+L735Q amino acid substitutions.

[0285] 41. The variant AAV capsid protein according to item 38, comprising an A35P amino acid substitution.

[0286] 42. The variant AAV capsid protein according to item 38, comprising N312K+N449D+N551S+I698V+L735Q amino acid substitutions.

[0287] 43. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion in the GH-loop of the capsid protein and optionally comprising one or more point mutations, wherein the peptide insertion is selected from the group consisting of NKTTNKD (SEQ ID NO: 14) and LANKTTNKDA (SEQ ID NO: 27).

[0288] 44. The variant AAV capsid protein according to item 43, comprising a V708I amino acid substitution.

[0289] 45. The variant AAV capsid protein according to item 44, comprising V708I+S109T, V708I+W694C, V708I+W606C or V708I+N312K+N449D+N551S+I698V+L735Q amino acid substitutions.

[0290] 46. ​​The variant AAV capsid protein according to item 43, comprising a 1698V amino acid substitution.

[0291] 47. The variant AAV capsid protein according to item 46, comprising N312K+N449D+N551S+I698V+L735Q amino acid substitutions.

[0292] 48. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion in the GH-loop of the capsid protein and optionally comprising one or more point mutations, wherein the peptide insertion is selected from the group consisting of TNKIGVT (SEQ ID NO: 15), LATNKIGVTA (SEQ ID NO: 28) and LATNKIGVTS (SEQ ID NO: 36).

[0293] 49. The variant AAV capsid protein according to item 48, comprising a V708I amino acid substitution.

[0294] 50. The variant AAV capsid protein according to item 49, comprising V708I+N449K, V708I+G222S or V708I+N312K+N449D+N551S+I698V+L735Q amino acid substitutions.

[0295] 51. The variant AAV capsid protein according to item 48, comprising N312K+N449D+N551S+I698V+L735Q amino acid substitutions.

[0296] 52. A variant adeno-associated virus (AAV) capsid protein comprising the sequence of SEQ ID NO: 62 or a sequence having at least 90% identity thereto, wherein the variant AAV capsid protein comprises the following amino acid substitutions relative to the AAV5 capsid: V229I+A490T+A581T.

[0297] 53. The variant AAV capsid protein according to item 52, further comprising a Y585S or V447F amino acid substitution relative to the AA5 capsid.

[0298] 54. A variant adeno-associated virus (AAV) capsid protein comprising the sequence of SEQ ID NO: 63 or a sequence having at least 90% identity thereto, wherein the variant AAV capsid protein comprises the following amino acid substitutions relative to the AAV5 capsid: V229I+A427D+A490T+A581T.

[0299] Examples

[0300] The following examples are presented to fully disclose and describe how to make and use the present invention to those of ordinary skill in the art, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the quantities used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be noted. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is degrees Celsius, and pressure is at or near atmospheric pressure.

[0301] General methods of molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd edition, (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th edition, (Ausubel et al., eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonvial Vectors for Gene Therapy (Wagner et al., eds., Academic Press 1999); Viral Vectors (Kapliff & Loewy, eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits, ed., Academic Press 1996). 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. The reagents, cloning vectors and kits for genetic manipulation mentioned in the present disclosure can be purchased from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich and ClonTech.

[0302] Example 1

[0303] Intravenous injection and tissue collection. For each round of selection, a single male cynomolgus macaque (Macaca fascicularis) aged 3-10 years and weighing at least 3 kg was dosed by intravenous injection into the great saphenous vein. The animal was anesthetized and dosed with 1-5 mL of the pool (in the first round, the pool was prepared using Figure 1 All variants generated by the mutagenesis techniques described in A were constructed; in each subsequent round, variants were isolated from the previous round) and, in some cases, pre-incubated with human IVIG for 30 min at 37°C.

[0304] Depending on the choice, at 14 ± 3 days or 21 ± 3 days, euthanasia was performed by trained veterinary personnel using 100 mg / kg sodium pentobarbital intravenous injection. Myocardial and / or skeletal muscle tissue from the quadriceps was removed and DNA was isolated from the tissue. In some cases, myocardial tissue was divided into several regions: atrium, ventricular septum, left papillary muscle, right papillary muscle, left ventricle and right ventricle.

[0305] Directed evolution. Figure 1 The directed evolution process is shown in A-1E. Briefly, a viral capsid library ( Figure 1 A) The virus is then packaged ( Figure 1 B) - each particle is composed of a mutant capsid surrounding the cap gene encoding that capsid - and purified. The capsid library is placed under in vivo selective pressure. The relevant tissue or cell material is harvested to isolate AAV variants that have successfully infected that target, and the successful viruses are recovered. Successful clones are enriched by repeated selection (Phase I-B). Figure 1 D) The selected cap genes are then exclusively redistributed and enriched through further selection steps to iteratively increase viral fitness (Phase 2- Figure 1 D). Variants identified during vector selection phases 1 and 2 demonstrated the ability to transduce primate myocytes ( Figure 1 E).

[0306] Successful recovery of the AAV capsid genome. Capsids recovered from each round of selection are used to package the injected library to initiate a subsequent round of selection. Recovery of the capsid gene from tissues indicates successful internalization of the library vector into the relevant tissue. Figure 2 Recovery of viral genomes from cardiac skeletal muscle tissue from a representative round of selection is shown. Boxed bands indicate successful recovery of viral genomes.

[0307] Sequencing analysis. During rounds 3-4 of selection that included selective pressure for intravenous delivery to cardiac muscle tissue or skeletal muscle tissue and rounds 1-2 of selection that included selective pressure for intravenous delivery to cardiac muscle tissue in the presence of neutralizing antibodies, individual clones within the pool were sequenced to determine the frequency of variants in the population. Variants were evaluated for the presence of motifs in the sequencing data. Variants were grouped into motifs based on the presence of uniform variants occurring in multiple sequences (e.g., specific point mutations in a consistent position within the capsid or specific peptide insertion sequences). Motifs that represented at least 5% of the sequenced population in two or more rounds of selection, or motifs that represented at least 10% of the sequenced population in one or more rounds of selection, are denoted in Figure 3A (4th round of sequencing analysis of selective pressure delivered intravenously to myocardial tissue), Figure 3B (Second round sequencing analysis of selective pressure delivered intravenously to myocardial tissue in the presence of neutralizing antibodies) and Figure 3C (Provided by 3rd round sequencing analysis of selective pressure delivered intravenously to skeletal muscle tissue.

[0308] Several representative clones identified as conferring increased infectivity to cardiac and / or skeletal muscle cells are listed in Table 1 below (each clone contains one or more substitutions and / or peptide insertions identified and is otherwise identical to SEQ ID NO: 2; the selection round, sequence number and frequency (in parentheses) for each clone are listed):

[0309] Table 1. Amino acid sequence modifications to the AAV VP1 capsid protein that confer increased infectivity to cardiac and / or skeletal muscle cells. The substitutions listed in column 2 are based on the amino acid sequence of wild-type AAV2, i.e., without the inserted peptide. "Myocardial+NAb" in column 5 indicates that the amino acid sequence modification should confer increased resistance to neutralization by anti-AAV antibodies in addition to increased infectivity to cardiac myocytes.

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] Also identified as capsids conferring increased infectivity to cardiomyocytes and increased resistance to neutralization by anti-AAV antibodies were the following chimeras:

[0317] A chimera having (i) amino acids 1-129 of AAV6 and (ii) amino acids 130-725 of AAV5 and having the following amino acid substitutions relative to AAV5: V229I+A490T+A581T (sequence of SEQ ID NO: 62).

[0318] A chimera having (i) amino acids 1-61 of AAV2, (ii) amino acids 62-129 of AAV6, and (iii) amino acids 130-725 of AAV5 and having the following amino acid substitutions relative to AAV5: V229I+A490T+A581T (sequence of SEQ ID NO: 63).

[0319] A chimera having (i) amino acids 1-129 of AAV6 and (ii) amino acids 130-725 of AAV5 and having the following amino acid substitutions relative to AAV5: V229I+A490T+A581T+Y585S

[0320] A chimera having (i) amino acids 1-129 of AAV6 and (ii) amino acids 130-725 of AAV5 and having the following amino acid substitutions relative to AAV5: V229I+A447F+A490T+A581T

[0321] The AAV variant virions disclosed herein may incorporate reasonable design parameters, features, modifications, advantages and variations that are apparent to one of ordinary skill in the art of engineering AAV viral vectors.

[0322] Example 2

[0323] The cellular tropism of recombinant AAV virions including the novel AAV variants LANKIQRTDA+V708I (SEQ ID NO: 43), LANKTTNKDA+V708I (SEQ ID NO: 48), and LATNKIGVTA+V708I (SEQ ID NO: 46) for cardiomyocytes was evaluated in vitro using cardiomyocytes generated from human embryonic stem cells (ESCs).

[0324] Standard methods were used to make recombinant AAV viral particles comprising an AAV1 capsid, an AAV2 capsid, an AAV9 capsid, a novel variant capsid LANKIQRTDA+V708I, a novel variant capsid LANKTTNKDA+V708I, or a novel variant capsid LATNKIGVTA+V708I; and a genome comprising a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (respectively, AAV1.CAG.EGFP, AAV2.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA+V708I.CAG.EGFP, LANKTTNKDA+V708I.CAG.EGFP, and LATNKIGVTA+V708I.CAG.GFP). Cardiomyocytes were generated from the human embryonic stem cell line ESI-017 by regulating Wnt signaling using small molecules. After 14 days of myocardial mesoderm induction, the culture was further enriched for cardiomyocytes by glucose deprivation. After approximately 24 days of differentiation, the majority of cells expressed the cardiomyocyte marker, cardiac troponin T (cTnT), and the ventricle-specific marker MLC-2V. The generated cardiomyocytes were evaluated for expression of the gap junction protein connexin43, membrane potential fluctuations, calcium handling, and contractile function to ensure that the generated cardiomyocytes reached a mature state prior to vector characterization.

[0325] For AAV1, AAV2, and AAV9, the expression of Fig. 6A ), flow cytometry ( Figure 6B ) and Western blot analysis ( Figure 6C -D) determined that the LANKIQRTDA+V708I, LANKTTNKDA+V708I, and LATNKIGVTA+V708I variants provided significantly improved transduction efficiency and transgene expression in human cardiomyocyte cultures. In addition, relative to AAV1, AAV2, and AAV9, as measured by immunofluorescence ( Fig. 6E ) determined that LANKIQRTDA+V708I, LANKTTNKDA+V708I, and LATNKIGVTA+V708I provided a faster onset of gene expression in human cardiomyocyte cultures. The number of infectious units per administered viral genome was several orders of magnitude higher for LANKIQRTDA+V708I and LANKTTNKDA+V708I relative to AAV8 and AAV9, which exhibit cardiac and skeletal muscle cell tropism. ( Fig. 10A ) This study demonstrates the superior ability of AAV capsid variants including NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), and TNKIGVT (SEQ ID NO: 15) to deliver genes to cardiomyocytes.

[0326] Example 3

[0327] The cellular tropism of recombinant AAV virions comprising a novel AAV variant, AAV6 / AAV5 chimera, of cardiomyocytes was assessed in vitro using cardiomyocytes generated from human embryonic stem cells (ESCs).

[0328] Recombinant AAV viral particles comprising an AAV1 capsid, an AAV8 capsid, an AAV9 capsid, or a novel variant capsid AAV6 / AAV5 chimera (having SEQ ID NO: 62) and a genome comprising a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (respectively, AAV1.CAG.EGFP, AAV8.CAG.EGFP, AAV9.CAG.EGFP, AAV6 / AAV5 chimera.CAG.EGFP). Cardiomyocytes were generated from the human embryonic stem cell line ESI-017 by modulating Wnt signaling using small molecules. After 14 days of cardiac mesoderm induction, the cultures were further enriched for cardiomyocytes by glucose deprivation. After approximately 24 days of differentiation, the majority of cells expressed the cardiomyocyte marker, cardiac troponin T (cTnT), and the ventricular-specific marker MLC-2V. The generated cardiomyocytes were evaluated for expression of the gap junction protein connexin43, membrane potential fluctuations, calcium handling, and contractile function to ensure that the generated cardiomyocytes reached a mature state prior to vector characterization.

[0329] For AAV1, AAV8, and AAV9, the expression of Fig. 7A ), flow cytometry ( Figure 7B ) and Western blot analysis ( Figure 7C -D) determined that AAV6 / AAV5 chimeras provide significantly improved transduction efficiency and transgene expression in human cardiomyocyte cultures. In addition, compared to AAV8, as measured by immunofluorescence ( Fig. 7E ) determined that AAV6 / AAV5 chimeras provided a faster onset of gene expression in human cardiomyocyte cultures. The number of infectious units per administered viral genome was several orders of magnitude higher for AAV6 / AAV5 chimeras relative to AAV8 and AAV9. ( Fig. 10A ) This study demonstrates the superior ability of AAV capsid variants comprising SEQ ID NO: 62 to deliver genes to cardiomyocytes.

[0330] Example 4

[0331] The cellular tropism of recombinant AAV virions including the novel AAV variants LANKIQRTDA+V708I, LANKTTNKDA+V708I, and AAV6 / AAV5 chimeras for skeletal muscle fibers was assessed in vitro using skeletal muscle fibers generated from primary human myoblasts.

[0332] Standard methods were used to make recombinant AAV viral particles including AAV8 capsid, AAV9 capsid, novel variant capsid LANKIQRTDA+V708I, novel variant capsid LANKTTNKDA+V708I or novel variant capsid AAV6 / AAV5 chimera, and a genome including a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (respectively, AAV8.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA+V708I.CAG.EGFP, LANKTTNKDA+V708I.CAG.EGFP and AAV6 / AAV5 chimera.CAG.GFP). Skeletal muscle fibers were generated from primary human skeletal myoblasts obtained from a healthy 51-year-old male (CookMyosites). Myoblasts were differentiated for 30 days to form mature multinucleated skeletal muscle fibers. The generated skeletal muscle fibers were evaluated for expression of myosin heavy chain (MHC) and dystrophin to ensure that the majority of generated skeletal muscle fibers reached a mature state prior to vector characterization.

[0333] For AAV8 and AAV9, seven days after infection, as measured by immunofluorescence ( Fig. 8A ) and flow cytometry ( Figure 8B ) determined that LANKIQRTDA+V708I, LANKTTNKDA+V708I, and AAV6 / AAV5 chimeras provided significantly improved transduction efficiency and transgene expression in human skeletal muscle fiber cultures. In addition, relative to AAV8 and AAV9, as measured by immunofluorescence ( Figure 8C ) determined that LANKIQRTDA+V708I and LANKTINKDA+V708I provided a faster onset of gene expression in human skeletal muscle fiber cultures. The number of infectious units per administered viral genome was several-fold higher for LANKIQRTDA+V708I, LANKTTNKDA+V708I, and the AAV6 / 5 chimera relative to AAV8 and AAV9 ( Fig. 10B ). This study demonstrates the superior ability of variants including NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), and SEQ ID NO: 62 to deliver genes to skeletal muscle fibers.

[0334] Example 5

[0335] The cell tropism of recombinant AAV virions including novel AAV variants LANKIQRTDA+V708I, LANKTTNKDA+V708I, and AAV6 / AAV5 chimeras for skeletal muscle progenitor cells was evaluated in vitro using skeletal muscle progenitor cells generated from fibroblast-derived human induced pluripotent stem cells (FB-iPSCs) or human embryonic stem cells (ESCs).

[0336] Recombinant AAV viral particles comprising an AAV9 capsid, a novel variant capsid LANKIQRTDA+V708I, a novel variant capsid LANKTTNKDA+V708I, or a novel variant capsid AAV6 / AAV5 chimera and a genome comprising a green fluorescent protein (EGFP) transgene operably linked to a CAG promoter (respectively, AAV8.CAG.EGFP, AAV9.CAG.EGFP, LANKIQRTDA+V708I.CAG.EGFP, LANKTTNKDA+V708I.CAG.EGFP, and AAV6 / AAV5 chimera.CAG.GFP) were made using standard methods. Skeletal muscle progenitor cells were generated from the human embryonic stem cell line ESI-017 (ESI-BIO) following the differentiation strategy described in Shelton et al., Methods 2016 with minor modifications. After approximately 40 days of differentiation, before the cultures were used for vector characterization, lineage restriction to skeletal muscle progenitors was confirmed by expression of PAX7 and MyoD in the majority of cells.

[0337] In contrast to AAV9, at six days post-infection, as measured by immunofluorescence ( Fig.9A ) and flow cytometry ( Fig. 9B ) determined that LANKIQRTDA+V708I, LANKTTNKDA+V708I, and AAV6 / AAV5 chimeras provided significantly improved transduction efficiency and transgene expression in human skeletal muscle progenitor cell cultures. This study demonstrates the superior ability of AAV capsid variants including NKIQRTD (SEQ ID NO: 13), NKTTNKD (SEQ ID NO: 14), and SEQ ID NO: 62 to deliver genes to skeletal muscle progenitor cells.

[0338] Example 6

[0339] Directed evolution was used to discover novel adeno-associated virus (AAV) variants by delivering superior genes to cardiac and skeletal muscle cells following intravenous (IV) administration, a route of administration that was significantly superior to other methods of gene delivery to human cardiac and skeletal muscle (Example 1). Cellular tropism in mice was assessed following intramuscular administration of recombinant AAV virions comprising novel AAV variants as representative examples of the ability of rAAV virions comprising AAV capsid variants containing NKIQRTD (SEQ ID NO: 13) to transduce muscle cells, the novel AAV variants comprising a V708I substitution and a peptide LANKIQRTDA (SEQ ID NO: 27) inserted between amino acids 587 and 588 (LANKIQRTDA+V708I; SEQ ID NO: 43).

[0340] Recombinant AAV virions (LANKIQRTDA+V708I.CAG.luciferase) comprising the novel variant capsid LANKIQRTDA+V708I and a genome comprising a luciferase transgene operably linked to a CAG promoter were produced using standard methods. B6 Albino (C57BL / 6) were injected intravenously via the tail vein with 2×10 12 vg, and transduction was assessed by vital luciferase imaging and by postmortem tissue luciferase activity. Vital imaging of luciferase at day 14 (left) and day 28 (right) after administration demonstrates that the novel AAV variant LANKIQRTDA+V708I capsid can transduce mouse cells in vivo ( Fig.11A Luciferase activity in the heart, diaphragm, and quadriceps at 56 days after administration indicated that the novel AAV variant LANKIQRTDA+V708I capsid could transduce myocardial and skeletal muscles of mice in vivo ( Fig. 11B ).

[0341] This study demonstrates gene delivery via one of several clinically acceptable routes of administration through variants comprising NKIQRTD (SEQ ID NO: 13). Similar efficacy can be achieved with other variants comprising this peptide insertion motif. Likewise, similar efficacy can be achieved with other variants identified using the same directed evolution method disclosed herein.

[0342] Example 7

[0343] Directed evolution was used to discover novel adeno-associated virus (AAV) variants by delivering superior genes to cardiac and skeletal muscle cells following intravenous (IV) administration, a route of administration that was significantly superior to other methods of gene delivery to human cardiac and skeletal muscle (Example 1). Cellular tropism in vivo in non-human primates (NHPs) was assessed following intramuscular administration of recombinant AAV virions comprising novel AAV variants as representative examples of the ability of rAAV variants comprising AAV capsid variants containing NKIQRTD (SEQ ID NO: 13) to transduce muscle cells, the novel AAV variants comprising a V708I substitution and a peptide LANKIQRTDA (SEQ ID NO: 27) inserted between amino acids 587 and 588 (LANKIQRTDA+V708I; SEQ ID NO: 43).

[0344] Recombinant AAV virions (LANKIQRTDA+V708I.CAG.GFP) comprising the novel variant capsid LANKIQRTDA+V708I and a genome comprising a green fluorescent protein (GFP) transgene operably linked to a CAG promoter were produced using standard methods. Three doses of the vector (1×10 11 vg) were injected into the vastus lateralis of cynomolgus macaques, and postmortem transduction of skeletal muscle cells was assessed by immunofluorescence imaging. Representative images of cross-sections of the proximal biopsy site stained with hematoxylin and eosin (H&E) and anti-GFP antibodies at 2x, 4x, and 20x magnification demonstrate that the novel AAV variant LANKIQRTDA+V708I capsid can transduce primate skeletal muscle in vivo ( Fig. 12A Representative images of longitudinal sections of distal biopsy sites stained with hematoxylin and eosin (H&E) and anti-GFP antibodies at 2x, 4x, and 20x magnification demonstrate that the novel AAV variant LANKIQRTDA+V708I capsid can transduce primate skeletal muscle cells in vivo ( Fig. 12B ).

[0345] This study demonstrates gene delivery via one of several clinically acceptable routes of administration through variants comprising NKIQRTD (SEQ ID NO: 13). Similar efficacy can be achieved with other variants comprising this peptide insertion motif. Likewise, similar efficacy can be achieved with other variants identified using the same directed evolution method disclosed herein.

[0346] The foregoing merely illustrates the principles of the present invention. It will be appreciated that one of ordinary skill in the art will be able to design various arrangements that, although not expressly described or shown herein, embody the principles of the present invention and are included within its spirit and scope. In addition, all embodiments and conditional language listed herein are primarily intended to assist the reader in understanding the principles of the present invention and the concepts provided by the inventors to further the development of the field, and should be considered not to limit such specific listed embodiments and conditions.

[0347] In addition, the statements of all the present invention's principles, aspects, and embodiments are intended to cover both their structural and functional equivalents together with their specific examples. In addition, it is expected that such equivalents include both currently known equivalents and future development equivalents, that is, any element that performs the same function regardless of structure. Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. On the contrary, the scope and spirit of the present invention are embodied by the appended claims.

Claims

1. A variant adeno-associated virus (AAV) capsid protein selected from the group consisting of: (i) an AAV capsid protein comprising a peptide insertion relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or NKTTNKD (SEQ ID NO: 14), wherein the insertion site is located between two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2 or a corresponding position in the capsid protein of another AAV serotype, and (ii) an AAV capsid protein comprising (a) an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the entire length of the amino acid sequence set forth in SEQ ID NO: 62, and (b) the following amino acid substitutions relative to AAV5 capsid (SEQ ID NO: 6): V229I+A490T+A581T, Wherein the variant AAV capsid protein confers increased infectivity of infectious recombinant AAV (rAAV) virions to the muscle cells compared to the infectivity of AAV virions comprising wild-type AAV capsid protein to the muscle cells.

2. A variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein, the peptide insertion corresponding to two adjacent amino acids at a position between amino acids 570 and 611 of VP1 of AAV2, wherein the insertion comprises the amino acid sequence Y1Y2X1X2X3X4X5X6X7Y3, and wherein 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. The variant AAV capsid protein according to claim 2, wherein 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. The variant AAV capsid protein of any one of claims 1 to 3, wherein the capsid protein comprises a peptide insertion, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or a corresponding position in the capsid protein of another AAV serotype.

5. The variant AAV capsid protein of any one of claims 1 to 4, wherein the AAV capsid protein comprises a peptide insertion, and wherein the peptide insertion comprises the amino acid sequence NKIQRTD (SEQ ID NO: 13) or LANKIQRTDA (SEQ ID NO: 26).

6. The variant AAV capsid protein of claim 5, wherein the AAV capsid protein comprises a V708I amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or a corresponding position in the capsid protein of another AAV serotype.

7. The variant AAV capsid protein of claim 6, wherein the AAV capsid protein further comprises one or more of an S109T amino acid substitution, an R588M amino acid substitution, and an A593 amino acid substitution relative to one or more corresponding positions in VP1 of AAV2 (SEQ ID NO: 2) or the capsid protein of another AAV serotype.

8. The variant AAV capsid protein of claim 6, wherein the AAV capsid protein further comprises an A35P amino acid substitution relative to VP1 of AAV2 (SEQ ID NO: 2) or one or more corresponding positions in the capsid protein of another AAV serotype.

9. The variant AAV capsid protein of claim 6, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85% identical, at least 90% identical, at least 95% identical, or 100% identical to the entire length of the amino acid sequence shown in SEQ ID NO:

43.

10. The variant AAV capsid protein of any one of claims 1 to 4, wherein the AAV capsid protein comprises a peptide insertion, and wherein the peptide insertion comprises the amino acid sequence NKTINKD (SEQ ID NO: 14) or LANKTTNKDA (SEQ ID NO: 27).

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