New AAV capsids with enhanced muscle and peripheral nerve gene transfer efficiency

Abstract

The disclosure relates, in some aspects, to modified adeno-associated virus (AAV) capsid proteins and recombinant adeno-associated viruses (rAAVs) comprising the same. In some aspects, the disclosure relates to isolated nucleic acids encoding modified AAV9 capsid proteins. In some embodiments, rAAVs and compositions described by the disclosure are useful for delivery of one or more transgenes to the muscle-tissue or peripheral nervous tissue of a subject.

Description

[0001] NEW AAV CAPSIDS WITH ENHANCED MUSCLE AND PERIPHERAL NERVE GENE TRANSFER EFFICIENCY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of the filing date of United States Provisional Application Serial Number 63 / 666,357, filed July 1, 2024, and entitled “NEW AAV CAPSIDS WITH ENHANCED MUSCLE AND PERIPHERAL NERVE GENE TRANSFER EFFICIENCY,” which is herein incorporated by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (U012070198WO00-SEQ-EAS.xml; Size: 65,946 bytes; and Date of Creation: June 25, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND OF INVENTION

[0007] AAV9 is one of the main platforms being used for in vivo gene therapy for genetic diseases affecting muscle.

[0008] SUMMARY OF INVENTION

[0009] The disclosure relates, in some aspects, to AAV capsid proteins with tropism for muscle tissue and neural tissue. The disclosure is based, in part, on modified AAV capsid proteins (e.g., capsid proteins having one or more amino acid insertions, deletions, or substitutions relative to a wild-type AAV capsid protein, such as AAV9) that are characterized by desirable functional properties, for example, highly efficient transduction of specific tissue / cell types. In some embodiments, AAV capsid proteins (and rAAVs comprising such capsid proteins) are useful for treatment of certain diseases and disorders, such as muscular dystrophies (e.g., Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, etc.) or peripheral nervous system disorders (e.g., Charcot- Marie-tooth disease, Dejerine-Sottas disease, multiple sclerosis, Friedreich’s ataxia, etc.).

[0010] Accordingly, in some aspects, the disclosure provides a peptide (e.g., a capsid protein) comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 1 (wild-type AAV9), and one or more amino acid insertions or substitutions at one or more positions selected from 450-490 relative to SEQ ID NO:1. In some embodiments, the amino acid sequence is not 100% identical to SEQ ID NO: 1. In some embodiments, one or more amino acid insertions comprises between 5 amino acids and 10 (e.g., 5, 6, 7, 8, 9, or 10) amino acids. In some embodiments, one or more amino acid insertions is 7 amino acids in length.

[0011] In some embodiments, one or more amino acid substitutions comprises between 5 amino acids and 10 (e.g., 5, 6, 7, 8, 9, or 10) amino acids. In some embodiments, one or more amino acid substitutions is 7 amino acids in length.

[0012] In some embodiments, the amino acid sequence of the peptide (e.g., capsid protein) is at least 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 (e.g., wild-type AAV9). In some embodiments, the amino acid sequence is not 100% identical to SEQ ID NO: 1.

[0013] In some embodiments, one or more amino acid insertions comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-25 (e.g., one or more of SEQ ID NOs: 2-25 are inserted at a position in Loop IV of an AAV9 capsid protein). In some embodiments, one or more positions in Loop IV of an AAV9 capsid protein (e.g., amino acid residues 450-490 of SEQ ID NO: 1) are substituted to produce a capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2-25. In some embodiments, the amino acid sequence of the peptide (e.g., capsid protein) comprises the sequence set forth in any one of SEQ ID NOs: 2- 25.

[0014] In some embodiments, one or more amino acids insertions are positioned between amino acid 455 and 456 of AAV9 capsid, SEQ ID NO:1. In some embodiments, one or more amino acids substitutions are positioned at amino acids 452 to 458 of AAV9 capsid, SEQ ID NO:1.

[0015] In some embodiments, a peptide comprises the amino acid sequence as set forth in any one of SEQ ID NO: 26-49.

[0016] In some aspects, the disclosure provides a nucleic acid encoding a peptide as described herein. In some aspects, the disclosure provides a vector comprising a nucleic acid encoding a peptide as described herein. In some embodiments, the vector is a plasmid. In some embodiments, the disclosure provides a host cell comprising a vector or nucleic acid as described herein.

[0017] In some aspects, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising a capsid protein comprising the peptide of any one of claims 1 to 11 or the amino acid sequence set forth in any one of SEQ ID NOs: 26-49; and an isolated nucleic acid comprising a transgene having a promoter operably linked to a nucleic acid sequence encoding a gene product, flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). In some aspects, the disclosure provides a method of delivering a transgene to a cell, the method comprising contacting the cell with an rAAV as described herein.

[0018] In some aspects, the disclosure provides a method for treating a muscle disease or neural disease of a subject, the method comprising administering an rAAV as described herein to the subject.

[0019] In some embodiments, transgene encodes a therapeutic gene product, therapeutic protein, or an inhibitory nucleic acid. In some embodiments, a therapeutic protein is a toxin, an enzyme, a growth factor, an interleukin, an interferons, an anti-apoptosis factor, a cytokine, an antidiabetic factor, an anti-apoptosis agent, a coagulation factor, an anti-tumor factor, or an antiproliferative protein. In some embodiments, an inhibitory nucleic acid is a shRNA, a dsRNA, a siRNA, a miRNA, or an artificial miRNA.

[0020] In some embodiments, a cell is a muscle cell. In some embodiments, a cell is a nerve cell. In some embodiments, a nerve cell is a peripheral nerve cell. In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is a human cell. In some embodiments, a cell is in a subject.

[0021] In some embodiments, a muscle disease is a skeletal muscle disease. In some embodiments, a skeletal muscle disease is muscular dystrophy, amyotrophic lateral sclerosis, myopathies, myotonic dystrophy, or dermatomyositis.

[0022] In some embodiments, a neurological disorder is a peripheral nervous system disorder. In some embodiments, a peripheral nervous system disorder is a Charcot-Marie-Tooth disease, Dejerine-Sottas disease, Multiple sclerosis, or Friedreich’s ataxia.

[0023] In some aspects, the disclosure provides a capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO: 26-49.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] FIG. 1 shows the analysis of the production efficiency of different capsid candidates.

[0026] FIG. 2A-2B shows the biodistribution of capsid candidates in the biceps brachii from two different cynomolgus macaques. FIG. 2A shows the data from a first cynomolgus macaque and FIG. 2B shows the data from a second cynomolgus macaque.

[0027] FIG. 3A-3B shows the biodistribution of capsid candidates in the triceps brachii from two different cynomolgus macaques. FIG. 3A shows the data from a first cynomolgus macaque and FIG. 3B shows the data from a second cynomolgus macaque. FIG. 4A-4B shows the biodistribution of capsid candidates in the hamstring (bicep femoris) from two different cynomolgus macaque. FIG. 4A shows the data from a first cynomolgus macaques and FIG. 4B shows the data from a second cynomolgus macaque.

[0028] FIG. 5A-5B shows the biodistribution of capsid candidates in the quadriceps (rectus femoris) from two different cynomolgus macaques. FIG. 5A shows the data from a first cynomolgus macaque and FIG. 5B shows the data from a second cynomolgus macaque.

[0029] FIG. 6A-6B shows the biodistribution of capsid candidates in the soleus from two different cynomolgus macaques. FIG. 6A shows the data from a first cynomolgus macaque and FIG. 6B shows the data from a second cynomolgus macaque.

[0030] FIG. 7A-7B shows the biodistribution of capsid candidates in the intercostal muscle from two different cynomolgus macaques. FIG. 7A shows the data from a first cynomolgus macaque and FIG. 7B shows the data from a second cynomolgus macaque.

[0031] FIG. 8A-8B shows the biodistribution of capsid candidates in the diaphragm muscle from two different cynomolgus macaques. FIG. 8A shows the data from a first cynomolgus macaque and FIG. 8B shows the data from a second cynomolgus macaques.

[0032] FIGs. 9A-9B shows the biodistribution of capsid candidates in the heart from two different cynomolgus macaques. FIG. 9A shows the data from a first cynomolgus macaque and FIG. 9B shows the data from a second cynomolgus macaque.

[0033] FIGs. 10A-10B shows the biodistribution of capsid candidates in the liver from two different cynomolgus macaques. FIG. 10A shows the data from a first cynomolgus macaque and FIG. 10B shows the data from a second cynomolgus macaque.

[0034] FIGs. 11A-1 IB shows the biodistribution of capsid candidates in the lung from two different cynomolgus macaques. FIG. 11A shows the data from a first cynomolgus macaque and FIG. 11B shows the data from a second cynomolgus macaque.

[0035] FIGs. 12A-12B shows the biodistribution of capsid candidates in the aorta (smooth muscle) from two different cynomolgus macaques. FIG. 12A shows the data from a first cynomolgus macaques and FIG. 12B shows the data from a second cynomolgus macaques.

[0036] FIGs. 13A-13B shows the biodistribution of capsid candidates in the pancreas from two different cynomolgus macaques. FIG. 13A shows the data from a first cynomolgus macaques and FIG. 13B shows the data from a second cynomolgus macaques.

[0037] FIGs. 14A-14B shows the biodistribution of capsid candidates in the kidney from two different cynomolgus macaques. FIG. 14A shows the data from a first cynomolgus macaques and FIG. 14B shows the data from a second cynomolgus macaques. FIGs. 15A-15G shows representative cynomolgus macaque brain coronal slices and locations, labeled 1-12, where biodistribution of capsid candidates was measured.

[0038] FIGs. 16A-16L shows the biodistribution of capsid candidates in the representative brain regions indicated in FIGs. 15A-15G from a first cynomolgus macaque. FIG. 16A shows the biodistribution of capsid candidates from location 1 as seen in FIG. 15 A. 16B shows the biodistribution of capsid candidates from location 2 as seen in FIG. 15B. 16C shows the biodistribution of capsid candidates from location 3 as seen in FIG. 15C. 16D shows the biodistribution of capsid candidates from location 4 as seen in FIG. 15C. 16E shows the biodistribution of capsid candidates from location 5 as seen in FIG. 15C. 16F shows the biodistribution of capsid candidates from location 6 as seen in FIG. 15D. 16G shows the biodistribution of capsid candidates from location 7 as seen in FIG. 15E. 16H shows the biodistribution of capsid candidates from location 8 as seen in FIG. 15F. 161 shows the biodistribution of capsid candidates from location 9 as seen in FIG. 15F. 16J shows the biodistribution of capsid candidates from location 10 as seen in FIG. 15F. 16K shows the biodistribution of capsid candidates from location 11 as seen in FIG. 15F. 16L shows the biodistribution of capsid candidates from location 12 as seen in FIG. 15G.

[0039] FIGs. 17A-17H shows the biodistribution of capsid candidates in the cervical spinal cord (FIG. 17A), thoracic spinal cord (FIG. 17B), cervical dorsal root ganglion (FIG. 17C), thoracic dorsal root ganglion (FIG. 17D), the sciatic nerve (FIG. 17E), the phrenic nerve (FIG. 17F), the optic nerve (FIG. 17G), and the retina (FIG. 17H) of a first cynomolgus macaque.

[0040] FIGs. 18A-18G shows representative cynomolgus macaque brain coronal slices and locations, labeled 1-12, where biodistribution of capsid candidates was measured.

[0041] FIGs. 19A-19L shows the biodistribution of capsid candidates in the representative brain regions indicated in FIGs. 18A-18G from a second cynomolgus macaque. FIG. 19A shows the biodistribution of capsid candidates from location 1 as seen in FIG. 18 A. FIG. 19B shows the biodistribution of capsid candidates from location 2 as seen in FIG. 18B. FIG. 19C shows the biodistribution of capsid candidates from location 3 as seen in FIG. 18C. FIG. 19D shows the biodistribution of capsid candidates from location 4 as seen in FIG. 18C. FIG. 19E shows the biodistribution of capsid candidates from location 5 as seen in FIG. 18C. FIG. 19F shows the biodistribution of capsid candidates from location 6 as seen in FIG. 18D. FIG. 19G shows the biodistribution of capsid candidates from location 7 as seen in FIG. 18E. FIG. 19H shows the biodistribution of capsid candidates from location 8 as seen in FIG. 18F. FIG. 191 shows the biodistribution of capsid candidates from location 9 as seen in FIG. 18F. FIG. 19J shows the biodistribution of capsid candidates from location 10 as seen in FIG. 18F. FIG. 19K shows the biodistribution of capsid candidates from location 11 as seen in FIG. 18F. FIG. 19L shows the biodistribution of capsid candidates from location 12 as seen in FIG. 18G.

[0042] FIGs. 20A-20H shows the biodistribution of capsid candidates in the cervical spinal cord (FIG. 20A), thoracic spinal cord (FIG. 20B), cervical dorsal root ganglion (FIG. 20C), thoracic dorsal root ganglion (FIG. 20D), the sciatic nerve (FIG. 20E), the phrenic nerve (FIG. 20F), the optic nerve (FIG. 20G), and the retina (FIG. 20H) of a second cynomolgus macaque.

[0043] DETAILED DESCRIPTION OF INVENTION

[0044] The disclosure relates, at least in part, to adeno-associated virus 9 (AAV9) capsid variants (e.g., modified AAV9 capsid proteins) that have an increased tropism for muscle tissue and / or neural tissue. In some aspects, the disclosure relates to AAV9-based capsid libraries comprising modified capsids having amino acid insertions or substitutions relative to wild-type AAV9 capsid protein (e.g., wild type AAV9 VP1, VP2, or VP3 protein). In some embodiments, a modified capsid protein of the library comprise one or more amino acid substitutions and / or amino acid insertions within a loop region (e.g., Loop IV) of the AAV9 capsid protein (e.g., wild type AAV9 VP1, VP2, or VP3 protein).

[0045] In some aspects, the disclosure relates to compositions (e.g., rAAVs comprising an AAV9 capsid protein variant described herein) and methods for delivering a transgene (e.g., a transgene encoding one or more gene products) to a target cell (e.g., muscle cell and / or nerve cell).

[0046] AAV9 Capsid Variants

[0047] Aspects of the disclosure relate to peptides and proteins. In some embodiments, the peptides are capsid proteins (e.g., modified capsid proteins). Capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which are transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with host cells. In some embodiments, a wild type AAV9 capsid VP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, an AAV9 VP2 capsid protein comprises the amino acid sequence comprising amino acids 138 to comprises the amino acid sequence comprising amino acids 203 to 736 of SEQ ID NO: 1 (SEQ ID NO: 52).

[0048] Aspects of the disclosure relate to peptides (e.g., capsid proteins) that have one or more amino acid substitutions and / or one or more amino acid insertions relative to a wild-type AAV capsid protein. In some embodiments, the wild-type capsid protein is AAV9 capsid protein. In some embodiments, peptides described herein comprise an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type AAV9 VP1 capsid protein (e.g., SEQ ID NO: 1). In some embodiments, peptides described herein comprise an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type AAV9 VP2 capsid protein (e.g., SEQ ID NO: 51). In some embodiments, peptides described herein comprise an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type AAV9 VP3 capsid protein (e.g., SEQ ID NO: 52). In some aspects, the disclosure relate to peptides (e.g., capsid proteins) having substantial homology to an AAV9 capsid protein. “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. The term "substantial homology", when referring to a nucleic acid, or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in about 90 to 100% of the aligned sequences. When referring to a polypeptide, or fragment thereof, the term “substantial homology” indicates that, when optimally aligned with appropriate gaps, insertions or deletions with another polypeptide, there is nucleotide sequence identity in about 90 to 100% of the aligned sequences. The term "highly conserved" means at least 80% identity, preferably at least 90% identity, and more preferably, over 97% identity. In some cases, highly conserved may refer to 100% identity. Identity is readily determined by one of skill in the art by, for example, the use of algorithms and computer programs known by those of skill in the art.

[0049] In some embodiments, a peptide having substantial homology to an AAV9 VP1 capsid protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a peptide having substantial homology to an AAV9 VP2 capsid protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, a peptide having substantial homology to an AAV9 VP3 capsid protein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 52.

[0050] In some embodiments, a peptide having substantial homology to an AAV9 VP1 capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, a peptide having substantial homology to an AAV9 VP2 capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, a peptide having substantial homology to an AAV9 VP3 capsid protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:52.

[0051] In some embodiments, a peptide having substantial homology to an AAV9 capsid protein (e.g., VP1, VP2, or VP3) comprises 5 to 10 (e.g., 5-6, 6-7, 7-8, 8-9, 9-10, 5-7, 6-8, 7-9, 8-10, 5- 8, 6-9, 7-10, 5-9, 6-10) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:1, 51, or 52. In some embodiments, a peptide having substantial homology to an AAV9 capsid protein comprises no more than 5 to 10 (e.g., 5-6, 6-7, 7-8, 8-9, 9-10, 5-7, 6-8, 7-9, 8-10, 5-8, 6-9, 7-10, 5-9, 6-10) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:1, 51, or 52. In some embodiments, a peptide having substantial homology to an AAV9 VP1 capsid protein comprises more than 10 (e.g., more than 20, more than 30, more than 40, more than 50, etc.) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, a peptide having substantial homology to an AAV9 VP2 capsid protein comprises more than 10 (e.g., more than 20, more than 30, more than 40, more than 50, etc.) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, a peptide having substantial homology to an AAV9 VP3 capsid protein comprises more than 10 (e.g., more than 20, more than 30, more than 40, more than 50, etc.) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in SEQ ID NO:52.

[0052] In some embodiments, a capsid protein having substantial homology to an AAV9 capsid protein (e.g., VP1, VP2, or VP3) comprises 7 amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in any one of SEQ ID NO: 1, 51, or 53. In some embodiments, a peptide comprises 7 amino acids inserted into wild-type AA9 VP1 capsid protein (SEQ ID NO: 1). In some embodiments, a peptide comprises 7 amino acids inserted into wild-type AA9 VP2 capsid protein (SEQ ID NO: 51). In some embodiments, a peptide comprises 7 amino acids inserted into wild-type AA9 VP3 capsid protein (SEQ ID NO: 52).

[0053] In some embodiments, a peptide (e.g., capsid protein) comprises 5 to 10 (e.g., 5-6, 6-7, 7-8, 8-9, 9-10, 5-7, 6-8, 7-9, 8-10, 5-8, 6-9, 7-10, 5-9, 6-10) amino acids substitutions relative to a wild-type AAV9 VP1 capsid protein (SEQ ID NO: 1). In some embodiments, a peptide comprises 7 amino acids substituted into a wild-type AA9 capsid protein (SEQ ID NO: 1). In some embodiments, the amino acids 452 to 458 of SEQ ID NO: 1 are substituted. In some embodiments, the amino acid sequence substituted out is NGSGQNQ (SEQ ID NO: 50). In some embodiments, amino acids 452 to 458 of SEQ ID NO: 1 are substituted with any one of SEQ ID NO: 2-25. In some embodiments, the amino acid sequence NGSGQNQ (SEQ ID NO: 50) is substituted with any one of SEQ ID NO: 2-25.

[0054] In some embodiments, a peptide (e.g., capsid protein) comprises 5 to 10 (e.g., 5-6, 6-7, 7-8, 8-9, 9-10, 5-7, 6-8, 7-9, 8-10, 5-8, 6-9, 7-10, 5-9, 6-10) amino acids substitutions relative to a wild-type AAV9 VP2 capsid protein (SEQ ID NO: 51). In some embodiments, a peptide comprises 7 amino acids substituted into a wild-type AA9 VP2 capsid protein (SEQ ID NO: 51). In some embodiments, the amino acids 315 to 321 of SEQ ID NO: 51 are substituted. In some embodiments, the amino acid sequence substituted out is NGSGQNQ (SEQ ID NO: 50). In some embodiments, amino acids 315 to 321 of SEQ ID NO: 1 are substituted with any one of SEQ ID NO: 2-25. In some embodiments, the amino acid sequence NGSGQNQ (SEQ ID NO: 50) is substituted with any one of SEQ ID NO: 2-25.

[0055] In some embodiments, a peptide (e.g., capsid protein) comprises 5 to 10 (e.g., 5-6, 6-7, 7-8, 8-9, 9-10, 5-7, 6-8, 7-9, 8-10, 5-8, 6-9, 7-10, 5-9, 6-10) amino acids substitutions relative to a wild-type AAV9 VP3 capsid protein (SEQ ID NO: 52). In some embodiments, a peptide comprises 7 amino acids substituted into a wild-type AA9 VP3 capsid protein (SEQ ID NO: 52). In some embodiments, the amino acids 250 to 256 of SEQ ID NO: 52 are substituted. In some embodiments, the amino acid sequence substituted out is NGSGQNQ (SEQ ID NO: 50). In some embodiments, amino acids 250 to 256 of SEQ ID NO: 52 are substituted with any one of SEQ ID NO: 2-25. In some embodiments, the amino acid sequence NGSGQNQ (SEQ ID NO: 50) is substituted with any one of SEQ ID NO: 2-25.

[0056] In some embodiments, the amino acid sequence substituted into an AAV9 capsid protein (e.g., VP1, VP2, or VP3) described herein comprise an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NO: 2-25. In some embodiments, the amino acid sequence substituted into an AAV9 capsid protein (e.g., VP1, VP2, or VP3) described herein comprise up to 5 (e.g., 1-5, 1-4, 2-51-3, 2-4, 3-5, 1-2, 3-4, 4-5, 1, 2, 3, 4, or 5) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in any one of SEQ ID NO: 2-25.

[0057] The positioning of amino acid insertions and / or substitutions relative to SEQ ID NO: 1 may vary. In some embodiments, the amino acid substitutions and / or insertions are positioned in a loop region of an AAV capsid protein, for example a loop region of AAV9 capsid protein (e.g., comprising the sequence set forth in SEQ ID NO: 1). AAV capsid proteins typically comprise nine (9) loop regions, numbered I-IX from N-terminus to C-terminus of the protein. In some embodiments, amino acid insertions and / or substitutions are positioned within loop IV of AAV9 protein. In some embodiments, loop IV of AAV9 protein comprises amino acid positions 449-468 of SEQ ID NO: 1. Since amino acid positions 449-468 of AAV9 VP1 capsid protein are present in AAV9 VP2 and VP3 capsid protein, it is understood that the insertion and substitution would be present in AAV9 capsid variant VP2 and VP3 proteins.

[0058] In some embodiments, the a AAV9 VP1 capsid protein comprises an insertion of the amino acid sequence consisting of any one of SEQ ID NOs: 2-25 inserted between amino acid positions 455 and 456 of SEQ ID NO: 1. In some embodiments, a AAV9 VP2 capsid protein comprises an insertion of the amino acid sequence consisting of any one of SEQ ID NOs: 2-25 between amino acid positions 318 and 319 of SEQ ID NO: 51. In some embodiments, a AAV9 VP3 capsid protein comprises an insertion of the amino acid sequence consisting of any one of SEQ ID NOs: 2-25 between amino acid positions 253 and 254 of SEQ ID NO: 51. In some embodiments, the amino acid sequence inserted into an AAV9 capsid protein (e.g., VP1, VP2, or VP3) described herein comprise an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NO: 2-25. In some embodiments, the amino acid sequence inserted into an AAV9 capsid protein (e.g., VP1, VP2, or VP3) comprises up to 5 (e.g., 1-5, 1-4, 2-5, 1-3, 2-4, 3-5, 1-2, 3-4, 4-5, 1, 2, 3, 4, or 5) amino acid substitutions, insertions, or deletions, relative to the amino acid sequence set forth in any one of SEQ ID NO: 2-25.

[0059] The disclosure relates, in some aspects, to the surprising discovery that rAAVs comprising peptides described by the disclosure (e.g., capsid proteins, such as modified AAV9 capsid proteins described herein) have higher tropism for certain cell types (e.g., nerve cells or muscle cells) relative to rAAVs having other AAV capsid proteins (e.g., wild-type AAV9 capsid protein). The increase in tropism of rAAVs comprising peptides of the disclosure relative to wild-type AAV9 (or other AAV capsid serotypes) may vary. In some embodiments, the tropism for muscle and / or nerve cells is increased by at least 5%, 10%, 20%, 40%, 50%, 75%, 100%, or more. In some embodiments, the tropism for muscle and / or nerve cells is increased by at least 2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 200 or more times. In some embodiments, an rAAV comprising a peptide (e.g., capsid protein as described herein) has increased biodistribution relative to a rAAV comprising a wild-type AAV9 capsid protein. The increase in biodistribution of rAAVs comprising peptides of the disclosure relative to wild-type AAV9 (or other AAV capsid serotypes) may vary. In some embodiments, the biodistribution is increased by at least 5%, 10%, 20%, 40%, 50%, 75%, 100%, or more.

[0060] In some embodiments a capsid protein (e.g., SEQ ID NO: 26) comprises the amino acid sequence DTVHQYR (SEQ ID NO: 2) inserted between amino acids 455 and 456 of SEQ ID NO: 1. In some embodiments, a AAV9 VP1 capsid protein variant comprises an amino acid sequence at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 26. In some embodiments, a AAV9 VP1 capsid protein variant comprises the amino acid sequence of SEQ ID NO: 26.

[0061] In some embodiments capsid protein MSK2 comprises the amino acid sequence

[0062] QRHHNTQ (SEQ ID NO: 3) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK2 comprises the amino acid sequence SEQ ID NO:27. In some embodiments capsid protein MSK3 comprises the amino acid sequence RCQEHRV (SEQ ID NO: 4) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK3 comprises the amino acid sequence SEQ ID NO:28.

[0063] In some embodiments capsid protein MSK4 comprises the amino acid sequence RGQEHRV (SEQ ID NO: 5) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK4 comprises the amino acid sequence SEQ ID NO:29.

[0064] In some embodiments capsid protein MSK5 comprises the amino acid sequence KYYRTTE (SEQ ID NO: 6) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK5 comprises the amino acid sequence SEQ ID NO:30.

[0065] In some embodiments capsid protein MSK6 comprises the amino acid sequence RMMQQDV (SEQ ID NO: 7) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK6 comprises the amino acid sequence SEQ ID NO:31.

[0066] In some embodiments capsid protein MSK7 comprises the amino acid sequence ISRPAVV (SEQ ID NO: 8) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK7 comprises the amino acid sequence SEQ ID NO:32.

[0067] In some embodiments capsid protein MSK8 comprises the amino acid sequence PGGHPGT (SEQ ID NO: 9) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK8 comprises the amino acid sequence SEQ ID NO:33.

[0068] In some embodiments capsid protein MSK9 comprises the amino acid sequence HVTHQHR (SEQ ID NO: 10) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK9 comprises the amino acid sequence SEQ ID NO:34.

[0069] In some embodiments capsid protein MSK10 comprises the amino acid sequence EHAHAHR (SEQ ID NO: 11) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK10 comprises the amino acid sequence SEQ ID NO:35. In some embodiments capsid protein MSK11 comprises the amino acid sequence SARHTQY (SEQ ID NO: 12) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK11 comprises the amino acid sequence SEQ ID NO:36.

[0070] In some embodiments capsid protein MSK12 comprises the amino acid sequence RMRTHDS (SEQ ID NO: 13) inserted between amino acids 455 and 456 of wild-type AAV9 (SEQ ID NO: 1). In some embodiments, MSK12 comprises the amino acid sequence SEQ ID NO:37.

[0071] In some embodiments capsid protein MSK13 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence QANVRPC (SEQ ID NO: 14). In some embodiments, MSK13 comprises the amino acid sequence SEQ ID NO:38.

[0072] In some embodiments capsid protein MSK14 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence VQAQAPA (SEQ ID NO: 15). In some embodiments, MSK14 comprises the amino acid sequence SEQ ID NO:39.

[0073] In some embodiments capsid protein MSK15 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence ARQPHIC (SEQ ID NO: 16). In some embodiments, MSK15 comprises the amino acid sequence SEQ ID NO:40.

[0074] In some embodiments capsid protein MSK16 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence PARQDRA (SEQ ID NO: 17). In some embodiments, MSK16 comprises the amino acid sequence SEQ ID NO:41.

[0075] In some embodiments capsid protein MSK17 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence PVSQPYM (SEQ ID NO: 18). In some embodiments, MSK17 comprises the amino acid sequence SEQ ID NO:42.

[0076] In some embodiments capsid protein MSK18 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence RGITHMP (SEQ ID NO: 19). In some embodiments, MSK18 comprises the amino acid sequence SEQ ID NO:43.

[0077] In some embodiments capsid protein MSK19 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence YVQSRSA (SEQ ID NO: 20). In some embodiments, MSK19 comprises the amino acid sequence SEQ ID NO:44.

[0078] In some embodiments capsid protein MSK20 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence GAQAVWR (SEQ ID NO: 21). In some embodiments, MSK20 comprises the amino acid sequence SEQ ID NO:45. In some embodiments capsid protein MSK21 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence YRHSQQQ (SEQ ID NO: 22). In some embodiments, MSK21 comprises the amino acid sequence SEQ ID NO:46.

[0079] In some embodiments capsid protein MSK22 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence MRSAPVV (SEQ ID NO: 23). In some embodiments, MSK22 comprises the amino acid sequence SEQ ID NO:47.

[0080] In some embodiments capsid protein MSK23 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence YRSQAHA (SEQ ID NO: 24). In some embodiments, MSK23 comprises the amino acid sequence SEQ ID NO:48.

[0081] In some embodiments capsid protein MSK24 comprises a substitution of amino acids 452 to 458 of wild-type AAV9 (SEQ ID NO: 1) with amino acid sequence QAHVQPR (SEQ ID NO: 25). In some embodiments, MSK24 comprises the amino acid sequence SEQ ID NO:49.

[0082] Nucleic acids

[0083] As used herein, the term "nucleic acid" refers to polymers of linked nucleotides, such as DNA, RNA, etc. In some embodiments, proteins and nucleic acids of the disclosure are isolated. In some embodiments, the DNA of a transgene is transcribed into a messenger RNA (mRNA) transcript. As used herein, the term “isolated” means artificially produced (e.g., an artificially produced nucleic acid, or an artificially produced protein, such as a capsid protein). As used herein with respect to nucleic acids, the term “isolated” means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which polymerase chain reaction (PCR) primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified but need not be. For example, a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. As used herein with respect to proteins or peptides, the term “isolated” refers to a protein or peptide that has been artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.) As used herein, a “transgene” is a nucleic acid sequence, which is not homologous to vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., inhibitory RNA, such as miRNA and miRNA inhibitors) or other gene product, of interest. In some embodiments, a transgene encodes a therapeutic protein or inhibitory nucleic acid (e.g., inhibitory RNA). Examples of therapeutic proteins include toxins, enzymes e.g., kinases, phosphorylases, proteases, acetylases, deacetylases, methylases, demethylases, etc.) growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, antiapoptosis agents, coagulation factors, anti-tumor factors, and anti-proliferative proteins. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue.

[0084] Thus, the disclosure embraces the delivery of vectors encoding one or more polypeptides or proteins, which are useful for the treatment or prevention of disease states in a mammalian subject. Exemplary therapeutic proteins include one or more polypeptides selected from the group consisting of growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors, enzymes, and anti-proliferative proteins. Other non-limiting examples of therapeutic proteins include BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, VEGF, TGF-B2, TNF, prolactin, somatotropin, XIAP1, IE-1, IE-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(187A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL- 16 IL-17, and IL-18.

[0085] The nucleic acids disclosed herein may comprise a transgene to be transferred to a subject to treat a disease associated with reduced expression, lack of expression or dysfunction of the native gene. Exemplary genes and associated disease states include, but are not limited to: glucose-6-phosphatase, associated with glycogen storage deficiency type 1A; phosphoenolpyruvate-carboxykinase, associated with Pepck deficiency; galactose- 1 phosphate uridyl transferase, associated with galactosemia; phenylalanine hydroxylase, associated with phenylketonuria; branched chain alpha-ketoacid dehydrogenase, associated with Maple syrup urine disease; fumarylacetoacetate hydrolase, associated with tyrosinemia type 1; methylmalonyl-CoA mutase, associated with methylmalonic acidemia; medium chain acyl Co A dehydrogenase, associated with medium chain acetyl Co A deficiency; ornithine transcarbamylase, associated with ornithine transcarbamylase deficiency; argininosuccinic acid synthetase, associated with citrullinemia; low density lipoprotein receptor protein, associated with familial hypercholesterolemia; UDP-glucouronosyltransferase, associated with Crigler- Najjar disease; adenosine deaminase, associated with severe combined immunodeficiency disease; hypoxanthine guanine phosphoribosyl transferase, associated with Gout and Lesch- Nyan syndrome; biotinidase, associated with biotinidase deficiency; beta-glucocerebrosidase, associated with Gaucher disease; beta-glucuronidase, associated with Sly syndrome; peroxisome membrane protein 70 kDa, associated with Zellweger syndrome; porphobilinogen deaminase, associated with acute intermittent porphyria; alpha- 1 antitrypsin for treatment of alpha- 1 antitrypsin deficiency (emphysema); erythropoietin for treatment of anemia due to thalassemia or to renal failure; vascular endothelial growth factor, angiopoietin-1, and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitor for the treatment of occluded blood vessels as seen in, for example, atherosclerosis, thrombosis, or embolisms; aromatic amino acid decarboxylase (AADC), and tyrosine hydroxylase (TH) for the treatment of Parkinson's disease; the beta adrenergic receptor, antisense to, or a mutant form of, phospholamban, the sarco(endo)plasmic reticulum adenosine triphosphatase-2 (SERCA2), and the cardiac adenylyl cyclase for the treatment of congestive heart failure; a tumor suppressor gene such as p53 for the treatment of various cancers; a cytokine such as one of the various interleukins for the treatment of inflammatory and immune disorders and cancers; dystrophin or minidystrophin and utrophin or miniutrophin for the treatment of muscular dystrophies; and, insulin for the treatment of diabetes. In some embodiment a disease is a disorder. In some embodiments, a disorder is a disease.

[0086] The following are further non-limiting examples of proteins that may be encoded by transgenes disclosed herein to treat a disease associated with reduced expression, lack of expression or dysfunction of the native gene: a-galactosidase, acid-glucosidase, adiopokines, adiponectin, alglucosidase alfa, anti-thrombin, Apo AV, ApoCII, apolipoprotein A-I (APOA1), arylsulfatase A, arylsulfatase B, ATP-binding cassette transporter Al (ABCA1), ABCD1, CCR5 receptor, erythropoietin, Factor VIII, Factor VII, Factor IX, Factor V, fetal hemoglobin, betaglobin, GPI-anchored HDL-binding protein (GPI-HBP) I, growth hormone, hepatocyte growth factor, imiglucerase, lecithin-cholesterol acyltransferase (LCAT), leptin, LDL receptor, lipase maturation factor (LMF) 1, lipoprotein lipase, lysozyme, nicotinamide dinucleotide phosphate (NADPH) oxidase, Rab escort protein- 1 (REP-1), retinal degeneration slow (RDS), retinal pigment epithelium- specific 65 (RPE65), rhodopsin, T cell receptor alpha or beta chains, thrombopoeitin, tyrosine hydroxylase, VEGF, von heldebrant factor, von willebrand factor, and X-linked inhibitor of apoptosis (XIAP). As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases "operatively positioned," "under control" or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

[0087] The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically active polypeptide product (e.g., a therapeutic protein or therapeutic minigene) or inhibitory RNA (e.g., shRNA, miRNA, amiRNA, miRNA inhibitor) from a transcribed gene.

[0088] Recombinant AA Vs

[0089] In some aspects, the disclosure provides isolated AAVs. As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially obtained or produced. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”. Recombinant AAVs (rAAVs) preferably have tissue- specific targeting capabilities, such that a transgene of the rAAV will be delivered specifically to one or more predetermined tissue(s). The AAV capsid is an important element in determining the “tropism”, or tissue-specific targeting capabilities of an AAV. Thus, an rAAV having a capsid appropriate for the tissue being targeted can be selected. In some embodiments, an rAAV comprises a peptide (e.g., capsid protein) having the amino acid sequence as set forth in any one of SEQ ID NOs: 26-49, or a protein having substantial homology thereto. In some embodiments, an rAAV comprises a peptide (e.g., capsid protein) having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 1, but not 100% identical, to SEQ ID NO:1. In some embodiments, an rAAV comprises a peptide (e.g., capsid protein) having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 26-49.

[0090] Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (See, for example, US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein (e.g., a nucleic acid having a sequence as set forth in any one of SEQ ID NOs: 26-49, or fragment thereof; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.

[0091] In some embodiments, the rAAV targets muscle tissue of a subject. Examples of muscle tissue include but are not limited to skeletal muscle, smooth muscle, and cardiac muscle. In some embodiments, the rAAV targets muscle cells. Examples of muscle cells include but are not limited to myocytes (e.g., cardiac myocytes, skeletal myocytes, etc.), myocardial cells, myofibril cells, sarcomere cells, etc.

[0092] In some embodiments, the rAAV targets nerve tissue of a subject. In some embodiments, the rAAV targets peripheral nerve tissue of a subject. Examples of peripheral nerve cells are spinal nerves (e.g., cervical nerves, thoracic nerves, lumbar nerves, sacral nerves, coccygeal nerves), cranial nerves (e.g., olfactory nerve, optic nerve, oculomotor nerve, trigeminal nerve, trochlear nerve, abducens nerve, facial nerve, vestibulocochlear nerve, glossopharyngeal nerve, vagus nerve, Accessory nerve, Hypoglossal nerve), and visceral or autonomic nerves. In some embodiments, peripheral nerves lie outside of the central nervous system (CNS).

[0093] In some embodiments, an rAAV further comprises a transgene. In some embodiments, a transgene encodes a therapeutic protein or therapeutic functional RNA. Examples of therapeutic proteins include toxins, enzymes e.g., kinases, phosphorylases, proteases, acetylases, deacetylases, methylases, demethylases, etc.) growth factors, interleukins, interferons, anti-apoptosis factors, cytokines, anti-diabetic factors, anti-apoptosis agents, coagulation factors, anti-tumor factors, and anti-proliferative proteins.

[0094] Viral Vectors

[0095] Viral vectors present a powerful tool for the delivery of plasmids and genetic material into cells. Adapting plasmid DNA for use with virus-mediated delivery has provided numerous advantages for research, including the delivery of genetic information in traditionally hard-to- transfect cells, such as neurons. Viruses naturally infect host cells and direct them to reproduce the viral genome. Scientists have taken advantage of this process by providing the virus with alternate genomes (e.g., plasmids encoding a nucleic acid or transgene), which can then be replicated once the virus has infected a host cell. In short, researchers can introduce plasmids into a host cell to generate recombinant virus.

[0096] For safety reasons, viral genomes used in research and drug development have been modified through the removal of certain genes that are required for viral replication. These genes are usually divided among numerous “accessory plasmids” which must also be present in the cell for a viral particle to be produced. The production of viral particles comprising nucleic acid(s) of interest, along with the viral genome, by a host cell is herein referred to as “packaging”. The process for the delivery and packaging of nucleic acids into viral genomes varies depending on the viral genome the nucleic acid is encoded in and will be discussed in greater detail for each viral vector below.

[0097] Recombinant adeno-associated virus (rAAV) particles are produced by introducing into a host cell, a cis-element nucleic acid comprising a transgene, a helper nucleic acid encoding adenoviral helper genes, and a packaging nucleic acid encoding Rep and / or Cap genes. A cis- element nucleic acid comprising a transgene may comprise a transgene flanked by adeno- associated virus (AAV) inverted terminal repeats (ITRs). In some embodiments, a helper nucleic acid encoding adenoviral helper genes comprises genes that mediate AAV replication (e.g., AAV E4, E2a and / or VA genes). In some embodiments, a packaging nucleic acid encodes one or more Rep genes. In some embodiments, a packaging nucleic acid encodes one or more Cap genes.

[0098] As used herein, the term “recombinant virus” or “recombinant viral particle” refers to a particle produced in a host cell which encapsulates nucleic acid produced from exogenous DNA inserted into the host cell genome is, has been introduced.

[0099] In some aspects, the disclosure provides transfected host cells. The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells. The skilled artisan will appreciate that in methods described by the disclosure, a host cell may be transfected with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more isolated nucleic acids.

[0100] The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). In some embodiments, an isolated nucleic acid as described by the disclosure comprises a region (e.g., a first region) comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR), or a variant thereof. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to a subject and / or delivered to a selected target cell. “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene, and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise, as disclosed elsewhere herein, one or more regions that encode one or more proteins and / or one or more binding sites for inhibitory nucleic acids (e.g., shRNA, miRNAs, etc.). The transgene may also comprise a region encoding, for example, a protein and / or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure.

[0101] Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., the rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2 / 2-66, AAV2 / 2-84, AAV2 / 2-125, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region (e.g., a first region) encoding an AAV2 ITR.

[0102] In some embodiments, the isolated nucleic acid further comprises one or more AAV ITRs. In some embodiments, an AAV ITR has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAVrh8, AAV9, AAVrhlO, AAVrh39, AAVrh43, AAV2 / 2-66, AAV2 / 2-84, AAV2 / 2-125, and variants thereof. In some embodiments, an AAV ITR is a mutant ITR (mTR) that lacks a functional terminal resolution site (TRS). The term “lacking a terminal resolution site” can refer to an AAV ITR that comprises a mutation (e.g., a sense mutation such as a non-synonymous mutation, or missense mutation) that abrogates the function of the terminal resolution site (TRS) of the ITR, or to a truncated AAV ITR that lacks a nucleic acid sequence encoding a functional TRS (e.g., a ATRS ITR). Without wishing to be bound by any particular theory, a rAAV vector comprising an ITR lacking a functional TRS produces a self-complementary rAAV vector, for example as described by McCarthy (2008) Molecular Therapy 16(10): 1648- 1656.

[0103] As used herein, the term “self-complementary AAV vector” (scAAV) refers to a vector containing a double-stranded vector genome generated by the absence of a terminal resolution site (TR) from one of the ITRs of the AAV. The absence of a TR prevents the initiation of replication at the vector terminus where the TR is not present. In general, scAAV vectors generate single-stranded, inverted repeat genomes, with a wild-type (wt) AAV TR at each end and a mutated TR (mTR) in the middle. In some embodiments, isolated nucleic acids comprise DNA sequences encoding RNA hairpin structures (e.g., shRNA, miRNA, and amiRNA) that can serve a function similar to a mutant inverted terminal repeat (mTR) during viral genome replication, generating self-complementary AAV vector (scAAV) genomes. For example, in some embodiments, the disclosure provides rAAV (e.g. self-complementary AAV; scAAV) vectors comprising a single- stranded self-complementary nucleic acid with inverted terminal repeats (ITRs) at each of two ends and a central portion comprising a promoter operably linked with a sequence encoding a hairpin-forming RNA (e.g., shRNA, miRNA, amiRNA, etc.). In some embodiments, the sequence encoding a hairpin-forming RNA (e.g., shRNA, miRNA, amiRNA, etc.) is substituted at a position of the self-complementary nucleic acid normally occupied by a mutant ITR.

[0104] “Recombinant AAV (rAAV) vectors” are typically composed of, at a minimum, a transgene, and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue. The instant disclosure provides a vector comprising a single, cis-acting wild-type ITR. In some embodiments, the ITR is a 5' ITR. In some embodiments, the ITR is a 3' ITR Generally, ITR sequences are about 145 bp in length. Preferably, substantially the entire sequences encoding the ITR(s) is used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). For example, an ITR may be mutated at its terminal resolution site (TR), which inhibits replication at the vector terminus where the TR has been mutated and results in the formation of a self-complementary AAV. Another example of such a molecule employed in the present disclosure is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' AAV ITR sequence and a 3' hairpin-forming RNA sequence. AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.

[0105] In some embodiments, the rAAVs of the disclosure are pseudotyped rAAVs. For example, a pseudotyped AAV vector containing the ITRs of serotype X encapsidated with the proteins of Y will be designated as AAVX / Y (e.g., AAV2 / 1 has the ITRs of AAV2 and the capsid of AAV1). In some embodiments, pseudotyped rAAVs may be useful for combining the tissue-specific targeting capabilities of a capsid protein from one AAV serotype with the viral DNA from another AAV serotype, thereby allowing targeted delivery of a transgene to a target tissue.

[0106] As described herein, the methods of producing rAAV particles involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of, AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.

[0107] In some embodiments, an AAV capsid protein is of an AAV serotype selected from the peptides (e.g., modified AAV9 capsids) disclosed herein. In some embodiments, an AAV capsid protein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 26-49.

[0108] The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, in the discussion of regulatory elements suitable for use with the transgene. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0109] The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAV of the disclosure may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct any embodiment of this disclosure are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present disclosure. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

[0110] Cells

[0111] A “host cell” refers to any cell that harbors, or is capable of harboring, a substance of interest or of packaging the nucleic acid of interest into a viral particle. Often a host cell is a mammalian cell. Examples of host cells include human cells, mouse cells, rat cells, dog cells, cat cells, hamster cells, monkey cells, insect cells, plant cells, or bacterial cells. Examples of insect cells include but are not limited to Spodoptera frugiperda (e.g., Sf9, Sf21), Spodoptera exigua, Heliothis virescens, Helicoverpa zea, Heliothis subflexa, Anticarsia gemmatalis, Trichopulsia ni (e.g., High-Five cells), Drosophila melanogaster (e.g., S2, S3), Antheraea eucalypti, Bombyx mori, Aedes alpopictus, Aedes aegyptii, and others. Examples of bacterial cells include, but are not limited to Escherichia coli, Corynebacterium glutamicum, and Pseudomonas fluorescens. Examples of yeast cells include but are not limited to Saccharomyces cerevisiae, Saccharomyces pombe, Pichia pastoris, Bacillus sp., Aspergillus sp., Trichoderma sp., and Myceliophthora thermophila Cl. Examples of plant cells include but are not limited to Nicotiana sp., Arabidopsis thaliana, Mays zea, Solanum sp., or Lemna sp.

[0112] In some embodiments, a host cell is a mammalian cell. Examples of mammalian cells include Henrietta Lacks tumor (HeLa) cells and baby hamster kidney (BHK-21) cells. In some embodiments, a host cell is a human cell, for example a HEK293T cell. A host cell may be used as a recipient of one or more viral transfer vectors and one or more accessory plasmids. The term includes the progeny of the original cell which has been transfected. Thus, a “host cell” as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

[0113] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0114] As used herein, the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

[0115] Administration

[0116] The rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. The rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, e.g., host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some embodiments a host animal does not include a human. In some embodiments, a subject is a mammal. In some embodiments, a subject is a human. Delivery of the rAAVs to a mammalian subject may be by, for example, intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In some embodiments, administration is performed using intra-articular injection. In some embodiments, administration is performed using direct injection to a joint. In some embodiments, the rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer the virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in certain instances, it may be desirable to deliver the virions to a joint of a subject. “Joint” is intended to include all cells and tissue of joint space (e.g., knee joint, shoulder joint, elbow joint, hip joint, ankle joint, spinal joint, finger joint, or toe joint). Thus, the term includes, but is not limited to, skin, muscle, bone cells, articular cartilage, meniscus, synovium, ligament tissues and the like. Recombinant AAVs may be delivered directly to a joint by injection into an artery, a vein, the bloodstream, the space within a joint, etc.

[0117] The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In some embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.

[0118] Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present disclosure.

[0119] Optionally, the compositions of the disclosure may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin. The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intraportal delivery to the liver), oral, inhalation (including intranasal and intratracheal delivery), intra- articular injection, intraocular, intravenous, intramuscular, subcutaneous, intradermal, intratumoral, intracranial (e.g., intrahippocampal), and other parental routes of administration. Routes of administration may be combined, if desired.

[0120] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary between animals or tissues. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some embodiments the rAAV is administered at a dose of 1010, 1011, 1012, 1013, 1014, or 1015genome copies per subject. In some embodiments the rAAV is administered at a dose of 1010, 1011, 1012, 1013, or 1014genome copies per kg. In some cases, a dosage between about 1011to 1012rAAV genome copies is appropriate. In certain embodiments, 1012rAAV genome copies is effective to target heart, liver, and pancreas tissues. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

[0121] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ~1013GC / ml or more). Methods for reducing aggregation of rAAVs are well-known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright FR, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference.) Formulations of pharmaceutically-acceptable excipients and carrier solutions are well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0122] Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active compound in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0123] In certain circumstances it will be desirable to deliver the rAAV-based therapeutic constructs in suitably formulated pharmaceutical compositions disclosed herein either subcutaneously, intraopancreatically, intranasally, parenterally, intravenously, intracranially (e.g., intrahippocampally), intramuscularly, intrathecally, or orally, intraperitoneally, or by inhalation. In some embodiments, the administration modalities as described in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each specifically incorporated herein by reference in its entirety) may be used to deliver rAAVs. In some embodiments, a preferred mode of administration is by portal vein injection.

[0124] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0125] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0126] Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0127] The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0128] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0129] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the present disclosure into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0130] Such formulations may be preferred for the introduction of pharmaceutically acceptable formulations of the nucleic acids or the rAAV constructs disclosed herein. The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Pat. No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

[0131] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

[0132] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500. ANG., containing an aqueous solution in the core. Alternatively, nanocapsule formulations of the rAAV may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0133] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Pat. No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Pat. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al., 1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) and feedback- controlled delivery (U.S. Pat. No. 5,697,899).

[0134] Treatment

[0135] In some aspects, the disclosure provides methods for treating a subject having a muscle disease or disorder. In some embodiments, a muscle disease is a skeletal muscle disease. Examples of skeletal muscle diseases include but are not limited to muscular dystrophy, amyotrophic lateral sclerosis, myopathies, myotonic dystrophy, or dermatomyositis.

[0136] In some aspects, the disclosure provides methods for treating a subject having a disease or disorder associated with nerve cells (e.g., a neurological disease or disorder). In some embodiments, a neurological disease or disorder is a peripheral nervous system disorder. Examples of peripheral nervous system disorders include but are not limited to Charcot-Marie- Tooth disease, Dejerine-Sottas disease, Multiple sclerosis, or Friedreich’s ataxia.

[0137] As used herein, a "subject" is interchangeable with a "subject in need thereof", both of which may refer to a subject having a muscular disease or a neurological disease, or a subject having an increased risk of developing such a disease or disorder relative to the population at large. A subject can be a human, non-human primate, rat, mouse, cat, dog, or other mammal.

[0138] As used herein, the terms “treatment”, “treating”, and “therapy” refer to therapeutic treatment and prophylactic or preventative manipulations. The terms further include ameliorating existing symptoms, preventing additional symptoms, ameliorating, or preventing the underlying causes of symptoms, preventing, or reversing causes of symptoms, for example, symptoms associated with muscle disease or neurological disease. Thus, the terms denote that a beneficial result has been conferred on a subject having a muscle disease or neurological disease, or with the potential to develop such a disorder. Furthermore, the term "treatment" is defined as the application or administration of an agent (e.g., therapeutic agent or a therapeutic composition) to a subject, or an isolated tissue or cell line from a subject, who may have a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

[0139] Therapeutic agents or therapeutic compositions may include a compound in a pharmaceutically acceptable form that prevents and / or reduces the symptoms of a particular disease (e.g., a muscle disease or neurological disease). For example, a therapeutic composition may be a pharmaceutical composition that prevents and / or reduces the symptoms of muscular dystrophy, amyotrophic lateral sclerosis, myopathies, myotonic dystrophy, dermatomyositis, Charcot-Marie-Tooth disease, Dejerine-Sottas disease, Multiple sclerosis, or Friedreich’s ataxia. It is contemplated that the therapeutic composition of the present invention will be provided in any suitable form. The form of the therapeutic composition will depend on a number of factors, including the mode of administration as described herein. The therapeutic composition may contain diluents, adjuvants, and excipients, among other ingredients as described herein.

[0140] EXAMPLES

[0141] Example 1: Comparison of Production Efficiency of AAV Capsids In Vitro

[0142] HEK293 cells were transiently transfected with AAV vector, containing either an insertion of amino acids or replacement of amino acids in loop IV of an AAV9 capsid protein. 72 hours post transfection the expression of the capsid protein was measured for its packaging efficiency relative to MY04A (FIG. 1).

[0143] Example 2: Comparison of AAV Capsid Expression Across Tissues In Vivo

[0144] Several AAV9-based capsid libraries carrying a randomized seven-amino acid insertion, or replacement of amino acids in loop IV were developed. These libraries were screened in human myotubes differentiated from human myoblasts from healthy donors and wild type mice. The resulting capsids from both screens were combined into a single secondary library and screened in non-human primates (cynomolgus macaques) and mice. The results from this in vivo screening yielded mutually exclusive capsid candidates from mice and non-human primates (cynomolgus macaques). Multiplex biodistribution was analyzed in the non-human primates (cynomolgus macaques) comparing the top candidates from the screen with AAV9, AAV8, AAVrh74 and AAV-MY0-4A (Tabebordbar et al., 2021, Cell 184, 1-20). Barcode frequency was analyzed at the RNA and DNA level for biceps brachii (FIGs. 2A-2B, Table 1, and Table 2), triceps brachii (FIGs. 3A-3B, Table 3, and Table 4), bicep femoris (FIGs. 4A-4B, Table 5, and Table 6), rectus femoris (FIGs. 5A-5B, Table 7 and Table 8), soleus (FIGs. 6A-6B, Table 9 , and Table 10), intercostal muscle (FIGs. 7A-7B, Table 11, and Table 12), diaphragm (FIGs. 8A- 8B, Table 13, and Table 14), heart (FIGs. 9A-9B Table 15, and Table 16), liver (FIGs. 10A-10B, Table 17, and Table 18), lungs (FIGs. 11A-11B, Table 19, and Table 20), the smooth muscle of the aorta (FIGs. 12A-12B, Table 21, and Table 22), pancreas (FIGs. 13A-13B, Table 23, and Table 24), kidney (FIGs. 14A-14B, Table 25, and Table 26), brain (FIGs. 16A-16L and FIGs. 19A-19L, and Tables 27-48), cervical spinal cord (FIGs. 17A and 20A, Table 49, and Table 50), thoracic spinal cord (FIGs. 17B and 20B, Table 51, and Table 52), cervical dorsal root ganglion (DRG) (FIGs. 17C and 20C, Table 53, and Table 54), thoracic DRG (FIGs. 17D and 20D, Table 55, and Table 56), sciatic nerve (FIGs. 17E and 20E, Table 57, and Table 58), phrenic nerve (FIGs. 17F and 20F, Table 59, and Table 60), optic nerve (FIGs. 17G and 20G, Table 61, and Table 62), and retina (FIGs. 17H and 20H, Table 63, and Table 64). The enrichment over the injected library was calculated and normalized to AAV9. Several candidates identified in the screen were more efficient for skeletal gene transfer than AAV9 and comparable to AAV-MYO- 4A.

[0145] The comparative biodistribution study in non-human primates has identified several candidates that are 3-5-fold more efficient than AAV9 as well as AAV8, and AAVrh74 for muscle gene transfer . In addition, at least one candidate revealed a dramatic increase enrichment in peripheral nerve compared to AAV9. In some embodiments, this capsid could be used in AAV gene therapy for diseases that affect peripheral nerve (e.g., Charcot-Marie-Tooth diseases).

[0146] Table 1: Relative Expression of AAV9-based Capsid Library in Biceps Brachii of Cynomolgus Macaque 1

[0147] Table 2: Relative Expression of AAV9-based Capsid Library in Biceps Brachii of

[0148] Cynomolgus Macaque 2

[0149] Table 3: Relative Expression of AAV9-based Capsid Library in Triceps Brachii of

[0150] Cynomolgus Macaque 1 Table 4: Relative Expression of AAV9-based Capsid Library in Triceps Brachii of

[0151] Cynomolgus Macaque 2 Table 5: Relative Expression of AAV9-based Capsid Library in Bicep Femoris of

[0152] Cynomolgus Macaque 1

[0153] Table 6: Relative Expression of AAV9-based Capsid Library in Bicep Femoris of

[0154] Cynomolgus Macaque 2

[0155] Table 7: Relative Expression of AAV9-based Capsid Library in Rectus Femoris of

[0156] Cynomolgus Macaque 1 Table 8: Relative Expression of AAV9-based Capsid Library in Rectus Femoris of

[0157] Cynomolgus Macaque 2

[0158] Table 9: Relative Expression of AAV9-based Capsid Library in Soleus of Cynomolgus Macaque 1

[0159] Table 10: Relative Expression of AAV9-based Capsid Library in Soleus of Cynomolgus

[0160] Macaque 2

[0161] Table 11: Relative Expression of AAV9-based Capsid Library in Intercostal Muscle of

[0162] Cynomolgus Macaque 1 Table 12: Relative Expression of AAV9-based Capsid Library in Intercostal Muscle of

[0163] Cynomolgus Macaque 2

[0164] Table 13: Relative Expression of AAV9-based Capsid Library in the Diaphragm of Cynomolgus Macaque 1

[0165] Table 14: Relative Expression of AAV9-based Capsid Library in the Diaphragm of

[0166] Cynomolgus Macaque 2

[0167] Table 15: Relative Expression of AAV9-based Capsid Library in the Heart of Cynomolgus

[0168] Macaque 1 Table 16: Relative Expression of AAV9-based Capsid Library in the Heart of Cynomolgus

[0169] Macaque 2

[0170] Table 17: Relative Expression of AAV9-based Capsid Library in the Liver of Cynomolgus

[0171] Macaque 1

[0172] Table 18: Relative Expression of AAV9-based Capsid Library in the Liver of Cynomolgus

[0173] Macaque 2

[0174] Table 19: Relative Expression of AAV9-based Capsid Library in the Lung of Cynomolgus

[0175] Macaque 1 Table 20: Relative Expression of AAV9-based Capsid Library in the Lung of Cynomolgus

[0176] Macaque 2

[0177] Table 21: Relative Expression of AAV9-based Capsid Library in the Smooth Muscle of the

[0178] Aorta of Cynomolgus Macaque 1

[0179] Table 22: Relative Expression of AAV9-based Capsid Library in in the Smooth Muscle of the Aorta of Cynomolgus Macaque 2

[0180] Table 23: Relative Expression of AAV9-based Capsid Library in in the Pancreas of

[0181] Cynomolgus Macaque 1 Table 24: Relative Expression of AAV9-based Capsid Library in in the Pancreas of

[0182] Cynomolgus Macaque 2

[0183] Table 25: Relative Expression of AAV9-based Capsid Library in in the Kidney of

[0184] Cynomolgus Macaque 1

[0185] Table 26: Relative Expression of AAV9-based Capsid Library in in the Kidney of

[0186] Cynomolgus Macaque 2

[0187] Table 27: Relative Expression of AAV9-based Capsid Library in the Brain location 1 (e.g., see FIG. 16A) of Cynomolgus Macaque 1 Table 28: Relative Expression of AAV9-based Capsid Library in the Brain location 1 (e.g., see FIG. 18A) of Cynomolgus Macaque 2

[0188] Table 29: Relative Expression of AAV9-based Capsid Library in the Brain location 2 (e.g., see FIG. 16B) of Cynomolgus Macaque 1

[0189] Table 30: Relative Expression of AAV9-based Capsid Library in the Brain location 2 (e.g., see FIG. 18B) of Cynomolgus Macaque 2

[0190] Table 31: Relative Expression of AAV9-based Capsid Library in the Brain location 3 (e.g., see FIG. 16C) of Cynomolgus Macaque 1 Table 32: Relative Expression of AAV9-based Capsid Library in the Brain location 3 (e.g., see FIG. 18C) of Cynomolgus Macaque 2

[0191] Table 33: Relative Expression of AAV9-based Capsid Library in the Brain location 4 (e.g., see FIG. 16C) of Cynomolgus Macaque 1

[0192] Table 34: Relative Expression of AAV9-based Capsid Library in the Brain location 4 (e.g., see FIG. 18C) of Cynomolgus Macaque 2

[0193] Table 33: Relative Expression of AAV9-based Capsid Library in the Brain location 5 (e.g., see FIG. 16C) of Cynomolgus Macaque 1 Table 34: Relative Expression of AAV9-based Capsid Library in the Brain location 5 (e.g., see FIG. 18C) of Cynomolgus Macaque 2

[0194] Table 35: Relative Expression of AAV9-based Capsid Library in the Brain location 6 (e.g., see FIG. 16C) of Cynomolgus Macaque 1

[0195] Table 36: Relative Expression of AAV9-based Capsid Library in the Brain location 6 (e.g., see FIG. 18C) of Cynomolgus Macaque 2

[0196] Table 37: Relative Expression of AAV9-based Capsid Library in the Brain location 7 (e.g., see FIG. 16D) of Cynomolgus Macaque 1 Table 38: Relative Expression of AAV9-based Capsid Library in the Brain location 7 (e.g., see FIG. 18D) of Cynomolgus Macaque 2

[0197] Table 39: Relative Expression of AAV9-based Capsid Library in the Brain location 8 (e.g., see FIG. 16E) of Cynomolgus Macaque 1

[0198] Table 40: Relative Expression of AAV9-based Capsid Library in the Brain location 8 (e.g., see FIG. 18E) of Cynomolgus Macaque 2

[0199] Table 41: Relative Expression of AAV9-based Capsid Library in the Brain location 9 (e.g., see FIG. 16F) of Cynomolgus Macaque 1 Table 42: Relative Expression of AAV9-based Capsid Library in the Brain location 9 (e.g., see FIG. 18F) of Cynomolgus Macaque 2

[0200] Table 43: Relative Expression of AAV9-based Capsid Library in the Brain location 10 (e.g., see FIG. 16F) of Cynomolgus Macaque 1

[0201] Table 44: Relative Expression of AAV9-based Capsid Library in the Brain location 10

[0202] (e.g., see FIG. 18F) of Cynomolgus Macaque 2

[0203] Table 45: Relative Expression of AAV9-based Capsid Library in the Brain location 11

[0204] (e.g., see FIG. 16F) of Cynomolgus Macaque 1 Table 46: Relative Expression of AAV9-based Capsid Library in the Brain location 11

[0205] (e.g., see FIG. 18F) of Cynomolgus Macaque 2

[0206] Table 47: Relative Expression of AAV9-based Capsid Library in the Brain location 12 (e.g., see FIG. 16F) of Cynomolgus Macaque 1

[0207] Table 48: Relative Expression of AAV9-based Capsid Library in the Brain location 12

[0208] (e.g., see FIG. 18F) of Cynomolgus Macaque 2

[0209] Table 49: Relative Expression of AAV9-based Capsid Library in the cervical spinal cord

[0210] (e.g., see FIG. 17A) of Cynomolgus Macaque 1 Table 50: Relative Expression of AAV9-based Capsid Library in the cervical spinal cord

[0211] (e.g., see FIG. 20A) of Cynomolgus Macaque 2

[0212] Table 51: Relative Expression of AAV9-based Capsid Library in the thoracic spinal cord (e.g., see FIG. 17B) of Cynomolgus Macaque 1

[0213] Table 52: Relative Expression of AAV9-based Capsid Library in the thoracic spinal cord

[0214] (e.g., see FIG. 20B) of Cynomolgus Macaque 2

[0215] Table 53: Relative Expression of AAV9-based Capsid Library in the cervical dorsal root ganglion (DRG) (e.g., see FIG. 17C) of Cynomolgus Macaque 1 Table 54: Relative Expression of AAV9-based Capsid Library in the cervical dorsal root ganglion (DRG) (e.g., see FIG. 20C) of Cynomolgus Macaque 2

[0216] Table 55: Relative Expression of AAV9-based Capsid Library in the thoracic DRG (e.g., see FIG. 17D) of Cynomolgus Macaque 1

[0217] Table 56: Relative Expression of AAV9-based Capsid Library in the thoracic DRG (e.g., see FIG. 20D) of Cynomolgus Macaque 2

[0218] Table 57: Relative Expression of AAV9-based Capsid Library in the sciatic nerve (e.g., see

[0219] FIG. 17E) of Cynomolgus Macaque 1 Table 58: Relative Expression of AAV9-based Capsid Library in the sciatic nerve (e.g., see

[0220] FIG. 20E) of Cynomolgus Macaque 2

[0221] Table 59: Relative Expression of AAV9-based Capsid Library in the phrenic nerve (e.g., see FIG. 17F) of Cynomolgus Macaque 1

[0222] Table 60: Relative Expression of AAV9-based Capsid Library in the phrenic nerve (e.g., see FIG. 20F) of Cynomolgus Macaque 2

[0223] Table 61: Relative Expression of AAV9-based Capsid Library in the optic nerve (e.g., see

[0224] FIG. 17G) of Cynomolgus Macaque 1 Table 62: Relative Expression of AAV9-based Capsid Library in the optic nerve (e.g., see

[0225] FIG. 20G) of Cynomolgus Macaque 2

[0226] Table 63: Relative Expression of AAV9-based Capsid Library in the retina (e.g., see FIG. 17H) of Cynomolgus Macaque 1

[0227] Table 64: Relative Expression of AAV9-based Capsid Library in the retina (e.g., see FIG.

[0228] 20H) of Cynomolgus Macaque 2

[0229] Sequences:

[0230] AAV9:

[0231] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0232] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAG PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0233] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 1)

[0234] DTVHQYR (SEQ ID NO: 2)

[0235] QRHHNTQ (SEQ ID NO: 3)

[0236] RCQEHRV (SEQ ID NO: 4)

[0237] RGQEHRV (SEQ ID NO: 5)

[0238] KYYRTTE (SEQ ID NO: 6)

[0239] RMMQQDV (SEQ ID NO: 7)

[0240] ISRPAVV (SEQ ID NO: 8)

[0241] PGGHPGT (SEQ ID NO: 9)

[0242] HVTHQHR (SEQ ID NO: 10) EHAHAHR (SEQ ID NO: 11)

[0243] SARHTQY (SEQ ID NO: 12)

[0244] RMRTHDS (SEQ ID NO: 13)

[0245] QANVRPC (SEQ ID NO: 14)

[0246] VQAQAPA (SEQ ID NO: 15)

[0247] ARQPHIC (SEQ ID NO: 16)

[0248] PARQDRA (SEQ ID NO: 17)

[0249] PVSQPYM (SEQ ID NO: 18)

[0250] RGITHMP (SEQ ID NO: 19)

[0251] YVQSRSA (SEQ ID NO: 20)

[0252] GAQAVWR (SEQ ID NO: 21)

[0253] YRHSQQQ (SEQ ID NO: 22)

[0254] MRSAPVV (SEQ ID NO: 23)

[0255] YRSQAHA (SEQ ID NO: 24)

[0256] QAHVQPR (SEQ ID NO: 25)

[0257] MSK1:

[0258] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0259] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0260] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0261] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0262] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0263] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0264] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0265] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGDTVHQYRQNQQ

[0266] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0267] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0268] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 26)

[0269] MSK2:

[0270] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0271] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0272] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0273] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0274] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0275] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0276] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQRHHNTQQNQQ

[0277] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0278] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0279] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 27)

[0280] MSK3:

[0281] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0282] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0283] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0284] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0285] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0286] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0287] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0288] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGRCQEHRVQNQQ

[0289] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0290] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0291] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 28)

[0292] MSK4:

[0293] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0294] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0295] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0296] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0297] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0298] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0299] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0300] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGRGQEHRVQNQQ

[0301] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0302] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0303] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 29)

[0304] MSK5:

[0305] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0306] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0307] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0308] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0309] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGKYYRTTEQNQQ

[0310] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0311] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0312] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 30)

[0313] MSK6:

[0314] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0315] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0316] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0317] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0318] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0319] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0320] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0321] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGRMMQQDVQNQ

[0322] QTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0323] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0324] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 31)

[0325] MSK7:

[0326] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0327] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0328] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0329] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0330] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0331] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0332] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0333] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGISRPAVVQNQQT

[0334] LKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLM

[0335] NPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYG

[0336] QVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLM GGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 32)

[0337] MSK8:

[0338] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0339] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0340] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0341] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0342] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0343] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGPGGHPGTQNQQ TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0344] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 33)

[0345] MSK9:

[0346] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0347] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0348] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0349] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0350] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0351] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0352] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0353] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGHVTHQHRQNQQ

[0354] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0355] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0356] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 34)

[0357] MSK10:

[0358] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0359] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0360] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0361] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0362] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0363] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0364] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0365] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGEHAHAHRQNQQ

[0366] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0367] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0368] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 35)

[0369] MSK11:

[0370] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0371] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0372] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0373] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0374] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0375] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0376] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0377] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGSARHTQYQNQQ

[0378] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 36)

[0379] MSK12:

[0380] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0381] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0382] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0383] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0384] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0385] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0386] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0387] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGRMRTHDSQNQQ

[0388] TLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSL

[0389] MNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATES

[0390] YGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSP LMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSK RWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 37)

[0391] MSK13:

[0392] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0393] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0394] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0395] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0396] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0397] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0398] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0399] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIQANVRPCQTLKFSVAG

[0400] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0401] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0402] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0403] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 38)

[0404] MSK14:

[0405] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0406] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0407] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0408] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0409] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0410] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0411] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0412] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIVQAQAPAQTLKFSVAG

[0413] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0414] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0415] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 39)

[0416] MSK15:

[0417] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0418] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0419] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0420] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0421] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0422] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0423] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0424] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIARQPHICQTLKFSVAGP

[0425] SNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMAS

[0426] HKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQS

[0427] AQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKH

[0428] PPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTS

[0429] NYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 40)

[0430] MSK16:

[0431] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0432] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0433] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0434] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0435] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0436] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0437] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0438] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIPARQDRAQTLKFSVAG

[0439] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0440] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0441] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0442] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0443] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 41)

[0444] MSK17:

[0445] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0446] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0447] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0448] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0449] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0450] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0451] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0452] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIPVSQPYMQTLKFSVAG

[0453] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0454] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0455] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 42)

[0456] MSK18:

[0457] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0458] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0459] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0460] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0461] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0462] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0463] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0464] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIRGITHMPQTLKFSVAG

[0465] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0466] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0467] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0468] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0469] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 43)

[0470] MSK19:

[0471] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0472] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0473] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0474] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0475] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0476] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0477] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0478] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIYVQSRSAQTLKFSVAG

[0479] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0480] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0481] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0482] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0483] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 44)

[0484] MSK20:

[0485] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0486] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0487] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0488] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0489] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0490] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0491] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0492] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIGAQAVWRQTLKFSVA

[0493] GPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAM

[0494] ASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNH

[0495] QSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGM KHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQY

[0496] TSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 45)

[0497] MSK21:

[0498] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0499] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0500] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0501] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0502] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0503] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0504] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0505] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIYRHSQQQQTLKFSVAG

[0506] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0507] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0508] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0509] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0510] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 46)

[0511] MSK22:

[0512] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0513] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0514] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0515] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0516] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0517] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0518] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0519] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIMRSAPVVQTLKFSVAG

[0520] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0521] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0522] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0523] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT

[0524] SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 47)

[0525] MSK23:

[0526] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0527] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0528] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0529] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0530] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0531] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0532] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0533] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIYRSQAHAQTLKFSVAG

[0534] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0535] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 48)

[0536] MSK24:

[0537] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG

[0538] NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG

[0539] NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL

[0540] NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH

[0541] CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNR

[0542] FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD

[0543] SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRT

[0544] GNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTIQAHVQPRQTLKFSVAG

[0545] PSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0546] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQ

[0547] SAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMK

[0548] HPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYT SNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL(SEQ ID NO: 49)

[0549] NGSGQNQ (SEQ ID NO: 50)

[0550] AAV9 VP2:

[0551] TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSG

[0552] VGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNN

[0553] HLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL

[0554] NFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVF

[0555] MIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSL

[0556] DRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVST

[0557] TVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTG

[0558] RDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMV

[0559] WQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKD

[0560] KLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEP

[0561] RPIGTRYLTRNL (SEQ ID NO: 51)

[0562] AAV9 VP3:

[0563] MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQ

[0564] ISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLF

[0565] NIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQY

[0566] GYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLM

[0567] NPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQ NNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNV

[0568] DADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDR DVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFI TQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTR YLTRNL (SEQ ID NO: 52)

Claims

CLAIMSWhat is claimed is:

1. A peptide comprising an amino acid sequence having at least 80% identity to the amino acid sequence set forth in SEQ ID NO: 1 (wild-type AAV9), and one or more amino acid insertions or substitutions at one or more positions selected from 449-468 relative to SEQ ID NO:1.

2. The peptide of claim 1, wherein the one or more amino acid insertions comprises between 5 amino acids and 10 amino acids.

3. The peptide of claim 1, wherein the one or more amino acid substitutions comprises between 5 amino acids and 10 amino acids.

4. The peptide of any one of claims 1 to 3, wherein the amino acid sequence is at least 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1.

5. The peptide of claim 2, wherein the one or more amino acid insertions is 7 amino acids in length.

6. The peptide of claim 3, wherein the one or more amino acid substitutions is 7 amino acids in length.

7. The peptide of any one of claims 1, 2, 4 and 5, wherein the amino acid insertion comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-25.

8. The peptide of any one of claims 1, 3, 4 and 6, wherein the amino acid sequence of the peptide comprises the sequence set forth in any one of SEQ ID NOs: 2-25.

9. The peptide of any one of claims 1, 2, 4, 5 and 7, wherein the amino acids insertions are between amino acid 455 and 456 of AAV9 capsid, SEQ ID NO:1.

10. The peptide of any one of claims 1, 3, 4, 6 and 8, wherein the amino acids substitutions are of amino acids 452 to 458 of AAV9 capsid, SEQ ID NO:1.

11. The peptide of any one of claims 1 to 10, wherein the peptide comprises the amino acid sequence as set forth in any one of SEQ ID NO: 26-49.

12. A nucleic acid encoding the peptide of any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. The vector of claim 13, wherein the vector is a plasmid.

15. A host cell comprising the plasmid of claim 14.

16. A recombinant adeno-associated virus (rAAV) comprising:(i) a capsid protein comprising the peptide of any one of claims 1 to 11 or the amino acid sequence set forth in any one of SEQ ID NOs: 26-49; and(ii) an isolated nucleic acid comprising a transgene having a promoter operably linked to a nucleic acid sequence encoding a gene product, flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).

17. A method of delivering a transgene to a cell, the method comprising contacting the cell with the rAAV of claim 16.

18. The method of claim 17, wherein the transgene is a therapeutic gene product, therapeutic protein, or an inhibitory nucleic acid.

19. The method of claim 18, wherein the therapeutic protein is a toxin, an enzyme, a growth factor, an interleukin, an interferons, an anti-apoptosis factor, a cytokine, an anti-diabetic factor, an anti-apoptosis agent, a coagulation factor, an anti-tumor factor, or an anti-proliferative protein.

20. The method of claim 18, wherein the inhibitory nucleic acid is a shRNA, a dsRNA, a siRNA, a miRNA, or an artificial miRNA.

21. The method of any one of claims 17 to 20, wherein the cell is a muscle cell.

22. The method any one of claims 17 to 20, wherein the cell is a nerve cell.

23. The method of claim 22, wherein the nerve cell is a peripheral nerve cell.

24. The method of any one of claims 17 to 23, wherein the cell is a mammalian cell.

25. The method of claim 24, wherein the mammalian cell is a human cell.

26. A method for treating a muscle disease or neural disease of a subject, the method comprising administering the rAAV of claim 17 to the subject.

27. The method of claim 26, wherein the disease is a muscle disease.

28. The method of claim 26, wherein the muscle disease is a skeletal muscle disease.

29. The method of claim 28, wherein the skeletal muscle disease is muscular dystrophy, amyotrophic lateral sclerosis, myopathies, myotonic dystrophy, or dermatomyositis.

30. The method of claim 26, wherein the disease is a neurological disorder.

31. The method of claim 30, wherein the neurological disorder is a peripheral nervous system disorder.

32. The method of claim 31, wherein the peripheral nervous system disorder is a Charcot- Marie-Tooth disease, Dejerine-Sottas disease, Multiple sclerosis, or Friedreich’s ataxia.

33. A capsid protein comprising the amino acid sequence set forth in any one of SEQ ID NO:

Images