EP4665743A2 - Rgd-containing peptides for delivering payloads - Google Patents

Rgd-containing peptides for delivering payloads

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Publication number
EP4665743A2
EP4665743A2 EP24714284.7A EP24714284A EP4665743A2 EP 4665743 A2 EP4665743 A2 EP 4665743A2 EP 24714284 A EP24714284 A EP 24714284A EP 4665743 A2 EP4665743 A2 EP 4665743A2
Authority
EP
European Patent Office
Prior art keywords
capsid protein
seq
aav capsid
sequence
peptide insertion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24714284.7A
Other languages
German (de)
French (fr)
Inventor
Adelaida Palla CACHORRO
Wei Tong CHEN
Ka Ying HUNG
Quan JIN
Anne-Sophie PARMENTIER-BATTEUR
Junghae Suh SPARROW
Yuan Yuan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biogen MA Inc
Original Assignee
Biogen MA Inc
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Filing date
Publication date
Application filed by Biogen MA Inc filed Critical Biogen MA Inc
Publication of EP4665743A2 publication Critical patent/EP4665743A2/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present disclosure provides technologies that can address certain limitations identified in existing targeted delivery of payloads.
  • the technologies provided herein are particularly useful for specifically delivering payloads to a target cell or tissue.
  • technologies provided here can also increase the potency of a payload.
  • the technologies provided herein are useful for delivering payloads to a target cell or tissue, e.g., muscle cells or tissue.
  • Page 1 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0006]
  • the present disclosure encompasses -targeting moieties comprising a peptide comprising an RGD-motif.
  • Targeting moieties disclosed herein include muscle-targeting moieties.
  • Also disclosed herein are recombinant adeno-associated virus (rAAV) particles comprising a variant AAV capsid comprising a targeting moiety, e.g., a muscle-targeting moiety disclosed herein.
  • rAAV adeno-associated virus
  • a targeting moiety e.g., a muscle-targeting moiety in a variant AAV capsid is also referred to as a “peptide insertion.”
  • a targeting moiety e.g., a muscle-targeting moiety, e.g., in a variant AAV capsid, provides muscle-cell tropism.
  • rAAV particles comprising a variant capsid having a peptide insertion disclosed herein binds to and/or recognizes a target on a cell, e.g., a muscle cell. Also disclosed herein are compositions comprising rAAV particles disclosed herein, and uses of the same.
  • a recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F; (III) RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX 1 X 2 RX 3 (SEQ ID NO: 1), wherein X 1 and X 2 are independently any amino acid and X 3 is Y, W, or F ; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX 1
  • X 3 is W or F. In some embodiments X3 is Y. In some embodiments X3 is W. In some embodiments X3 is F. [0009] In some embodiments, a peptide insertion comprises a sequence provided in Table 2. In some embodiments, a peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F ; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX1X2R
  • X3 is W or F. In some embodiments X3 is Y. In some embodiments X3 is W. In some embodiments X3 is F. [0011] In some embodiments, a peptide insertion consists of a sequence provided in Table 2. In some embodiments, a peptide insertion consists of the sequence of RGDPQRW (SEQ ID NO: 802).
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3QX
  • X3 is W or F. In some embodiments, X 3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. Page 3 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0013]
  • a peptide insertion comprises a sequence provided in Table 3. In some embodiments, a peptide insertion does not comprise the sequence of RGDYQAV (SEQ ID NO: 1764). In some embodiments, a peptide insertion does not comprise the sequence of RGDYQTL (SEQ ID NO: 1814). In some embodiments, a peptide insertion does not comprise RGDYQEL (SEQ ID NO: 2008).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3Q
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0015] In some embodiments, a peptide insertion consists of a sequence provided in Table 3.
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDHX1X
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0017] In some embodiments, a peptide insertion comprises a sequence provided in Table 4. In some embodiments, a peptide insertion does not comprise the sequence of RGDHASW (SEQ ID NO: 191). In some embodiments, a peptide insertion does not comprise the sequence of RGDHSGW (SEQ ID NO: 322). In some embodiments, a peptide insertion does not comprise the sequence of RGDHSTW (SEQ ID NO: 333).
  • a peptide insertion does not comprise the sequence of RGDHTQW (SEQ ID NO: 349). In some Page 4 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, a peptide insertion does not comprise the sequence of RGDHQNF (SEQ ID NO: 283).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consist of the sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consist of the sequence of RGDHX1X2
  • X3 is W or F. In some embodiments, X 3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. [0019] In some embodiments, a peptide insertion consists of a sequence provided in Table 4.
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDPX1X
  • X3 is W or F. In some embodiments, X 3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. [0021] In some embodiments, a peptide insertion comprises a sequence provided in Table 5. In some embodiments, a peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consist of the sequence of RGDPX 1 X 2 X 3 (SEQ ID NO: 4), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid Page 5 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental A
  • X 3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0023] In some embodiments, a peptide insertion consists of a sequence provided in Table 5. In some embodiments, a peptide insertion consists of the sequence of RGDPQRW (SEQ ID NO: 802).
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3X1
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0025] In some embodiments, a peptide insertion comprises a sequence provided in Table 6.
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3X
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0027] In some embodiments, a peptide insertion consists of a sequence provided in Table 6.
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV Page 6 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0029] In some embodiments, a peptide insertion comprises a sequence provided in Table 7. In some embodiments, a peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0030] In some embodiments, a peptide insertion does not comprise the sequence of RGDVQNF (SEQ ID NO: 2011). In some embodiments, a peptide insertion does not comprise the sequence of RGDHQNF (SEQ ID NO: 283).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX1
  • X3 is W or F. In some embodiments, X 3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. [0032] In some embodiments, a peptide insertion consists of a sequence provided in Table 7. In some embodiments, a peptide insertion consists of the sequence of RGDPQRW (SEQ ID NO: 802).
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX 3
  • X 3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0034] In some embodiments, a peptide insertion comprises a sequence provided in Table 8. In some embodiments, a peptide insertion does not comprise the sequence of Page 7 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) RGDYVSV (SEQ ID NO: 1948). In some embodiments, a peptide insertion does not comprise the sequence of RGDYSSV (SEQ ID NO: 1882).
  • a peptide insertion does not comprise the sequence of RGDYHSF (SEQ ID NO: 1623). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTSM (SEQ ID NO: 1906). In some embodiments, a peptide insertion does not comprise the sequence of RGDYASL (SEQ ID NO: 2015). In some embodiments, a peptide insertion does not comprise the sequence of RGDYNSL (SEQ ID NO: 2016). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTSV (SEQ ID NO: 2018). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTST (SEQ ID NO: 2021).
  • a peptide insertion does not comprise the sequence of RGDYTSL (SEQ ID NO: 2023).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • VR variable region
  • X 3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0036] In some embodiments, a peptide insertion consists of a sequence provided in Table 8. In some embodiments, a peptide insertion does not consists of the sequence of RGDYVSV (SEQ ID NO: 1948).
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of LRGDX1X
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. Page 8 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0038] In some embodiments, a peptide insertion comprises a sequence provided in Table 9.
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of LRGDX1
  • X3 is W or F. In some embodiments, X 3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. [0040] In some embodiments, a peptide insertion consists of a sequence provided in Table 9.
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. [0042] In some embodiments, a peptide insertion comprises a sequence provided in Table 10.
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX1GLX2 (SEQ ID NO:
  • X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X 3 is W. In some embodiments, X 3 is F. Page 9 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0044] In some embodiments, a peptide insertion consist of a sequence provided in Table 10.
  • a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and
  • a peptide insertion comprises a sequence provided in Table 11. In some embodiments, a peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide comprises the sequence of RGDYREV (SEQ ID NO: 1829).
  • a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDYX1X2
  • a peptide insertion consists of a sequence provided in Table 11. In some embodiments, a peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide consists of the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide consists of the sequence of RGDYREV (SEQ ID NO: 1829).
  • This disclosure further provides a rAAV particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence provided in Table 1; and (ii) the peptide insertion site is in a variable region of a Page 10 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence provided in Table 1; and (ii) the peptide insertion site is in a variable
  • variant AAV capsid protein comprising a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) the peptide insertion comprises a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX 3 QX 1 X 2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X 3 is
  • an insertion site is located between two adjacent amino acids in the variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein).
  • an insertion site is located between two non-adjacent amino acids in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein).
  • a peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of a parental AAV capsid protein (e.g., an AAV9 capsid protein).
  • a parental AAV capsid protein e.g., an AAV9 capsid protein
  • a peptide insertion is in VR-VIII of a parental AAV capsid Page 11 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein (e.g., an AAV9 capsid protein).
  • a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2, or VP3 of an AAV9 capsid protein or the corresponding positions in a capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein.
  • VR-VIII comprises amino acids 580 to 601 of a VP1, VP2, or VP3 of an AAV9 capsid protein or the corresponding positions in a capsid protein (e.
  • a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2, or VP3 of an AAV9 capsid protein.
  • a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein.
  • an insertion of the heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein).
  • an insertion of the heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein).
  • an insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding position in the capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein.
  • a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a VP e.g., VP1, VP2, and/or VP3
  • a variant AAV capsid protein comprises: (1) a peptide insertion Page 12 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) comprising any one sequence provided in any one of Tables 1-11, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a targeting moiety e.g., a muscle- targeting moiety, conjugated to a payload.
  • a targeting moiety e.g., a muscle-targeting moiety
  • a targeting moiety e.g., a muscle-targeting moiety
  • a targeting moiety e.g., a muscle-targeting moiety, encapsidates a payload, e.g., as described herein.
  • a targeting moiety e.g., a muscle-targeting moiety
  • a payload e.g., as described herein.
  • a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1825.
  • a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1551.
  • a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1829.
  • an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein.
  • an isolated cell transduced with an rAAV particle disclosed herein is also disclosed herein.
  • a cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein.
  • This disclosure provides a composition comprising a targeting moiety, e.g., a muscle-targeting moiety, and a payload, wherein the targeting moiety, e.g., a muscle-targeting moiety, comprises a peptide comprising a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and
  • a composition comprising a targeting moiety, e.g., a muscle-targeting moiety, and a payload, wherein the targeting moiety, e.g., a muscle-targeting moiety, comprises a peptide comprising a sequence provided in any one of Tables 1-11.
  • a peptide insertion in a targeting moiety e.g., a muscle-targeting moiety, comprises a sequence of SEQ ID NO: 1825.
  • a peptide insertion in a targeting moiety, e.g., a muscle-targeting moiety comprises a sequence of SEQ ID NO: 1551.
  • a peptide comprises the sequence of SEQ ID NO: 1829.
  • a targeting moiety e.g., a muscle-targeting moiety
  • a targeting moiety is conjugated to the payload.
  • a targeting moiety e.g., a muscle-targeting moiety
  • a viral protein e.g., an AAV capsid protein.
  • a targeting moiety e.g., a muscle-targeting moiety, is part of, e.g., incorporated into, a vector.
  • a targeting moiety e.g., a muscle-targeting moiety
  • a pharmaceutical composition comprising: (a) a rAAV particle disclosed herein; and (b) a pharmaceutically acceptable excipient.
  • a method of delivering a payload to a muscle cell comprising administering a pharmaceutical composition disclosed herein to a muscle cell.
  • a muscle cell is in vitro.
  • a muscle cell is in vivo.
  • a muscle cell is from a subject that has, or has been determined to have, a muscle disorder.
  • Page 14 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0075]
  • This disclosure provides a method of treating a subject having a muscle disorder and/or ameliorating a symptom of a muscle disorder in a subject, the method comprising administering to the subject a pharmaceutical composition disclosed herein.
  • a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • a pharmaceutical composition is administered via a route of administration chosen from: intramuscular, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, intra-cisterna magna, or limb perfusion.
  • a route of administration chosen from: intramuscular, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, intra-cisterna magna, or limb perfusion.
  • a subject is a human.
  • a variant AAV capsid protein confers increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a variant AAV capsid protein confers at least 5-fold, at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a variant AAV capsid protein confers about 5-fold, about 10-fold, about 15-fold, about 20 fold, about 25-fold, about 30-fold, about 40-fold, or about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a variant AAV capsid protein confers about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30 fold, about 5-fold to about 25-fold, about 5-fold to about 20-fold, about 5-fold to about 15-fold, about 5-fold to about 10- fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, 20-fold to about 50-fold, 25- fold to about 50-fold, 30-fold to about 50-fold, or 40-fold to about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof.
  • a peptide insertion site is located at or between amino acids 581 and 593 of VP1, VP2 or VP3 of AAV9 or the corresponding position in the capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein.
  • a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein.
  • a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site.
  • one or more modifications are within about 10 amino acids upstream or downstream of the location of a peptide insertion site. In some embodiments, one or more modifications are within about 5 amino acids upstream or downstream of the location of a peptide insertion site.
  • one or more modifications are located in a variable Page 16 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) region of parental AAV capsid protein.
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • one or more modifications are located in an AAV9 capsid protein variable region, e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX, or any combination thereof.
  • an AAV9 capsid protein variable region e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX, or any combination thereof.
  • one or more modifications are located in: VR-VIII of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein, VR-IV of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein, or VR-V of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein, or any combination thereof.
  • VR-VIII of a VP e.g., VP1, VP2, and/or VP3 of an AAV9 capsid protein
  • VR-IV of a VP e.g., VP1, VP2, and/or VP3 of an AAV9 capsid protein
  • VR-V of a VP e.g., VP1, VP2, and/or VP3
  • a VR-IV comprises amino acids 451-475 of VP1, VP2 or VP3 of an AAV9 capsid protein.
  • VR-V comprises amino acids 488-506 of VP1, VP2 or VP3 of an AAV9 capsid protein.
  • one or more modifications comprises an insertion, deletion, mutation, or a combination thereof.
  • a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region.
  • one or more modifications reduces glycan binding.
  • a glycan is galactose.
  • one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another Page 17 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501
  • a parental AAV capsid protein is an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74capsid protein.
  • a parental AAV capsid protein is an AAV9 capsid protein.
  • a variant capsid has 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% identity relative to a parental AAV capsid protein.
  • percent identity is determined by comparing the sequence of the variant capsid without the peptide insertion, with a parental AAV capsid protein (e.g., an AAV9 capsid protein).
  • a variant capsid protein and a parental AAV capsid protein have 100% identity when: (a) the peptide insertion in the variant capsid protein is not taken into account in the sequence comparison; and (b) the variant capsid protein does not have one or more modifications other than the peptide insertion.
  • a variant capsid protein and a parental AAV capsid protein have less than 100% identity when: (a) the peptide insertion in the variant capsid protein is not taken into account in the sequence comparison; and (b) the variant capsid protein comprises one or more modifications other than the peptide insertion.
  • a parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001. Page 18 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0098] In some embodiments, a parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002. [0099] In some embodiments, a parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003. [0100] In some embodiments, a parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2050.
  • a parental AAV capsid protein is an AAV4 capsid protein of SEQ ID NO: 2051.
  • a parental AAV capsid protein is an AAV5 capsid protein of SEQ ID NO: 2004.
  • a parental AAV capsid protein is an AAV6 capsid protein of SEQ ID NO: 2005.
  • a parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2052.
  • a parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006.
  • a parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2053. [0107] In some embodiments, a parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2054. [0108] In some embodiments, a parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2055. [0109] In some embodiments, a parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2056. [0110] In some embodiments, a parental AAV capsid protein is an AAVrh74 capsid protein of SEQ ID NO: 2057.
  • a payload is a polypeptide that is encoded by a nucleic acid sequence within the rAAV particle.
  • a polypeptide is or comprises a CRISPR-Cas protein.
  • a CRISPR-Cas protein is chosen from: a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment thereof.
  • the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the CRISPR-Cas protein.
  • a CRISPR-Cas protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain.
  • a CRISPR-Cas protein is a nuclease.
  • a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleic acid molecule.
  • a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule.
  • a polypeptide is or comprises a Zinc finger protein, or a variant or fragment thereof.
  • a Zinc finger protein is chosen from: a Zinc finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeted zinc-finger DNA-binding unit optionally fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or combination of any of the foregoing.
  • a Zinc finger protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain.
  • a polypeptide is or comprises a Transcription Activator- Like Effector (TAL) protein, or a variant or fragment thereof.
  • TAL Transcription Activator- Like Effector
  • a TAL comprises: a TAL effector DNA binding domain (e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.), a Transcription Activator-Like Effector Nuclease (TALEN), e.g., a TAL effector DNA binding domain fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or combination of any of the foregoing.
  • a TAL protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain.
  • a polypeptide is or comprises a base editor, or a variant or fragment thereof.
  • a base editor comprises a deaminase, an adenosine deaminase enzyme (ABE), a cytosine deaminase enzyme (CBE), an APOBEC1, an APOBEC3A, an APOBEC3G, an evoAPOBEC, a BE4-YE1, a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine deaminases (TadA*), an E.
  • coli tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA binding domain monomer, a base editing enzyme that is RNA guided, a DNA glyosylase inhibitor, one or more DNA glycosylase inhibitor domains, a 5-methylcytosine deaminase, a cytidine deaminase domain, an adenine deaminase domain, an adenosine base editor (ABE), a Target-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax., a circularly permuted base editor, an adenosine deaminase enzyme (ADAR), a RNA editing for programmable adenosine to inosine replacement (REPAIR), a leveraging endogenous ADAR for programmable editing of RNA (LEAPER) or a variant or fragment or
  • the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the base editor.
  • a polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising the same.
  • a prime editor and/or system comprising the same comprises: a reverse transcriptase, a prime editing enzyme, an editing enzyme that includes a reverse transcriptase domain, an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Murine Leukemia Virus (MLV) Reverse Transcriptase, a HIV- 1 reverse transcriptase, a bacterial reverse transcriptase, a reverse transcriptase associated with a DNA binding domain and/or protein, a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase), a prime editing 1 system (PE1), a prime editing 2 system (PE2), a prime editing 3 system (PE3), a prime editing 3b system (PE3b) or a variant or fragment or combination of any of the foregoing.
  • AMV Avian Myeloblastosis Virus
  • MMV Murine Leukemia
  • the payload also comprises a prime editing gRNA (pegRNA) or an extended sgRNA that interacts with the prime editor.
  • a polypeptide is or comprises a meganuclease, or a variant or fragment thereof.
  • a meganuclease is chosen from: a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His- Page 21 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Cyst box family meganuclease, or HNH family endonuclease, an I-SeeI, an I-CeuI, a PI-PspI, a PI-SceI, an I-SceIV, an I-CsmI, an I-PanI, an I-SceII, an I-PpoI, an I-SceIII, an I-CreI, an I-TevI, an I-TevII an I-TevIII or a variant or fragment or combination of any of the foregoing.
  • a polypeptide is associated with a muscle disorder, or a glycogen or sugar storage disorder.
  • a muscle disorder is chosen from: X- linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • a polypeptide is an enzyme.
  • an enzyme is a lysosomal enzyme or an adenosine deaminase enzyme.
  • a polypeptide is an antibody.
  • a polypeptide is a secreted protein.
  • a payload is an RNA molecule.
  • an RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, circular RNA, a snRNA, or an aptamer.
  • an RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder.
  • a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • a payload is a DNA molecule, e.g., a donor DNA molecule that is integrated into a host genome via homologous recombination.
  • a DNA molecule comprises a nucleic acid sequence of up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc.
  • a nucleotide sequence encoding a payload comprises a promoter.
  • a promoter is or comprises a muscle-specific promoter.
  • a muscle-specific promoter is chosen from: MHCK7, CK8, desmin, tMCK, dMCK, or CK6, a variant or fragment of any of the foregoing.
  • a promoter is or comprises a dual muscle-liver promoter.
  • a dual muscle-liver promoter is SPc5-12 or a variant or fragment thereof.
  • a peptide sequence disclosed herein e.g., a peptide comprising an RGD motif as disclosed in any one of Tables 1- 11, can be used as a targeting moiety, e.g., a muscle-targeting moiety, to deliver a payload.
  • a targeting moiety e.g., a muscle-targeting moiety
  • a targeting moiety e.g., a muscle-targeting moiety
  • a viral protein e.g., an AAV capsid protein
  • FIG.1 is a graph showing enhanced skeletal muscle transduction in cynomolgus macaque with RGDxxRW variants.
  • FIG.2 is a schematic of the transgene cassette used for the Round 3 library. Gene expression is initiated by a ubiquitous CAG promoter driving transcription of a histone-2B Page 23 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (H2B) enhanced green fluorescent protein (EGFP) coding sequence with a unique barcode (BC) in the 3’ UTR.
  • H2B enhanced green fluorescent protein
  • BC unique barcode
  • log2FC log2 fold-change
  • FIG.5 is a graph showing that top-performing Round 3 capsids in cynomolgus macaque also achieved enhanced skeletal muscle transduction in C57BL/6J mice.
  • the log2 fold- change (log 2 FC) enrichment values represent the average of all analyzed muscle tissue in the two species. Error bars represent standard error of the mean.
  • FIGS.7A-7J are graphs showing transduction enhancement observed across multiple muscle tissue regions in cynomolgus macaque. Dotted line represents AAV9 transduction level.
  • FIGS.8A-8C show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture.
  • AAV9 and two RGD capsid variants (with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits) were packaged with a CAG-mCherry reporter.
  • Individual purified capsids were added to differentiated primary myotubes from healthy human donors. Images were taken at 1 Page 24 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) day and 5 days post-transduction.
  • FIG.8A is in color
  • FIG.8B is in greyscale
  • FIG.8C is in black and white.
  • FIGS.9A-9D show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture.
  • Three independent primary myoblast cell lines hSKMDC_con1, hSKMDC_con2, hSKMDC_con3 were differentiated to myotubes (as described in Example 3).
  • FIGS.10A-10C show that AAV particles displaying RGD-containing peptides efficiently transduced immortalized human myotubes (ImmSkMDC_AB1190) in culture.
  • AAV9 and capsid variant RGDYREV (SEQ ID NO: 1829; with the specified peptide inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits) were packaged with a CBA- mCherry-Fluc2 dual reporter. Quantification of mCherry fluorescence (FIG.10B) and luciferase activity (FIG.10C) was performed 5 days after transduction with the AAV particles at the indicated doses (1E4 vg/cell, 3E4 vg/cell or 1E5 vg/cell).
  • FIGS.11A-11F show that AAV particles displaying RGD-containing peptides transduced 3D myobundles more efficiently than AAV particles having a wildtype AAV9 capsid.
  • FIGS.11A-11B are in color
  • FIGS.11C-11D are in greyscale
  • FIGS.11E-11F are in black and white.
  • FIGS.12A-12C provide a visualization of mCherry expression in transduced mouse quadriceps.
  • FIG.12A is in color
  • FIG.12B is in greyscale
  • FIG.12C is in black and white.
  • FIGS.13A-13C are a series of images showing mCherry expression in transduced striated muscle via IHC of tissues from mice treated with AAV particles displaying RGD-containing peptides compared to mice treated with AAV particles having a wild type AAV9 capsid at 4 weeks post-injection.
  • AAV9 and RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variants were packaged with a CAG- mCherry reporter.
  • FIGS.14A-14J show that administration of a low dose of AAV particles displaying a RGDYREI (SEQ ID NO: 1825) peptide or a RGDYERI (SEQ ID NO: 1551) peptide have similar or enhanced mCherry mRNA expression compared to administration of a high dose of AAV particles having a wildtype AAV9 capsid in samples from mice administered the AAV particles.
  • Non-muscle targets lung, kidney, brain, and liver
  • RGDYREI SEQ ID NO: 1825
  • RGDYERI SEQ ID NO: 1551
  • FIGS.15A-15F show the quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles having a wildtype AAV9 capsid, or AAV particles displaying a RGDYREI (SEQ ID NO: 1825) peptide or a RGDYERI (SEQ ID NO: 1551) peptide with a CAG-mCherry reporter.
  • the specified peptides were inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • Vector genome values were normalized to the RPP30 gene with a standard curve determined from a pUC57 plasmid control.
  • FIG.15A tibialis anterior
  • FIG.15B gastrocnemius muscle
  • FIG.15C diaphragm
  • FIG.15D brain
  • FIG. 15E kidney
  • FIG.15F liver.
  • FIGS.16A-16D show that AAV particles displaying a RGDYREV (SEQ ID NO: 1829) peptide have higher hindlimb transduction compared to AAV particles having a wildtype AAV9 capsid in mice.
  • RGDYREV was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • FIG.16A shows representative images of bioluminescence imaging 14 days after IV injection of AAV particles having capsids containing a dual reporter (CBA- mCherry-Fluc2) at 5E12 vg/kg. Red circles represent regions of interest (ROIs) for measurement.
  • FIG.17 shows qPCR quantification of vector genome biodistribution in NHPs dosed with AAV particles having a wild type AAV9 capsid or AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter.
  • RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • Y-axis denotes vector genome copy number per diploid genome (vg/dg).
  • FIGS.18A-18B are graphs showing RT-qPCR quantification of mCherry mRNA levels in NHPs dosed with AAV particles having a wildtype AAV9 capsid or AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • FIG.18A shows quantification from tissues collected in both AAV9 and RGDYREI capsid groups, where y-axis denotes relative mCherry mRNA levels compared to those from AAV9 transduction in the same tissues.
  • FIG.18B shows quantification from tissues collected solely from the RGDYREI Page 27 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) capsid group, where y-axis denotes relative mCherry levels normalized to that from AAV9 transduction in gastrocnemius.
  • FIGS.19A-19C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • FIG.19A is in color
  • FIG.19B is in greyscale
  • FIG.19C is in black and white.
  • FIGS.20A-20C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter.
  • RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • NHPs #1 and #2 were male, and NHP #3 was female.
  • FIGS.21A-21C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits.
  • FIG.21A is in color
  • FIG.21B is in greyscale
  • FIG.22C is in black and white.
  • FIG.22A is in color
  • FIG.22B is in greyscale
  • FIG.22C is in black and white.
  • 5’ and 3’ are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence.
  • 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment
  • 5’ indicates a segment of the nucleic acid that is upstream of another segment.
  • 3’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence.
  • 5’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence.
  • nucleic acid will be in the 5’ to 3’ direction of translation.
  • about or approximately As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • Adeno-associated virus As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in whole or in part, of the family Parvoviridae and the Page 29 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) genus Dependoparvovirus. AAV is a small replication-defective, nonenveloped virus.
  • AAV includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotypes 4, AAV serotypes 5, AAV serotypes 6, AAV serotypes 7, AAV serotypes 8, AAV serotypes 9, AAV serotypes 10, AAV serotypes 11, AAV serotypes 12, AAV serotype 13, AAVrh74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, non-human primate AAV, e.g., from rhesus monkeys, and any variant of any of the foregoing.
  • Wild-type AAV is replication deficient and requires co- infection of cells by a helper virus, e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate.
  • a helper virus e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate.
  • Ad2 helper refers to the Adenovirus serotype 2 (Ad2) helper virus (e.g., wildtype or recombinantly engineered Ad2 helper virus) and various Ad2 helper genes and/or Ad2 helper polypeptides, including, but not limited, to E1a, E1b, E2a, E4Orf6, VA RNA, and any variant or fragment of any of the foregoing.
  • Ad2 helper virus e.g., wildtype or recombinantly engineered Ad2 helper virus
  • Ad2 helper viruses e.g., wildtype or recombinantly engineered Ad2 helper virus
  • Ad2 helper viruses e.g., wildtype or recombinantly engineered Ad2 helper virus
  • Ad2 helper genes and/or Ad2 helper polypeptides including, but not limited, to E1a, E1b, E2a, E4Orf6, VA RNA, and any variant or fragment of any of the foregoing.
  • an Ad2 helper vector e.g., plasmid
  • Ad2 helper polypeptides e.g., one, two, three, or four of E1 (e.g., E1a and/or E1b), E2A, E4, or VA RNA) necessary to generate functional rAAV particles.
  • the Ad2 helper vector is transfected into an E1 complementing cell line (e.g., HEK293).
  • the nucleotide sequence of an Ad2 helper vector and Ad2 helper virus genes can be derived from the Adenovirus 2 genome (Genbank Accession No. J01917.1).
  • Ad5 helper refers to the Adenovirus serotype 5 (Ad5) helper virus (e.g., wildtype or recombinantly engineered Ad5 helper virus) and various Ad5 helper genes and/or Ad5 helper polypeptides, including, but not limited, to E1a, E1b, E2a, E4Orf6, and/or VA RNA.
  • Ad5 helper virus e.g., wildtype or recombinantly engineered Ad5 helper virus
  • Ad5 helper genes and/or Ad5 helper polypeptides including, but not limited, to E1a, E1b, E2a, E4Orf6, and/or VA RNA.
  • an Ad5 helper vector (e.g., plasmid) comprises Ad5 helper genes (e.g., one, two, three, or four of E1 (e.g., E1a and/or E1b), E2A, E4, or VA RNA) necessary to generation functional rAAV particles.
  • the Ad5 helper vector is transfected into an E1 complementing cell line (e.g., HEK293).
  • the nucleotide sequence of an Ad5 helper vector and Ad5 helper genes can be derived from the Adenovirus 5 genome (Genbank Accession No. AY601635).
  • Administration refers to the administration of a composition comprising rAAV particles as described herein to a subject.
  • Administration may be by any appropriate route.
  • administration may be local or systemic administration (e.g., to a mammal, e.g., to a human, e.g., a patient).
  • a composition of the disclosure may be administered by injection or infusion by any route.
  • a composition may be administered by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-cisterna magna, or intrathecal injection or infusion.
  • Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal.
  • bronchial e.g., bronchial instillation
  • buccal enteral
  • interdermal intra-arterial
  • intradermal intragastric
  • intramedullary intramuscular
  • intranasal intraperitoneal
  • Bioreactor refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture).
  • the bioreactor can be of any size and/or any shape so long as it is useful for culturing a cell culture (e.g., a mammalian cell culture).
  • Cap polypeptide refers to the structural proteins that form a functional AAV capsid, which can in turn package DNA and infect or transduce a target cell.
  • a Cap polypeptide comprises a variant AAV capsid as disclosed herein.
  • Cap polypeptides will comprise all of the AAV capsid subunits, but less than all of the capsid subunits may be present as long as a functional capsid is produced.
  • the nucleic acid sequence encoding Cap polypeptides will be present on a single vector (e.g., plasmid).
  • the Cap polypeptide comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAVrh74 Cap polypeptide, or a variant of any of the foregoing.
  • cell density refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area. For example, cell density may be represented as viable cells (vc)/cm 2 of culture medium or vc/mL.
  • Culture As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium.
  • Fragment As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure.
  • a fragment consists of such a discrete portion.
  • a fragment consists of or comprises a characteristic structural element or moiety found in the whole.
  • a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide.
  • monomeric units e.g., nucleic acids
  • a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide.
  • the whole material or entity may in some embodiments be referred to as the “parent” of the whole.
  • Gene refers to a DNA sequence that codes for a product (e.g., an RNA product and/or a polypeptide product).
  • a gene includes coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or effect one or more aspects of gene expression (e.g., inducible expression, etc.). [0165] Gene therapy: As used herein, the term “gene therapy” refers to insertion or deletion of specific genomic DNA sequences to treat or prevent a disorder or condition for which such therapy is sought.
  • the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells).
  • Target cells may be from a mammal and/or may be cells in a mammalian subject. Mammals include but are not limited to humans, dogs, Page 32 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) cats, cows, sheep, pigs, llamas, etc.
  • heterologous DNA is transferred to target cells.
  • the heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced.
  • the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product, or it may encode a product, such as a peptide or RNA that in some manner mediates or modulates, directly or indirectly, expression of a therapeutic product.
  • Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced.
  • the heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression.
  • Such an inhibitor or repressor or other modulator can be a polypeptide, peptide, or nucleic acid (e.g., DNA or RNA).
  • Gene therapy may include in vivo or ex vivo techniques.
  • viral and non-viral based gene transfer methods can be used to introduce a nucleic acid encoding a polypeptide of interest or to introduce a therapeutic nucleic acid into mammalian cells or target tissues.
  • Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as poloxamers or liposomes.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Host Cell refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced.
  • host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence).
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical.
  • Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Page 34 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0168]
  • Improve, increase, inhibit, or reduce As used herein the terms “improve”, “increase,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample, e.g., of a culture medium) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • an appropriate reference measurement may be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for survival and/or minimal growth.
  • the solution can also comprise components that enhance survival and/or growth above the minimal rate, including hormones and growth factors.
  • the solution can be formulated to a pH and concentration of one or more salts that are optimal for cellular survival and/or proliferation.
  • the medium can also be a “defined medium” or “chemically defined medium,” e.g., a serum-free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure.
  • Muscle targeting moiety refers to a peptide containing an RGD-motif which is effective in targeting a muscle cell or muscle tissue.
  • a muscle-targeting moiety can target a muscle cell or tissue by: (i) contacting a muscle cell or muscle tissue (e.g., binding to one or more receptors expressed on a muscle cell or tissue); (ii) contacting a cell in contact with a muscle cell or tissue (e.g., binding to one or more receptors expressed on a cell in contact with a muscle cell or tissue); (iii) delivering a payload to a muscle cell or tissue; or (iv) any combination of (i)-(iii).
  • delivering a payload to a muscle cell or muscle tissue comprises transducing a Page 35 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) muscle cell or muscle tissue.
  • delivering a payload to a muscle cell or muscle tissue results in expression of (e.g., detectable expression of) a payload in a muscle cell or muscle tissue.
  • a muscle cell comprises a skeletal muscle cell, a cardiac muscle cell, a smooth muscle cell, or a muscle stem cell, e.g., a muscle satellite cell.
  • a muscle targeting moiety can be conjugated to a payload.
  • a muscle targeting moiety can be incorporated into a vector, e.g., a viral vector or a non-viral vector.
  • a muscle targeting moiety can be inserted in an AAV capsid to form a variant AAV capsid as disclosed herein.
  • Nucleic acid includes any nucleotides, analogs thereof, and polymers thereof.
  • polynucleotide as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA).
  • RNA refers to the primary structure of the molecules and, thus, include double- and single- stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides.
  • RNA poly- or oligo-ribonucleotides
  • DNA poly- or oligo- deoxyribonucleotides
  • RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases
  • nucleic acids derived from sugars and/or modified sugars and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as “internucleotide linkages”).
  • the term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges.
  • nucleic acids containing ribose moieties examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties.
  • the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units.
  • the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.
  • an RNA comprises a short hairpin RNA (shRNA), small interfering RNA Page 36 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (siRNA), mRNA, snRNA, CRISPR/Cas guide RNA, microRNA (miRNA), and/or a precursor thereof.
  • shRNA short hairpin RNA
  • siRNA small interfering RNA Page 36 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • siRNA small interfering RNA Page 36 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • siRNA small interfering RNA Page 36 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • mRNA snRNA
  • CRISPR/Cas guide RNA CRISPR/Cas guide RNA
  • miRNA microRNA
  • Payload refers to a nucleic acid sequence of interest (e.g., comprising a sequence that encodes a target payload, such as a target polypeptide or RNA) that is desired to be introduced into a cell, tissue, organ, organism, and/or system comprising cells; or a polypeptide.
  • a target payload can be a heterologous protein with a therapeutic purpose, e.g., an enzyme or antibody.
  • the target payload can be a heterologous nucleic acid with a therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR/Cas guide RNA, or a precursor thereof.
  • the target payload can be selected from any heterologous protein or nucleic acid of interest.
  • encode or “encodes” means directs the expression of or processed into.
  • a nucleic acid encodes a polypeptide sequence if it directs the expression of that polypeptide sequence.
  • a nucleic acid precursor e.g., a pri- miRNA or pre-miRNA
  • a further processed version of the nucleic acid e.g., mature miRNA
  • composition refers to a composition comprising rAAV particles that is suitable for administration to a human or animal subject.
  • a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers.
  • the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen.
  • a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to achieve a desired therapeutic effect when administered to a subject or population in need thereof (e.g., by a statistically significant probability).
  • a pharmaceutical composition may be specially formulated for administration in solid or liquid form.
  • a pharmaceutical composition is formulated for administration by parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection.
  • a pharmaceutical composition is formulated as a sterile solution or suspension, e.g., in a sustained- release formulation.
  • Pharmaceutical compositions of the disclosure may be formulated for administration by injection or infusion (e.g., subcutaneous, intramuscular, intravenous or Page 37 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) epidural injection or infusion).
  • compositions may be formulated for administration by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-cisterna magna, or intrathecal injection or infusion.
  • a pharmaceutical composition is intended and suitable for administration to a human subject.
  • a pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free).
  • Formulations of the pharmaceutical compositions may include, but are not limited to, formulations for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; topical application, such as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
  • formulations for oral administration such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue
  • topical application such as
  • Polypeptide generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity.
  • Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art.
  • proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.
  • the term “peptide” is generally used to refer to Page 38 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.
  • Recombinant is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes
  • one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).
  • Recombinant AAV (rAAV) particle A “recombinant AAV particle”, or “rAAV particle,” as used herein, refers to an infectious, replication-defective viral particle comprising an AAV protein shell encapsulating a payload that is flanked on both sides by ITRs.
  • An AAV particle is produced in a suitable host cell (e.g., a HEK293 cell).
  • the host cell is transfected with at least one vector encoding one or more helper polypeptides (e.g., Ad2 helper polypeptides), at least one Rep polypeptide, at least one Cap polypeptide, and at least one payload (e.g., for polypeptide expression or a therapeutic nucleic acid), such that the host cell is capable of producing the Rep and Cap polypeptides necessary for packing the rAAV particle.
  • rAAV particles may be used for subsequent gene delivery.
  • Rep polypeptide The term “Rep polypeptide”, as used herein, refers to the AAV non-structural proteins that mediate AAV replication for the production of AAV particles.
  • RGD motif refers to a peptide comprising the amino acids R, G, and D in consecutive order. Peptides comprising an RGD- motif are provided in Tables 1-11.
  • seeding refers to the process of providing a cell culture to a vessel (e.g., a bioreactor or culture flask).
  • a vessel e.g., a bioreactor or culture flask
  • the process of providing a cell culture may include propagation of the cells in another bioreactor or vessel before providing to the bioreactor or other vessel. The cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel.
  • seeding refers to providing any number of cells, including a single cell.
  • Subject refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog).
  • a human subject is an adult, adolescent, or pediatric subject.
  • a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a neurological disease or disorder or a cancer or a tumor listed herein.
  • a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition.
  • a subject displays one or more symptoms of a disease, disorder or condition.
  • a subject does not display a particular symptom (e.g,. clinical manifestation of disease) or characteristic of a disease, disorder, or condition.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition.
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • Titer refers to the quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or Page 40 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume.
  • transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells (e.g., mammalian cells).
  • transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and/or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and/or transfection based on cationic polymers (e.g., DEAE-dextran or polyethylenimine).
  • viral-based transfection is also referred to herein as transduction.
  • Treating refers to providing treatment, e.g., providing any type of medical or surgical management of a subject.
  • the treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition.
  • Prevent refers to causing a disease, disorder, condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals.
  • Treating can include administering an agent to the subject following the development of one or more symptoms or manifestations indicative of a condition, disease, or disorder, e.g., in order to reverse, alleviate, reduce the severity of, and/or inhibit or prevent the progression of the condition and/or to reverse, alleviate, reduce the severity of, and/or inhibit or one or more symptoms or manifestations of the condition.
  • a composition comprising rAAV particles of the disclosure can be administered to a subject who has developed a disorder or is at increased risk of developing such a disorder relative to a member of the general population.
  • a composition of the disclosure can be administered prophylactically or before development of any symptom or manifestation of the condition. Typically, in this case, the subject will be at risk of developing the condition.
  • Variant As used herein in the context of molecules, e.g., nucleic acids, or proteins, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In Page 41 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule.
  • any biological or chemical reference molecule has certain characteristic structural elements.
  • a variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule.
  • a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function;
  • a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space.
  • a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone).
  • moieties e.g., carbohydrates, lipids, phosphate groups
  • a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.
  • a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid.
  • a reference polypeptide or nucleic acid has one or more biological activities.
  • a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.
  • a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions.
  • a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference.
  • a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference.
  • a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference.
  • a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference.
  • a reference polypeptide or nucleic acid is one found in nature.
  • Vector refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into a cell.
  • one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments may be packaged into a viral capsid and can be transferred into another cell and/or organism.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's Page 43 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.
  • VP refers to an AAV VP1 capsid protein, an AAV VP2 capsid protein, an AAV VP3 capsid protein, or variants or fragments or combinations of any of the foregoing.
  • capsid protein is used interchangeably herein with VP.
  • the numbering used herein in describing exemplary locations of peptide insertions in VP1, VP2 or VP3 are used relative to AAV VP1 numbering.
  • VP1, VP2 and VP3 of the AAV9 capsid protein correspond to amino acids 1 to 736 of VP1, amino acids 138 to 736 of VP1 and amino acids 203 to 736 of VP1, respectively.
  • reference to a peptide insertion between positions 588 and 589 in an AAV capsid variant refers to positions 588 and 589 in VP1, VP2 or VP3 relative to VP1 numbering.
  • Those with knowledge in the pertinent field would be able to readily ascertain the corresponding position in VP2 and VP3, e.g., by comparing the sequences of VP1, VP2 and VP3 of the parental AAV capsid proteins using methods known in the field such as sequence alignment.
  • a VP capsid protein is a VP1 capsid protein. In some embodiments, a VP capsid protein is a VP2 capsid protein. In some embodiments, a VP capsid protein is a VP3 capsid protein. In some embodiments, a VP protein comprises a peptide insertion disclosed herein.
  • Variant AAV capsid protein refers to a VP capsid protein (e.g., a VP1, VP2, or VP3) comprising a peptide insertion relative to a corresponding parental AAV capsid protein (e.g., a parental VP1, VP2, or VP3).
  • a VP capsid protein e.g., a VP1, VP2, or VP3
  • a parental VP1, VP2, or VP3 e.g., a parental VP1, VP2, or VP3
  • rAAV recombinant adeno- associated virus
  • rAAVs Recombinant adeno-associated Page 44 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) viruses
  • rAAVs Recombinant adeno-associated Page 44 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) viruses
  • rAAVs have emerged as some of the most promising vectors for in vivo gene therapy, and are currently under clinical evaluation for a number of disorders including muscular disorders.
  • Naturally occurring AAV capsids sub-optimally target skeletal muscle, and require extremely high doses to achieve minimum effective transgene expression. This poses daunting manufacturing challenges as well as safety concerns.
  • AAV muscle tropism can be obtained by inserting a short peptide onto an AAV capsid to direct said AAV capsid to a muscle cell.
  • rAAV particles comprising a variant capsid having a peptide insertion disclosed herein bind to and/or recognize a target on a muscle cell.
  • rAAV particles comprising a variant capsid comprising a peptide insertion disclosed herein can enhance vector attachment, internalization, and/or payload expression in muscle cells.
  • AAV9 Adeno-associated viruses
  • ssDNA single-stranded DNA
  • AAV9 is one of the human AAV serotypes that has enhanced transduction efficiency in cardiac and skeletal muscle, liver tissue, pancreatic tissue, and the eye compared to other serotypes (DiMattia 2012).
  • the AAV wild-type genome contains at least three genes, rep, cap and X (Buning and Srivastava, (2019) Molecular Therapy: Methods & Clinical Development vol.12 pages 248- 265).
  • the cap gene encodes for viral proteins VP1, VP2, and VP3, and assembly-activating protein (AAP). All three VP proteins are capsid monomers. Transcription of the cap gene results in two messenger RNA: a messenger RNA which encodes VP1 and a messenger RNA which encodes VP2 and VP3 (as described in Warrington KH et al., (2004) Journal of Virology volume 78(12) pages 6595-6609).
  • VP1, VP2, and VP3 are present at ratios of 1:1:10, respectively.
  • the Page 45 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) VP3 region is observed in all capsid structures of AAV serotypes that have been studied (DiMattia 2012).
  • VPs comprise beta strands, alpha helical regions, and structurally variable regions (VRs) in the surface loops which connect the beta strands. Without wishing to be bound by any particular theory, it is believed that differences in sequence and/or conformations of VRs contribute to the variability in cellular tropism, differences in tissue transduction efficiently, and/or antigenic reactivity among different AAV serotypes.
  • differences in VR sequence and/or structure among different AAV serotypes allow for differential recognition of cell surface glycans and/or tissue specific protein or lipid receptor interaction for internalization.
  • Wild type AAV9 (WT AAV9) has nine variable regions VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX (DiMattia 2012, see also Table 3 therein).
  • AAV9 VR-I encompasses amino acid positions 262-269.
  • AAV VR-II encompasses amino acid positions 327-332 and has a role, e.g., in genome packaging.
  • AAV9 VR-III encompasses amino acid positions 382-386.
  • AAV9 VR-IV encompasses amino acid positions 452-460 and has a role, e.g., in liver transduction and/or a delayed blood clearance phenotype.
  • AAV9 VR-V encompasses amino acid positions 488-505 and has a role, e.g., in LamR receptor binding, liver and/or muscle-specific transduction, and/or a delayed blood clearance phenotype.
  • AAV9 VR-VI encompasses amino acid positions 527-539 and has a role, e.g., in LamR receptor binding, and/or a delayed blood clearance phenotype.
  • AAV9 VR-VII encompasses amino acid positions 545-558 and has a role, e.g., in liver transduction and/or delayed blood clearance phenotype.
  • AAV9 VR- VIII encompasses amino acid positions 581-593 and has a role, e.g., in LamR receptor binding and/or transduction.
  • AAV9 VR-IX encompasses amino acid positions 704-714 and has a role, e.g., in heart tropism, melanoma tropism and/or altered tropism.
  • a rAAV particle disclosed herein is a recombinant AAV (rAAV) particle.
  • a rAAV particle comprises a variant AAV9 capsid protein comprising a peptide insertion disclosed herein.
  • a peptide insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of a parental AAV capsid protein.
  • a parental AAV capsid protein comprises the sequence of a wildtype AAV capsid protein, or a sequence having at least 95% identity to the sequence of a wildtype AAV capsid protein, or a sequence having no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein.
  • a parental AAV capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to the sequence of a wildtype AAV capsid protein.
  • a parental AAV capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein.
  • a parental AAV capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to the sequence of a wildtype AAV capsid protein and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a parental AAV capsid protein is other than an AAV9 capsid protein and comprises one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) at a position of VP1, VP2 or VP3 corresponding to position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein.
  • one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.
  • one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein;
  • a parental AAV capsid protein is chosen from: an AAV9 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, or an AAVrh74 capsid protein.
  • a parental AAV capsid protein comprises: an AAV9 capsid protein.
  • an AAV9 capsid protein comprises: the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • tissue e.g., a liver-detargeting mutation.
  • Exemplary mutations including liver de-targeting mutations are disclosed in Pulichla N. et al., (2011) Molecular Therapy volume 19, pages 1070-1078, the entire contents of which are hereby incorporated by reference.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, e.g., a W503R mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 595, e.g., a W595C mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 457, e.g., a N457H mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 574, e.g., a T574S mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 592, e.g., a Q592L mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 498, e.g., a N498Y or an N498I mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 602, e.g., a L602F mutation.
  • a mutation that alters a Page 49 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) binding profile of a parental AAV capsid protein comprises a mutation at position 468, e.g., a P468T mutation.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 500, e.g., a E500D mutation.
  • one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region.
  • one or more mutations reduces glycan binding.
  • a glycan is galactose.
  • one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein;
  • a parental AAV capsid protein comprises an AAV1 capsid protein. In some embodiments, an AAV1 capsid protein sequence is provided in SEQ ID NO: 2002. [0210] In some embodiments, a parental AAV capsid protein comprises an AAV2 capsid protein. In some embodiments, an AAV2 capsid protein sequence is provided in SEQ ID NO: 2003. [0211] In some embodiments, a parental AAV capsid protein comprises an AAV3B capsid protein. In some embodiments, an AAV3B capsid protein sequence is provided in SEQ ID NO: 2050.
  • a parental AAV capsid protein comprises an AAV4 capsid protein. In some embodiments, an AAV4 capsid protein sequence is provided in SEQ ID NO: 2051. [0213] In some embodiments, a parental AAV capsid protein comprises an AAV5 capsid protein. In some embodiments, an AAV5 capsid protein sequence is provided in SEQ ID NO: 2004. [0214] In some embodiments, a parental AAV capsid protein comprises an AAV6 capsid protein. In some embodiments, an AAV6 capsid protein sequence is provided in SEQ ID NO: 2005.
  • a parental AAV capsid protein comprises an AAV7 capsid protein. In some embodiments, an AAV7 capsid protein sequence is provided in SEQ ID NO: 2052. [0216] In some embodiments, a parental AAV capsid protein comprises an AAV8 capsid protein. In some embodiments, an AAV8 capsid protein sequence is provided in SEQ ID NO: 2006. [0217] In some embodiments, a parental AAV capsid protein comprises an AAV10 capsid protein. In some embodiments, an AAV10 capsid protein sequence is provided in SEQ ID NO: 2053. [0218] In some embodiments, a parental AAV capsid protein comprises an AAV11 capsid protein.
  • an AAV11 capsid protein sequence is provided in SEQ ID NO: 2054.
  • a parental AAV capsid protein comprises an AAV12 capsid protein.
  • an AAV12 capsid protein sequence is provided in SEQ ID NO: 2055.
  • a parental AAV capsid protein comprises an AAV13 capsid protein.
  • an AAV13 capsid protein sequence is provided in SEQ ID NO: 2056. Page 51 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0221]
  • a parental AAV capsid protein comprises an AAVrh74 capsid protein.
  • an AAVrh74 capsid protein sequence is provided in SEQ ID NO: 2057.
  • a peptide insertion is in VR-I of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-II of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-III of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-IV of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-V of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-VI of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-VII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-VIII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a parental AAV capsid protein e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a peptide insertion is in VR-IX of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a parental AAV capsid protein e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74.
  • a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74capsid protein
  • VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding positions in the capsid proteins of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein.
  • a parental AAV capsid protein is an AAV9 capsid protein
  • VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein.
  • a peptide insertion is in a VP (e.g., VP1, VP2, and/or VP3) of a parental AAV capsid protein.
  • a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of a parental AAV capsid protein (e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74).
  • a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein.
  • a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein. [0237] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein. [0238] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein. [0239] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein.
  • a peptide insertion site is located between two adjacent amino acids in VR-VIII of a parental AAV capsid protein.
  • a peptide insertion site is located between two non- adjacent amino acids in VR-VIII of a parental AAV capsid protein.
  • insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein.
  • insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV9 capsid protein, e.g., as compared to a WT AAV9 capsid protein.
  • an AAV9 WT capsid protein sequence is provided in SEQ ID NO: 2001.
  • a variant AAV9 capsid protein has 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% identity relative to a WT AAV9 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV1 capsid protein, e.g., as compared to a WT AAV1 capsid protein.
  • an AAV1 WT capsid protein is provided in SEQ ID NO: 2002.
  • a variant AAV1 capsid protein has 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% identity relative to a WT AAV1 capsid.
  • a rAAV particle disclosed herein comprises a variant AAV2 capsid protein, e.g., as compared to a WT AAV2 capsid protein.
  • an AAV2 WT capsid protein is provided in SEQ ID NO: 2003.
  • a variant AAV2 capsid protein has 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% identity relative to a WT AAV2 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV3B capsid protein, e.g., as compared to a WT AAV3B capsid protein.
  • an AAV3B WT capsid protein is provided in SEQ ID NO: 2050.
  • a variant AAV3B capsid protein has 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% identity relative to a WT AAV3B capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV5 capsid protein, e.g., as compared to a WT AAV5 capsid protein.
  • an AAV5 WT capsid protein is provided in SEQ ID NO: 2004.
  • a variant AAV5 capsid protein has 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% identity relative to a WT AAV5 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV6 capsid protein, e.g., as compared to a WT AAV6 capsid protein.
  • an AAV6 WT capsid protein is provided in SEQ ID NO: 2005.
  • a variant AAV6 capsid protein has 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% identity relative to a WT AAV6 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV8 capsid protein, e.g., as compared to a WT AAV8 capsid protein.
  • an AAV8 WT capsid protein is provided in SEQ ID NO: 2006.
  • a variant AAV8 capsid protein has 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% identity relative to a WT AAV8 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV4 capsid protein, e.g., as compared to a WT AAV4 capsid protein.
  • an AAV4 WT capsid protein is provided in SEQ ID NO: 2051.
  • a variant AAV4 capsid protein has 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% identity relative to a WT AAV4 capsid protein.
  • Page 55 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • a rAAV particle disclosed herein comprises a variant AAV7 capsid protein, e.g., as compared to a WT AAV7 capsid protein.
  • an AAV7 WT capsid protein is provided in SEQ ID NO: 2052.
  • a variant AAV7 capsid protein has 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% identity relative to a WT AAV7 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV10 capsid protein, e.g., as compared to a WT AAV10 capsid protein.
  • an AAV10 WT capsid protein is provided in SEQ ID NO: 2053.
  • a variant AAV10 capsid protein has 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% identity relative to a WT AAV10 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV11 capsid protein, e.g., as compared to a WT AAV11 capsid protein.
  • an AAV11 WT capsid protein is provided in SEQ ID NO: 2054.
  • a variant AAV11 capsid protein has 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% identity relative to a WT AAV11 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV12 capsid protein, e.g., as compared to a WT AAV12 capsid protein.
  • an AAV12 WT capsid protein is provided in SEQ ID NO: 2055.
  • a variant AAV12 capsid protein has 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% identity relative to a WT AAV12 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV13 capsid protein, e.g., as compared to a WT AAV13 capsid protein.
  • an AAV13 WT capsid protein is provided in SEQ ID NO: 2056.
  • a variant AAV13 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least Page 56 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV13 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAVrh74 capsid protein, e.g., as compared to a WT AAVrh74 capsid protein.
  • an AAVrh74 WT capsid protein is provided in SEQ ID NO: 2057.
  • a variant AAVrh74 capsid protein has 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% identity relative to a WT AAVrh74 capsid protein.
  • Additional modifications to an AAV9 capsid protein are possible including, for example, variants disclosed in International Patent Application WO 2003/052052 filed on November 12, 2002, the entire contents of which are hereby incorporated by reference.
  • a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in WO 2003/052052.
  • modifications to an AAV9 capsid protein including: Pulichla N. et al., (2011) Mol Ther.19(6): pp.1070–1078; Wang D. et al., (2016) Mol Ther Methods Clin Dev. (9): pp.234-246; Adachi K. et al., (2014) Nat. Comm. (5): art. 3075; or Bell CL.
  • a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in any of the reports referenced herein.
  • AAV9 capsid variants with peptide insertion [0260] Among other things, disclosed herein, are AAV9 capsid protein variants having one or more peptide insertions, e.g., as disclosed herein.
  • a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein.
  • a peptide Page 57 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of AAV9.
  • a peptide insertion is in VR-I of an AAV9 capsid protein.
  • a peptide insertion is in VR-II of an AAV9 capsid protein.
  • a peptide insertion is in VR-III of an AAV9 capsid protein.
  • a peptide insertion is in VR-IV of an AAV9 capsid protein.
  • a peptide insertion is in VR-V of an AAV9 capsid protein.
  • a peptide insertion is in VR-VI of an AAV9 capsid protein.
  • a peptide insertion is in VR-VII of an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a peptide insertion is in VR-IX of an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein.
  • a peptide insertion site is located between amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein.
  • a peptide insertion site is located between amino acids 580-585, amino acids 585-590, amino acids 590-595, or amino acids 595-601 of VP1, VP2 or VP3 of AAV9. In some embodiments, a peptide insertion site is located between amino acids 580-581, between amino acids 581-582, between amino acids 582-583, between amino acids 583-584, between amino acids 584-585, between amino acids 585-586, between amino acids 586-587, between amino acids 587-588, between amino acids 588-589, between amino acids 589-590, between amino acids 590-591, between amino acids 591-592, between amino acids 592-593, between amino acids 593-594, between amino acids 594-595, between amino acids 595-596, Page 58 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) between amino acids 596-597, between amino acids 597-598, between amino acids 598-599
  • a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein.
  • a peptide insertion is located between amino acids 588 and 589 of a VP1 of an AAV9 capsid protein.
  • a peptide insertion is located between amino acids 588 and 589 of a VP2 of an AAV9 capsid protein.
  • a peptide insertion is located between amino acids 588 and 589 of a VP3 of an AAV9 capsid protein.
  • a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein.
  • a peptide insertion site is located between two adjacent amino acids in VR-VIII of an AAV9 capsid protein [0276] In some embodiments, a peptide insertion site is located between two non- adjacent amino acids in VR-VIII of an AAV9 capsid protein. [0277] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.
  • insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising a peptide insertion disclosed herein, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a peptide Page 59 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) insertion comprises a sequence provided in any one of Tables 1-11.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 1, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a VP e.g., VP1, VP2, and/or VP3
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 2, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 3, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 4, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 5, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 6, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 7, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 8, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 9, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 10, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 11, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein.
  • a rAAV particle disclosed herein comprises: (1) a variant AAV capsid protein comprising a consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) one or more sequences of a VP (e.g., VP1, VP2, or VP3) of an AAV9 capsid protein.
  • a peptide insertion does not comprise an additional sequence N-terminal of a peptide sequence provided in any one of Tables 1-11.
  • a peptide insertion does not comprise an additional sequence C-terminal of a peptide sequence provided in any one of Tables 1-11.
  • a peptide insertion does not comprise an additional sequence N-terminal and C-terminal of a peptide sequence provided in any one of Tables 1-11.
  • All of the peptides provided in Tables 1-11 have a more than 5-fold enhanced skeletal muscle transduction compared to AAV9. The fold change data is presented in categories A, B, C and D.
  • Peptides in category A have a more than 5 fold enhanced skeletal muscle transduction compared to AAV9
  • peptides in category B have a more than 10 fold enhanced skeletal muscle transduction compared to AAV9
  • peptides in category C have a more than 15 fold enhanced skeletal muscle transduction compared to AAV9
  • peptides in category D have a more than 20 fold enhanced skeletal muscle transduction compared to AAV9.
  • Table 1 Exemplary peptide insertions.
  • a peptide insertion in an AAV capsid protein disclosed herein does not comprise RGDRDAL (SEQ ID NO: 975).
  • a peptide insertion in an AAV capsid protein disclosed herein is not YGVRGDRDAL (SEQ ID NO: 2025).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1X2R[W/F] (SEQ ID NO: 2026), wherein X1 and X2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 2.
  • Table 2 Exemplary peptides encompassed by SEQ ID NO: 1.
  • Peptide SEQ ID NO Fold change over AAV9 Page 65 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • Peptide SEQ ID NO Fold change over AAV9 8 62 D ein disclosed herein comprises a sequence of RGDX 3 QX 1 X 2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]QX1X2 (SEQ ID Page 66 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) NO: 2027), wherein X 1 and X 2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 3. [0291] Table 3: Exemplary peptides encompassed by SEQ ID NO: 2.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDYQAV (SEQ ID NO: 1764).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 QX 1 X 2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDYQAV (SEQ ID NO: 1764).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 QX 1 X 2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYQTL (SEQ ID NO: 1814).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are Page 69 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not SKDRGDYQTL (SEQ ID NO: 2007).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 QX 1 X 2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYQEL (SEQ ID NO: 2008).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not LSRRGDYQEL (SEQ ID NO: 2009).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYQAV (SEQ ID NO: 1764), SKDRGDYQTL (SEQ ID NO: 2007) or LSRRGDYQEL (SEQ ID NO: 2009).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2[W/F] (SEQ ID NO: 2028), wherein X 1 and X 2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 4. [0300] Table 4: Exemplary peptides encompassed by SEQ ID NO: 3.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X 1 and X 2 are Page 72 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X 3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHASW (SEQ ID NO: 191).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHASW (SEQ ID NO: 191).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHSGW (SEQ ID NO: 322).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHSGW (SEQ ID NO: 322).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHSTW (SEQ ID NO: 333).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHSTW(SEQ ID NO: 333).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHTQW (SEQ ID NO: 349).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are Page 73 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X 3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHTQW (SEQ ID NO: 349).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX 1 X 2 X 3 (SEQ ID NO: 3), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHQNF (SEQ ID NO: 283).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not ANTRGDHQNF (SEQ ID NO: 2010).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHASW (SEQ ID NO: 191), RGDHSGW (SEQ ID NO: 322), RGDHSTW (SEQ ID NO: 333), RGDHTQW (SEQ ID NO: 349) or ANTRGDHQNF (SEQ ID NO: 2010).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPX 1 X 2 X 3 (SEQ ID NO: 4), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPX1X2[W/F] (SEQ ID NO: 2029), wherein X 1 and X 2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 5. [0313] Table 5: Exemplary peptides encompassed by SEQ ID NO: 4.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]X1X2V (SEQ ID NO: 2030), wherein X1 and X2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 6. [0315] Table 6: Exemplary peptides encompassed by SEQ ID NO: 5.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 1 QX 2 X 3 (SEQ ID NO: 6), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 1 QX 2 [W/F] (SEQ ID NO: 2031), wherein X 1 and X 2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 7. [0317] Table 7: Exemplary peptides encompassed by SEQ ID NO: 6.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDVQNF (SEQ ID NO: 2011).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not NGVRGDVQNF (SEQ ID NO: 2012).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 1 QX 2 X 3 (SEQ ID NO: 6), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDHQNF (SEQ ID NO: 283).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 1 QX 2 X 3 (SEQ ID NO: 6), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not ANTRGDHQNF (SEQ ID NO: 2010).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not NGVRGDVQNF (SEQ ID NO: 2012) or ANTRGDHQNF(SEQ ID NO: 2010).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]X 1 SX 2 (SEQ ID NO: 2032), wherein X1 and X2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 8. [0324] Table 8: Exemplary peptides encompassed by SEQ ID NO: 7.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are Page 83 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYVSV (SEQ ID NO: 1948).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYVSV (SEQ ID NO: 1948).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYSSV (SEQ ID NO: 1882).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYSSV (SEQ ID NO: 1882).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYHSF (SEQ ID NO: 1623).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not RGDYHSF (SEQ ID NO: 1623).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTSM (SEQ ID NO: 1906).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are Page 84 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not STVRGDYTSM (SEQ ID NO: 2013).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not QERRGDYTSM (SEQ ID NO: 2014).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYASL (SEQ ID NO: 2015).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYASL (SEQ ID NO: 2015).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYNSL (SEQ ID NO: 2016).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not SNSRGDYNSL (SEQ ID NO: 2017).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTSV (SEQ ID NO: 2018).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are Page 85 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not STVRGDYTSV (SEQ ID NO: 2019).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not SNSRGDYTSV (SEQ ID NO: 2020).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTST (SEQ ID NO: 2021).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not SAMRGDYTST (SEQ ID NO: 2022).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTSL (SEQ ID NO: 2023).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F with the proviso that the peptide insertion is not TSQRGDYVSL (SEQ ID NO: 2024).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX 3 X 1 SX 2 (SEQ ID NO: 7), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYSSV (SEQ ID NO: 1882), RGDYASL (SEQ ID NO: 2015), RGDYVSV (SEQ ID NO: 1948), RGDYHSF (SEQ ID NO: 1623), STVRGDYTSM (SEQ ID NO: 2013), QERRGDYTSM (SEQ ID NO: 2014), SNSRGDYNSL (SEQ ID NO: 2017), STVRGDYTSV Page 86 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (SEQ ID NO: 2019), SNSRGDYTSV (SEQ ID NO: 2020), SAMRGDYTST (SEQ ID NO:
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of LRGDX 1 X 2 X 3 (SEQ ID NO: 8), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of LRGDX1X2[W/F] (SEQ ID NO: 2033), wherein X 1 and X 2 are independently any amino acid.
  • a peptide insertion comprises a sequence provided in Table 9. [0347] Table 9: Exemplary peptides encompassed by SEQ ID NO: 8.
  • I otein disclosed herein comprises a sequence of RGDX 1 GLX 2 (SEQ ID NO: 9), wherein X 1 is Y, W, or F, and X 2 is any amino acid.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]GLX (SEQ ID NO: 2034) wherein X is any amino acid.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYGLX (SEQ ID NO: 2035), wherein X is any amino acid.
  • a peptide insertion comprises a sequence provided in Table 10.
  • Table 10 Exemplary peptides encompassed by SEQ ID NO: 9.
  • Peptide SEQ ID NO Fold change over AAV9 Page 87 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • a peptide insertion in an AAV capsid protein comprises any one of the peptides disclosed in Table 11.
  • Table 11 Additional exemplary peptides.
  • P eptide SEQ ID N O Fold change over AAV9 [0352] In rotein disclosed herein comprises a sequence of RGDPIRW (SEQ ID NO: 772).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPQRW (SEQ ID NO: 802).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPSPW (SEQ ID NO: 833).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDSERW (SEQ ID NO: 1088).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYESR (SEQ ID NO: 1556).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYLNT (SEQ ID NO: 1696).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYLSV (SEQ ID NO: 1713).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2063), wherein X1 is E, X2 is E or R, and X3 is V or I.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2068), wherein X1 is R, X2 is E or R, and X3 is V or I.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX 1 X 2 X 3 (SEQ ID NO: 2064), wherein X 1 is E or R, X 2 is E, and X3 is V or I.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX 1 X 2 X 3 (SEQ ID NO: 2067), wherein X 1 is E or R, X 2 is R, and X3 is V or I.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX 1 X 2 X 3 (SEQ ID NO: 2066), wherein X 1 is E or R, X 2 is E or R, and X3 is V.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX 1 X 2 X 3 (SEQ ID NO: 2065), wherein X 1 is E or R, X 2 is E or R, and X 3 is I.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYERI (SEQ ID NO: 1551).
  • a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYERI (SEQ ID NO: 1551).
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2047 as shown below: Page 89 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0368] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWG
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I with the proviso that the peptide insertion is not NATRGDYREI (SEQ ID NO: 2069).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X 3 is V or I with the proviso that the peptide insertion is not RRGDYREIPL (SEQ ID NO: 2059).
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYREI (SEQ ID NO: 1825).
  • a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYREI (SEQ ID NO: 1825).
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2046 as shown below: [0373] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL Page 90 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWL
  • a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI does not include an N residue at the third position immediately upstream (or 5’) of the peptide insertion.
  • a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI does not include a R residue immediately upstream (or 5’) of the peptide insertion.
  • a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI does not include a P residue immediately downstream (or 3’) of the peptide insertion.
  • a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI does not include an L residue at the second position immediately downstream (or 3’) of the peptide insertion.
  • a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYREV (SEQ ID NO: 1829).
  • a Page 91 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYREV (SEQ ID NO: 1829).
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2058 as shown below: [0381] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLG
  • one or more modifications are within about 10 amino acids, e.g., within about 5 amino acids, upstream or downstream of the location of the peptide insertion site. [0383] In some embodiments, one or more modifications are located in a variable region IV (VR-IV) of a VP1, VP2, or VP3 of an AAV9 capsid protein, or a variable region V (VR-V) of VP1, VP2, or VP3 of an AAV9 capsid protein, or both. [0384] In some embodiments, VR-IV of a VP1, VP2, or VP3 of an AAV9 capsid protein comprises amino acids 451-475 of a VP1, VP2, or VP3 of an AAV9 capsid protein.
  • VR-V of a VP1, VP2, or VP3 of an AAV9 capsid protein comprises amino acids 488-506 of a VP1, VP2, or VP3 of an AAV9 capsid protein.
  • Page 92 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • one or more modifications comprises an insertion, deletion, mutation, or a combination thereof.
  • a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more modifications reduces glycan binding.
  • a glycan is galactose.
  • one or more modifications is at or between amino acids: (a) 271 and 272 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises any one of the peptide insertions disclosed herein;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 1; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is Page 93 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X 1 and X 2 are independently any amino acid and X 3 is Y, W, or F;
  • a peptide Page 94 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 2; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile Page 97 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 3; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 Page 98 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein;
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 4; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • Page 100 of 248 11821383v1 Attorney Docket No.: 2011256-1804 one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a Page 101 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 5; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein Page 102 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: Page 103 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • (1) a peptide insertion comprises a peptide insertion provided in Table 6; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% Page 104 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the Page 105 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 7;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as Page 106 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein;
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 8;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% Page 108 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of LRGDX 1 X 2 X 3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein;
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR- VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as Page 109 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 9;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity Page 110 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDX 1 GLX 2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid;
  • a peptide insertion site is in a VR- VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 10;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid Page 112 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDYX1X2I (SEQ ID NO: 10), wherein X 1 is E or R, and X 2 is E or R;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein;
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, Page 113 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a peptide insertion provided in Table 11;
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid Page 114 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2065), wherein X1 is E or R, X2 is E or R, and X3 is I;
  • a peptide insertion site is in a VR- VIII of a parental AAV capsid protein;
  • a parental AAV capsid protein comprises an Page 115 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) AAV9 capsid protein.
  • a peptide insertion is in VR-VIII of an AAV9 capsid protein.
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDYERI (SEQ ID NO: 1551); (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental Page 116 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDYREI (SEQ ID NO: 1825); (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion consists of the sequence of RGDYREI (SEQ ID NO: 1825).
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein.
  • a peptide insertion comprises a sequence of RGDYREV (SEQ ID NO: 1829);
  • a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and
  • a parental AAV capsid protein comprises an AAV9 capsid protein.
  • a peptide insertion consists of the sequence of RGDYREV (SEQ ID NO: 1829).
  • a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3.
  • a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein.
  • an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.
  • an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.
  • one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues).
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation.
  • one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof.
  • a mutation that alters a Page 119 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) binding profile of a parental AAV capsid protein comprises a W503R mutation.
  • AAV capsid variants disclosed herein have enhanced muscle-tropism.
  • an AAV capsid variant disclosed herein is characterized in that when administered to a cell or tissue or subject, an AAV capsid variant confers increased infectivity and/or transduction of a muscle cell compared to infectivity and/or transduction of a muscle cell by a control AAV particle comprising a corresponding parental AAV capsid protein.
  • a variant AAV capsid protein confers at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a variant AAV capsid protein confers about 5-fold, about 10-fold, about 15-fold, about 20 fold, about 25-fold, about 30-fold, about 40-fold, or about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a variant AAV capsid protein confers about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30 fold, about 5-fold to about 25-fold, about 5-fold to about 20-fold, about 5-fold to about 15-fold, about 5-fold to about 10- fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, 20-fold to about 50-fold, 25- fold to about 50-fold, 30-fold to about 50-fold, or 40-fold to about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • a variant AAV capsid is an AAV9 variant capsid.
  • Page 120 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • a rAAV particle comprising an AAV capsid variant disclosed herein and a heterologous nucleic acid comprising a nucleotide sequence encoding a payload is characterized in that when administered to a cell or tissue or subject, delivery of a payload is enhanced to a muscle cell as compared to delivery of a similar payload with an otherwise similar AAV particle without an AAV capsid variant disclosed herein.
  • a muscle cell is chosen from: a skeletal muscle cell, a cardia muscle cell, a smooth muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof.
  • Payloads for use in AAV particles comprising an AAV9 capsid variant [0422]
  • a rAAV particle comprising an AAV capsid variant disclosed herein can also comprise a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.
  • a payload is a polypeptide.
  • a payload polypeptide is chosen from: a CRISPR-Cas protein, a Zinc finger protein, a TAL, a base editor, a prime editor, a meganuclease, or any combination thereof.
  • a polypeptide is or comprises a CRISPR-Cas protein.
  • a CRISPR-Cas protein is chosen from: a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment thereof.
  • the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the CRISPR-Cas protein.
  • a CRISPR-Cas protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain.
  • a CRISPR-Cas protein is a nuclease.
  • a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleic acid molecule.
  • a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule.
  • a polypeptide is or comprises a Zinc finger protein, or a variant or fragment thereof.
  • a Zinc finger protein is chosen from : a Zinc Page 121 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeted zinc-finger DNA-binding unit with a nuclease domain (e.g., Fok1 nuclease domain) fusion protein, or a variant or fragment or combination of any of the foregoing.
  • a Zinc finger protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain.
  • a polypeptide is or comprises a Transcription Activator- Like Effector (TAL) protein, or a variant or fragment thereof.
  • a TAL comprises: a TAL effector DNA binding domain (e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.), a Transcription Activator-Like Effector Nuclease (TALEN), e.g., a TAL effector DNA binding domain fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or combination of any of the foregoing.
  • TAL effector DNA binding domain e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.
  • TALEN Transcription Activator-Like Effector Nuclease
  • TAL effector DNA binding domain e.g., a TAL effector DNA binding domain fused with a nuclease domain (e
  • a TAL protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain.
  • a polypeptide is or comprises a base editor, or a variant or fragment thereof.
  • a base editor comprises a deaminase, an adenosine deaminase enzyme (ABE), a cytosine deaminase enzyme (CBE), an APOBEC1, an APOBEC3A, an APOBEC3G, an evoAPOBEC, a BE4-YE1, a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine deaminases (TadA*), an E.
  • ABE adenosine deaminase enzyme
  • CBE cytosine deaminase enzyme
  • an APOBEC1A an APOBEC3A
  • an APOBEC3G an evoAPOBEC
  • a BE4-YE1 a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine dea
  • coli tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA binding domain monomer, a base editing enzyme that is RNA guided, a DNA glyosylase inhibitor, one or more DNA glycosylase inhibitor domains, a 5-methylcytosine deaminase, a cytidine deaminase domain, an adenine deaminase domain, an adenosine base editor (ABE), a Target-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax., a circularly permuted base editor, an adenosine deaminase enzyme (ADAR), a RNA editing for programmable adenosine to inosine replacement (REPAIR), a leveraging endogenous ADAR for programmable editing of RNA (LEAPER) or a variant or fragment or
  • the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the base editor.
  • a polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising the same.
  • a prime editor and/or system comprising the same comprises: a reverse transcriptase, a prime editing enzyme, Page 122 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) an editing enzyme that includes a reverse transcriptase domain, an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Murine Leukemia Virus (MLV) Reverse Transcriptase, a HIV- 1 reverse transcriptase, a bacterial reverse transcriptase, a reverse transcriptase associated with a DNA binding domain and/or protein, a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase), a prime editing 1 system (PE1), a prime editing 2 system (PE2), a prime editing 3 system (PE3), a prime editing 3b system (PE3b) or a variant or fragment or
  • AMV
  • the payload also comprises a prime editing gRNA (pegRNA) or an extended sgRNA that interacts with the prime editor.
  • a polypeptide is or comprises a meganuclease, or a variant or fragment thereof.
  • a meganuclease is chosen from: a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His- Cyst box family meganuclease, or HNH family endonuclease, an I-SeeI, an I-CeuI, a PI-PspI, a PI-SceI, an I-SceIV, an I-CsmI, an I-PanI, an I-SceII, an I-PpoI, an I-SceIII, an I-CreI, an I-TevI, an I-TevII an I-TevIII or a variant or fragment or combination of any of the foregoing.
  • a polypeptide is associated with a muscle disorder, or a glycogen or sugar storage disorder.
  • a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • a polypeptide is an enzyme.
  • an enzyme is a lysosomal enzyme or an adenosine deaminase enzyme.
  • a polypeptide is an antibody.
  • a polypeptide is a secreted protein. Page 123 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • a payload is an RNA molecule.
  • an RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, a snRNA, circular RNA or an aptamer, or combinations thereof.
  • an RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder.
  • a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchene muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • a payload is a DNA molecule.
  • a DNA molecule comprises a nucleic acid sequence of up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc.
  • a nucleotide sequence encoding a payload comprises a promoter.
  • a promoter is a muscle-specific promoter.
  • a muscle-specific promoter is chosen from: MHCK7, CK8, desmin, tMCK, dMCK, or CK6.
  • a promoter is a dual muscle-liver promoter.
  • a dual muscle-liver promoter is SPc5-12.
  • Uses of AAV particles comprising an AAV9 capsid variant [0441]
  • the present disclosure provides methods of delivering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles as described herein to a cell or tissue, e.g., a muscle cell or tissue.
  • the present disclosure also provides methods of treating a subject with a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles produced using a method or system described herein.
  • a method for treating a muscle disorder in a subject comprising administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein.
  • a composition e.g., a pharmaceutical composition
  • a method for amelioration a symptom of a muscle disorder in a subject comprising administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein.
  • a muscle disorder is chosen from: (a) a muscular dystrophy, e.g., X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker Muscular dystrophy, a Limb-Girdle muscular dystrophy, an Emery Dreifuss muscular dystrophy, a myotonic dystrophy, or Facioscapulohumeral muscular dystrophy (FSHD); (b) a neuro-muscular disease, e.g., Charcot-Marie-Tooth disease, a Myotonic Dystrophy, Nemaline Myopathy, or Facioscapulohumeral muscular dystrophy (FSHD); (c) a sugar or glycogen storage disease, e.g., MPS type III disease or Pompe disease; (d) an expanded repeat disease, e.g., a Myotonic Dystrophy, or Facioscapulohumeral muscular dystrophy (FSHD), or (e) a muscular dystrophy, e.
  • a composition comprising a plurality of rAAV particles as described herein is administered to a subject suffering from or at risk of a disease, disorder, or condition.
  • a composition e.g., a pharmaceutical composition
  • a composition comprising a plurality of rAAV particles as described herein is administered in combination with one or more additional therapeutics agents to a subject.
  • a composition e.g., a pharmaceutical composition
  • a composition comprising a plurality of rAAV particles as described herein is contacted with an organ, tissue, or cells ex vivo.
  • genes and kits of the present invention may be used for the evaluation and/or monitoring of gene therapy.
  • gene therapy comprises administration of a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein.
  • samples for evaluating and/or monitoring gene therapy may be obtained prior to the initiation of gene therapy.
  • Page 125 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, samples are obtained after a first gene therapy treatment or dose.
  • samples are obtained after the conclusion of gene therapy. In some embodiments, samples are obtained at specific time points, intervals, or any other metric of time before, during, or after gene therapy is performed.
  • Method of transfecting host cells using AAV particles comprising an AAV9 capsid variant [0447] The present disclosure, among other things, provides methods for transfection of a host cell comprising: combining nucleic acids with a transfection reagent and introducing the mix to host cells under conditions that lead to transfection of the host cells with the nucleic acids.
  • nucleic acids used in a method disclosed herein comprise one or more vectors.
  • nucleic acids disclosed herein comprise one or more vectors encoding: (i) at least one payload flanked by an AAV inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one AAV Rep polypeptide, (iii) at least one AAV Cap polypeptide, and/or (iv) at least one Adenoviral helper polypeptide.
  • ITR AAV inverted terminal repeat
  • a host cell e.g., a mammalian host cell, e.g., a HEK293
  • a host cell can be transfected with: at least one helper polypeptide (e.g., at least one Ad2 helper polypeptides), at least one Rep polypeptide or a fragment thereof, at least one Cap polypeptide or a fragment thereof, and at least one payload (e.g., for polypeptide expression or an inhibitory or guide nucleic acid).
  • a transfection method disclosed herein is or comprises transient transfection.
  • a transient transfection method is a suspension transient transfection (sTT).
  • a transient transfection method is an adherent transient transfection.
  • the disclosure provides transfected host cells comprising two, three, or four vectors as described herein.
  • the method comprises transfecting a host cell with three vectors.
  • the three vectors comprise: (i) a first vector encoding at least one payload flanked by an AAV ITR on either side of the at least one payload, (ii) a second vector encoding at least one AAV Rep polypeptide and at least one AAV Cap polypeptide, and (iii) a third vector encoding at least one Adenoviral helper polypeptide.
  • the method comprises transfecting a host cell with two vectors.
  • the two vectors comprises (i) a first vector encoding at least one AAV Cap polypeptide and at least one payload flanked by an AAV ITR on either side of the at least one payload; and (ii) a second vector encoding at least one Adenoviral helper polypeptide and at least one AAV Rep polypeptide.
  • Transfection methods disclosed herein comprise transfection of nucleic acids (e.g., comprising one or more vector) with any transfection reagent known to a skilled person for introducing nucleic acid molecules into host cells (e.g., mammalian cells, such as HEK293).
  • a transfection reagent comprises a lipid, a polymer, or a combination thereof.
  • a transfection reagent is a reagent that can form a complex with the nucleic acids.
  • a transfection reagent comprise a polymer, a lipid, or both a polymer and a lipid.
  • a transfection reagent is or comprises a polymer. In some embodiments, a transfection reagent is or comprises lipid. In some embodiments, a transfection reagent comprises a polymer and a lipid. [0456] In some embodiments, a transfection reagent is or comprises a polymer, e.g., a cationic polymer. In some embodiments, a transfection reagent comprises polyethyleneimine (PEI), FectoVIR, TransIT-VirusGEN, or a combination thereof. In some embodiments, a transfection reagent is or comprises polyethyleneimine (PEI). [0457] In some embodiments, host cells are transfected with PEI.
  • PEI polyethyleneimine
  • host cells are transfected with a weight (wt.) ratio of DNA to transfection reagent (e.g., PEI) of about 1:1 to about 1:2, about 1:1 to about 1:5, or about 1:1 to about 1:10, e.g., about 1:0.05, about 1:1, about 1:1.25, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.
  • a wt. ratio of DNA to transfection reagent is dependent on cell culture density (e.g., of adherent or suspension host cells).
  • a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is used in a method of Page 127 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) transfection disclosed herein.
  • a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is about 1:1:1.
  • a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is not about 1:1:1.
  • a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is about 1:0.5:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 5:10:1, about 1:0.5:2, about 1:0.5:10, about 1:0.5:5, about 0.5:5:1, about 1:10:20, about 1:2:1, about 1:3:1, about 1:4:1, about 1:5:1, about 1:6:1, about 1:7:1, about 1:8:1, about 1:9:1, about 1:10:1, about 10:1:1, about 9:1:1, about 8:1:1, about 7:1:1, about 6:1:1, about 6:1:1, about 4:1:
  • a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is about 1:0.5:1 to about 1:0.5:10; about 1:1:1 to about 1:1:10; about 0.5:1:1 to about 5:1:1; about 1:1:1 to about 1:10:1; or about 1:1:1 to about 10:1:1.
  • Host Cells [0461] The present disclosure, among other things, provides host cells for transfection with at least one vector as described herein for production of rAAV particles.
  • a host cell includes a progeny cell of an original cell transfected with at least one vector described herein.
  • a progeny cell of a parental cell may not be substantially identical in morphology or genomic content as a parent cell due to natural, accidental, or deliberate mutation.
  • Components for a host cell to produce rAAV particles may be provided in trans on at least one vector.
  • a stable host cell may comprise at least one polypeptide to produce rAAV particles using methods known to those of skill in the art.
  • a stable host cell comprises at least one polypeptide under control of an inducible promoter.
  • a stable host cell comprises at least one polypeptide under control of a constitutive promoter.
  • a stable host cell e.g., a HEK293 cell
  • Other stable host cells may be generated by one of skill in the art using routine methods.
  • Exemplary host cells include prokaryotes or eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E.
  • the host cell is a mammalian cell. In some embodiments, the host cell is a human, monkey, ape, hamster, rat, or mouse cell.
  • the host cell is selected from a kidney cell (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, or BHK), CHO cell (e.g., CHO Kl, DXB-11 CHO, or Veggie-CHO), COS cell (e.g., COS-7), retinal cell, Vero cell, CV1 cell, HepG2 cell, WI38 cell, MRC 5 cell, Colo205 cell, HB 8065 cell, HL-60 cell (e.g., BHK21), Jurkat cell, Daudi cell, A431 cell (epidermal), CV-1 cell, U937 cell, 3T3 cell, L cell, C127 cell, SP2/0 cell, NS-0 cell, MMT 060562 cell, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, or a cell line derived from an aforementioned cell.
  • a kidney cell e.g., HEK293, 293 EB
  • the host cell comprises a kidney cell (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, or BHK). In certain embodiments, the host cell comprises a HEK293 cell. In some embodiments, the host cell (e.g., a HEK 293 cell) comprises or expresses an E1 polypeptide. In some embodiments, the host cell does not comprise or express an E1 polypeptide. In some embodiments, the host cell comprises a CHO cell (e.g., CHO-K, DXB-11 CHO, or Veggie-CHO). In certain embodiments, the host cell comprises a CHO-K cell.
  • CHO-K e.g., CHO-K, DXB-11 CHO, or Veggie-CHO
  • host cells are or comprise suspension cells.
  • Page 129 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0467]
  • host cells e.g., adherent or suspended host cells
  • host cells e.g., adherent host cells
  • vc viable cells
  • vc/cm 2 a density of at least about 1.0 x 10 4 viable cells (vc)/cm 2 , e.g., at a density of about 1.0 x 10 4 vc/cm 2 to about 2.0 x 10 4 vc/cm 2 , e.g., about 1.0 x 10 4 vc/cm 2 , about 1.1 x 10 4 vc/cm 2 , about 1.2 x 10 4 vc/cm 2 , about 1.3 x 10 4 vc/cm 2 , about 1.4 x 10 4 vc/cm 2 , about 1.5 x 10 4 vc/cm 2 , about 1.6 x 10 4 vc/cm 2 , about 1.7 x 10 4 vc/cm 2 , about 1.8 x 10 4 vc/cm 2 , about 1.9 x 10 4 vc
  • host cells e.g., suspended host cells
  • host cells are seeded at a density of at least 1.0 x 10 6 vc/cm 2 +/- 15%, e.g., at a density of 1.0 x 10 6 vc/cm 2 +/- 15% to 2.0 x 10 6 vc/cm 2 +/- 15%, e.g., 1.0 x 10 6 vc/cm 2 +/- 15%, 1.1 x 10 6 vc/cm 2 +/- 15%, 1.2 x 10 6 vc/cm 2 +/- 15%, 1.3 x 10 6 vc/cm 2 +/- 15%, 1.4 x 10 6 vc/cm 2 +/- 15%, 1.5 x 10 6 vc/cm 2 +/- 15%, 1.6 x 10 6 vc/cm 2 +/- 15%, 1.7 x 10 6 vc/cm 2 +/- 15%, 1.8 x 10 6 vc
  • vectors can be used in methods of producing rAAV particles described herein.
  • Non-limiting examples of vectors include plasmids, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, and viral vectors (e.g., vectors suitable for gene therapy).
  • a vector genetic element may be delivered by any suitable method known in the art, e.g., 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.).
  • a vector encodes at least one helper polypeptide. In some embodiments, a vector encodes at least one Rep polypeptide and/or at least one Cap polypeptide. In some embodiments, a vector encodes at least one payload (e.g., for expression of polypeptide or as an inhibitory or guide nucleic acid). In some embodiments, a vector encodes at least one helper polypeptide and at least one Rep polypeptide. In some embodiments, a vector encodes at least one Cap polypeptide and at least one payload.
  • a vector can include conventional control elements operably linked to a nucleic acid encoding any polypeptide or payload described herein, in a manner that permits transcription, translation and/or expression in a cell transfected with a vector described herein.
  • Page 130 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • constitutive promoters include, but are not limited to, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with CMV enhancer), an SV40 promoter, and a dihydrofolate reductase promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors, such as temperature, or the presence of a specific physiological state (e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only).
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
  • inducible promoters regulated by exogenously supplied promoters include a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system, an ecdysone insect promoter, a tetracycline-repressible system, a tetracycline-inducible system, a RU486-inducible system, and a rapamycin-inducible system.
  • MT zinc-inducible sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system ecdysone insect promoter
  • a tetracycline-repressible system a tetracycline-inducible system
  • RU486-inducible system a rapamycin-inducible system.
  • a native promoter or fragment thereof for a nucleic acid encoding any polypeptide or payload described herein may be used.
  • other native expression control elements such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression.
  • the present disclosure provides vectors (e.g., plasmids) encoding at least one helper polypeptide.
  • AAV is a helper-dependent DNA parvovirus, which belongs to the genus Dependovirus.
  • a helper vector can comprise nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication, which may include, but are not limited to, activation of gene transcription, stage specific mRNA splicing, DNA replication, synthesis of at least one Cap polypeptide, and/or capsid assembly.
  • Viral-based helper polypeptides can be derived from any known helper viruses such as adenovirus, herpesvirus, vaccinia virus, or a combination thereof.
  • a helper vector e.g., a plasmid
  • a helper vector for culturing of the host cell can comprise sufficient helper polypeptides to permit packaging of the recombinant AAV vector into the AAV capsid polypeptides.
  • a helper vector comprises an Ad2 helper vector.
  • a nucleic acid sequence of an Ad2 helper vector is derived from an Adenovirus 2 genome (Genbank Accession No. J01917.1).
  • a helper vector comprises an Ad5 helper vector.
  • a nucleic acid sequence of an Ad5 helper vector is derived from an Adenovirus 5 genome (Genbank Accession No. AY601635).
  • Helper polypeptides can comprise at least one, two, three, or four of E1, E2A, E4, or VA RNA.
  • E1 comprises E1a and/or E1b.
  • one or both of E2A and VA RNA increase stability and/or efficiency of AAV mRNA translation, such as for cap gene transcripts.
  • E4 facilitates DNA replication.
  • E1a comprises a transactivator (e.g., regulating activity of at least one Ad gene, AAV rep gene, and/or AAV cap gene).
  • E1b comprises a viral mRNA transport.
  • Helper polypeptides are described in further detail in Coura and Nardi, A role for adeno-associated viral vectors in gene therapy, Genetics and Molecular Biology, 31(1): 1-11 (2008), which is hereby incorporated by reference in its entirety.
  • a helper vector comprises a selection marker. Exemplary selection markers include, but are not limited to, antibiotic resistance genes.
  • an antibiotic resistance gene is not a gene encoding penicillin.
  • an antibiotic resistance gene is not a gene encoding a penicillin-derivative.
  • an antibiotic resistance gene comprises an antibiotic resistance gene chosen from kanamycin, puromycin, neomycin, hygromycin, blasticidin, gentamycin, Gr18, or zeocin. In certain embodiments, an antibiotic resistance gene comprises an antibiotic resistance gene for kanamycin.
  • nucleic acids encoding helper polypeptides are oriented in the same direction (e.g., 5’ to 3’) on a helper vector. In some embodiments, nucleic acids encoding helper polypeptides are transcribed in the same direction from a helper vector. In certain embodiments, helper polypeptides comprise VA RNA and E4 oriented in the same direction on a helper vector.
  • helper polypeptides comprise E4 and E2A oriented in the same direction on a helper vector.
  • helper polypeptides comprise VA RNA, E4, and E2A oriented from 5’ to 3’ in direction on a helper vector.
  • a helper vector does not comprise a nucleic acid sequence encoding a Fiber protein or a fragment thereof (e.g., does not comprise a nucleic acid sequence of Genbank Accession No. AP_000226.1 or a fragment thereof).
  • Vector encoding Rep and/or Cap Polypeptides provides vectors (e.g., plasmids) encoding at least one Rep polypeptide and/or at least one Cap polypeptide (e.g., a variant Cap disclosed herein).
  • Production of rAAV particles can include culturing of a host cell with at least one Rep polypeptide and at least one Cap polypeptide (e.g., a variant Cap disclosed herein).
  • Rep proteins e.g., one, two, three, or four Rep78, Rep68, Rep52, and Rep40
  • a vector comprises a nucleic acid sequence encoding one, two, three, or four of Rep78, Rep68, Rep52, or Rep40, or a variant of any of the foregoing.
  • a Rep polypeptide comprises a nucleic acid sequence derived from an AAV2 serotype.
  • a nucleic acid sequence encoding a Rep polypeptide may be derived from the AAV2 genome (as found in Accession No. NC_001401).
  • a Rep polypeptide comprises an AAV2 Rep polypeptide operably linked to a p5 and/or p19 promotor (as found in Accession No. NC_001401).
  • a Page 133 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Rep polypeptide comprises an amino acid sequence of YP_680422.1 or a fragment thereof.
  • a promoter is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide.
  • a promoter operably linked to a nucleic acid sequence encoding at least one Rep polypeptide comprises a p5 and/or p19 promoter.
  • a wildtype promoter of AAV2 or a variant thereof is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide.
  • a promoter e.g., a p5 promoter regulating expression of at least one Rep polypeptide is located in a different location on a vector than a wildtype promoter of AAV2 or a variant thereof.
  • a promoter e.g., a p5 promoter
  • a promoter is located 3’ of a nucleic acid encoding at least one Rep polypeptide.
  • a promoter e.g., a p5 promoter
  • Cap polypeptides e.g., VP1, VP2, and VP3 are structural proteins comprising a Capsid.
  • a vector comprises a nucleic acid sequence encoding one, two, or three of VP1, VP2, and VP3, e.g., comprising a variant AAV capsid protein disclosed herein.
  • a vector comprises a nucleic acid sequence encoding at least one Cap polypeptide (e.g., a variant AAV capsid polypeptide disclosed herein) and at least one Rep polypeptide.
  • a vector encodes at least one Cap polypeptide (e.g., a variant AAV capsid polypeptide disclosed herein) and a separate vector encodes at least one Rep polypeptide.
  • a nucleic acid sequence encoding a Cap polypeptide comprising an AAV variant capsid disclosed herein may further comprise a nucleic acid sequence derived from a known AAV genome sequence including, but not limited to: AAV9/hu14 provided as SEQ ID NO: 123 in U.S. Patent 7,906,111; AAV1 Accession No. NC_002077 or AF063497; AAV2 Accession No. NC_001401; AAV5 Accession No. Y18065 or AF085716; Accession No. AAV6 NC_001862; or AAV8 Accession No NC_006261.1.
  • a nucleic acid sequence encoding a Cap polypeptide is derived from an AAV genome sequence or a variant thereof as described in US Patent No. 7,906,111, which is hereby incorporated by reference in its entirety.
  • a nucleic acid sequence encoding a Cap polypeptide is derived from an AAV genome sequence or a variant thereof as described in Page 134 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) International Publication No.
  • a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV2 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV2 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV5 serotype, or a variant thereof.
  • a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV8 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV9 serotype, or a variant thereof. [0485] In some embodiments, a promoter is operably linked to a nucleic acid sequence encoding at least one Cap polypeptide comprising an AAV variant capsid disclosed herein.
  • a wildtype promoter of AAV2, AAV5, AAV8, AAV9 is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide.
  • a p40 promoter is operably linked to a nucleic acid sequence encoding at least one Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises.
  • Vector encoding Payload [0486] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one payload.
  • a payload sequence is generally a sequence of interest that is desired to be introduced into a cell, tissue, organ, or organism.
  • a payload is flanked by inverted terminal repeats (ITRs).
  • the AAV sequences of a rAAV vector typically comprise cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp.155-168 (1990), which is hereby incorporated by reference in its entirety).
  • ITR sequences are typically about 145 bp in length.
  • one or both of a 5’ITR or a 3’ ITR nucleic acid sequence are modified relative to a known ITR nucleic acid sequence.
  • a payload is a heterologous protein with a therapeutic purpose, e.g., an enzyme, cytokine, antibody, receptor, fusion protein, or chimeric polypeptide.
  • a payload is linked to a secretion signal sequence for secretion of an expressed polypeptide from a host cell.
  • a payload is a heterologous nucleic acid with a therapeutic purpose, e.g., a miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR/Cas guide RNA, or a precursor thereof.
  • a payload can be selected from any heterologous protein or nucleic acid of interest.
  • a payload sequence comprises one or more aptamer-binding domains or polypeptide-binding domains (e.g., transcription factor binding domains).
  • a vector will also typically include other regulatory elements (e.g., promoters, introns, and/or enhancers) to regulate expression or amount of a payload in a cell or tissue.
  • a payload sequence can be of any length, e.g., between 2 and 5,100 nucleotides in length or any integer value there between.
  • a nucleic acid sequence encoding a payload comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides,
  • a nucleic acid sequence encoding a payload comprises between about 50 and about 5,100 nucleotides in length, between about 100 and about Page 136 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 5,100 nucleotides in length, between about 500 and about 5,100 nucleotides in length, between about 1,000 and about 5,100 nucleotides in length, and/or between about 2,000 and about 5,100 nucleotides in length.
  • Culture vessels and culturing parameters [0490] The present disclosure, among other things, provides methods for culturing of a host cell with at least one vector described herein for production of rAAV particles.
  • a wide variety of growth media may be used in accordance with the present invention.
  • cells may be grown in one of a variety of chemically defined media, wherein the components of the media are both known and controlled.
  • cells may be grown in a complex medium, in which not all components of the medium are known and/or controlled.
  • a culture of host cells can be prepared in any medium suitable for a particular cell type being cultured.
  • a host cell medium comprises, e.g., inorganic salts, carbohydrates (e.g., sugars, such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B group vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, or biotin), fatty acids (e.g., cholesterol or steroids), proteins (e.g., albumin, transferrin, fibronectin, or fetuin), serum (e.g., albumins, growth factors, or growth inhibitors, such as, fetal bovine serum, newborn calf serum, or horse serum), trace elements (e.g., zinc, copper, selenium, or tricarboxylic acid intermediates), hydrolysates (e.g., derived from plant or animal sources), or combinations thereof.
  • carbohydrates e.g., sugars, such as glucose, galactose, maltose, or fructose
  • Exemplary media can include, but is not limited to, Dulbecco's Modified Eagle's Medium ([DMEM], Sigma), FreeStyleTM F17 Expression Medium (ThermoFisher), DMEM/F12 medium (Invitrogen), CD OptiCHOTM medium (Invitrogen), CD EfficientFeedTM media (Invitrogen), Cell Boost (HyCloneTM) media (GE Life Sciences), BalanCDTM CHO Feed (Irvine Scientific), BD RechargeTM (Becton Dickinson), Cellvento FeedTM (EMD Millipore), Ex-cell CHOZN FeedTM (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHOTM (Kerry), Zap-CHOTM (Invitria), ActiCHOTM (PAA/GE Healthcare), Minimal Essential Medium (Sigma), or RPMI-1640 (Sigma).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Media can be supplemented as necessary Page 137 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) with hormones and/or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, or phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine or thymidine), antibiotics (e.g., kanamycin, puromycin, neomycin, hygromycin, blasticidin, gentamycin, Gr18, or zeocin), trace elements, lipids (e.g., linoleic or other fatty acids), or glucose or an equivalent energy source.
  • growth factors e.g., insulin, transferrin, or epidermal growth factor
  • salts e.g., sodium chloride, calcium, magnesium, or phosphate
  • buffers e.
  • the media for culturing host cells comprises glutamine or a glutamine dipeptide. In some embodiments, the media for culturing host cells comprises a surfactant. In some embodiments, the nutrient media is serum-free media, a protein-free media, or a chemically defined media. Any other necessary supplements can also be included at appropriate concentrations that would be known to those skilled in the art. [0493] After culturing of host cells as described herein, a plurality of rAAV particles are recovered. In some embodiments, rAAV particles are recovered by lysing host cells and recovering rAAV particles from lysate, e.g., after centrifugation.
  • rAAV particles are recovered from culture supernatant.
  • a lysis solution for host cells comprises chemical reagents, e.g., detergents (e.g., sodium dodecyl sulfate (SDS), ethyl trimethyl ammonium bromide, Triton X-100, bile salts, such as cholate, or zwitterionic detergents, such as CHAPS).
  • a lysis solution for host cells comprises a salt (e.g., NaCl) and a high pH (e.g., a pH of greater than about 7).
  • rAAV particles are purified using purification methods, such as chromatography (e.g., affinity chromatography or ion-exchange chromatography (e.g., cation exchange chromatography)) or filtration (e.g., UF/DF filtration)).
  • purification methods such as chromatography (e.g., affinity chromatography or ion-exchange chromatography (e.g., cation exchange chromatography)) or filtration (e.g., UF/DF filtration)).
  • chromatography e.g., affinity chromatography or ion-exchange chromatography (e.g., cation exchange chromatography)
  • filtration e.g., UF/DF filtration
  • a large-scale preparation of host cells is at least 3 liters +/- 15% of culture media, 10 liters +/- 15% of culture media, e.g., between 50 liters +/- 15% to 1000 liters +/- 15% of culture media or between 50 liters +/- 15% to 2000 liters+/- 15% of culture media, e.g., at least 20 liters +/- 15%, 30 liters +/- 15%, 40 liters +/- 15%, 50 liters +/- 15%, 55 liters +/- 15%, 60 liters +/- 15%, 65 liters +/- 15%, 70 liters +/- 15%, 75 liters +/- 15%, 80 liters +/- 15%, 85 liters +/- 15%, 90 liters +/- 15%, 95 liters +/- 15%, 100 liters +/- 15%, 200 liters +/- 15%, 300 liters +/- 15%, 400
  • a large-scale preparation of host cells is at least 5 m 2 +/- 15% of culture media, e.g., between 5 m 2 +/- 15% to 500 m 2 +/- 15% of culture media, e.g., at least 5 m 2 +/- 15%, 10 m 2 +/- 15%, 15 m 2 +/- 15%, 20 m 2 +/- 15%, 25 m 2 +/- 15%, 20 m 2 +/- 15%, 35 m 2 +/- 15%, 40 m 2 +/- 15%, 45 m 2 +/- 15%, 50 m 2 +/- 15%, 55 m 2 +/- 15%, 60 m 2 +/- 15%, 65 m 2 +/- 15%, 75 m 2 +/- 15%, 80 m 2 +/- 15%, 85 m 2 +/- 15%, 90 m 2 +/- 15%, 95 m 2 +/- 15%, 100 m 2 +/- 15%, 150 m
  • a host cell can be cultured in a cell culture vessel or a bioreactor.
  • a cell culture vessel is suitable for/used for culturing adherent cells.
  • a cell culture vessel is suitable for/used for culturing suspension cells.
  • Exemplary cell culture vessels include 35mm, 60mm, 100mm, or 150mm dishes, multi-well plates (e.g., 6- well, 12-well, 24-well, 48-well, or 96 well plates), or flasks (e.g., T-flasks, e.g., T-25, T-75, or T- 160 flasks), or shaker flasks.
  • a host cell is cultured in a bioreactor.
  • a bioreactor is suitable for/used for culturing adherent cells.
  • a bioreactor is suitable for/used for culturing suspension cells.
  • a bioreactor can be, e.g., a continuous flow batch bioreactor, a perfusion bioreactor, a batch process bioreactor, or a fed batch bioreactor.
  • An exemplary bioreactor is a fixed bed bioreactor, e.g., an iCELLis bioreactor (used for culturing adherent cells).
  • a bioreactor can be maintained under conditions Page 139 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sufficient to produce rAAV particles. Culture conditions can be modulated to optimize yield, purity, or structure of rAAV particles.
  • a bioreactor comprises a plurality of host cells.
  • host cells in a bioreactor comprise viable cells (vc).
  • a bioreactor comprises at least about 1 x 10 6 , about 1 x 10 7 , about 1 x 10 8 , about 1 x 10 9 , about 1 x 10 10 , about 1 x 10 11 , about 1 x 10 12 , about 1 x 10 13 , or about 1 x 10 14 host cells (e.g., viable host cells).
  • a bioreactor comprises between 1 x 10 6 to 1 x 10 14 host cells; between 1 x 10 6 to 0.5 x 10 14 host cells; between 1 x 10 6 to 1 x 10 13 host cells; between 1 x 10 6 to 0.5 x 10 13 host cells; between 1 x 10 6 to 1 x 10 12 host cells; between 1 x 10 6 to 0.5 x 10 12 host cells; between 1 x 10 6 to 1 x 10 11 host cells; between 1 x 10 6 to 0.5 x 10 11 host cells; between 1 x 10 6 to 1 x 10 10 host cells; between 1 x 10 6 to 1 x 10 10 host cells; between 1 x 10 6 to 0.5 x 10 10 host cells; between 1 x 10 6 to 1 x 10 9 host cells; between 1 x 10 6 to 0.5 x 10 9 host cells; between 1 x 10 6 to 1 x 10 8 host cells; between 1 x 10 6 to 0.5 x 10 8 host cells; between 1 x 10 6 to 1 x 10 7 host cells; between
  • a bioreactor comprises about 0.5 million host cells/mL, about 1 million host cells/mL, about 1.5 million host cells/mL, about 2 million host cells/mL, about 2.5 million host cells/mL, about 3 million host cells/mL, about 3.5 million host cells/mL, about 4 million host cells/mL, about 4.5 million host cells/mL, about 5 million host cells/mL, about 5.5 million host cells/mL, about 6 million host cells/mL, about 7 million host cells/mL, about 8 million host cells/mL, about 9 million host cells/mL, about 10 million host cells/mL.
  • host cells in a bioreactor comprise viable cells (vc).
  • a bioreactor comprises at least about 1 liter, about 2 liters, about 3 liters, about 10 liters, about 20 liters, about 30 liters, about 40 liters, about 50 liters, about 55 liters, about 60 liters, about 65 liters, about 70 liters, about 75 liters, about 80 liters, about 85 liters, about 90 liters, about 95 liters, about 100 liters, about 200 liters, about 300 liters, Page 140 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) about 400 liters, about 500 liters, about 600 liters, about 700 liters, about 800 liters, about 900 liters, about 1000 liters, about 1500 liters, about 2000 liters or about 3000 liters of culture media.
  • a bioreactor is maintained under conditions that promote growth of a host cell, e.g., at a temperature (e.g., 37°C) and gas concentration (e.g., 5% - 10% CO2) that is permissive for growth of the host cell.
  • a bioreactor can perform one or more of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), cleaning, and/or sterilization.
  • Exemplary bioreactor units may contain multiple reactors within a unit, e.g., a unit can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or more bioreactors. Any suitable bioreactor diameter and/or shape can be used. In some embodiments, suitable reactors can be round, e.g., cylindrical. In some embodiments, suitable reactors can be square, e.g., rectangular. rAAV Particle Production [0503] The present disclosure, among other things, rAAV particles produced using methods described herein. Generally, rAAV particles produced using methods described herein may be of any AAV serotype.
  • AAV serotypes generally have different tropisms to infect and/or transduce different tissues.
  • an AAV serotype is selected based on a tropism.
  • a rAAV particle may comprise or be based on a serotype selected from any of the following serotypes, and variants thereof, including, but not limited to: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and AAVrh74.
  • a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV1 serotype or a variant thereof.
  • a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV2 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV3B serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV4 serotype Page 141 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV5 serotype or a variant thereof.
  • a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV6 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV7 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV8 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV9 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV10 serotype or a variant thereof.
  • a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV11 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV12 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV13 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAVrh74 serotype or a variant thereof.
  • a plurality of rAAV particles are produced with methods described herein at a higher titer, e.g., such there is improved rAAV particle production.
  • the improved production comprises a higher yield of the plurality of rAAV particles relative to a plurality of rAAV particles produced with a helper vector comprising a nucleic acid sequence of an antibiotic resistance gene other than KanR (e.g., an Ampicillin resistance gene).
  • a high titer is relative to AAV particles produced from a reference helper vector (e.g., an Ad5 vector, e.g., an Ad5 vector described herein), e.g., under otherwise identical conditions.
  • a high titer is greater than 7.0 x 10 9 vg/mL +/- 15%, e.g., when cultured in suspension. In some embodiments, a high titer is greater than about 7.0 x 10 9 vg/mL, e.g., greater than about 7.5 x 10 9 vg/mL, 8.0 x 10 9 vg/mL, 8.5 x 10 9 vg/mL, 9.0 x 10 9 vg/mL, 1.0 x 10 10 vg/mL, 1.5 x 10 10 vg/mL, 2.0 x 10 10 vg/mL, 2.5 x 10 10 vg/mL, 3.0 x 10 10 vg/mL, 3.5 x 10 10 vg/mL, 4.0 x 10 10 vg/mL, 4.5 x 10 10 vg/mL, 5.0 x 10 10 vg/mL, 5.5
  • a high titer of rAAV particles is at least about 7.0 x 10 9 vg/cm 2 , about 7.5 x 10 9 vg/cm 2 , about 8.0 x 10 9 vg/cm 2 , about 8.5 x 10 9 vg/cm 2 , about 9.0 x 10 9 vg/cm 2 , about 9.5 x 10 9 vg/cm 2 , about 1.0 x 10 10 vg/cm 2 , about 1.5 x 10 10 vg/cm 2 , or higher, e.g., when cultured in a bioreactor, e.g., a fixed bed bioreactor.
  • a high titer of rAAV particles is greater than 5.0 x 10 13 vg/m 2 +/- 15%, e.g., greater than 6.0 x 10 13 vg/m 2 +/- 15, 7.0 x 10 13 vg/m 2 +/- 15, 8.0 x 10 13 vg/m 2 +/- 15, 9.0 x 10 13 vg/m 2 +/- 15, 1.0 x 10 14 vg/m 2 +/- 15, 2.0 x 10 14 vg/m 2 +/- 15, 3.0 x 10 14 vg/m 2 +/- 15, 4.0 x 10 14 vg/m 2 +/- 15, 5.0 x 10 14 vg/m 2 +/- 15, 6.0 x 10 14 vg/m 2 +/- 15, 7.0 x 10 14 vg/m 2 +/- 15, 8.0 x 10 14 vg/m 2 +/- 15, 9.0 x 10 14 vg/m 2 +////////
  • a plurality of rAAV particles described herein is harvested after at least 3 days of culturing. In some embodiments, a plurality of rAAV particles described herein is harvested after at least about 3 days to about 10 days of culturing, e.g., about 3 days to about 7 days, about 3 days to about 5 days, about 4 days to about 9 days, about 4 days to about 8 days, or about 4 days to about 6 days of culturing, e.g., after at least about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or longer after culturing.
  • a plurality of rAAV particles produced with methods described herein is substantially free of one or both of a helper adenovirus or a herpes virus.
  • a plurality of rAAV particles is substantially free of one or both of a helper adenovirus or a herpes virus, e.g., a purity of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more free of one or both of a helper adenovirus or a herpes virus [0511]
  • the foregoing methods for producing recombinant vectors are not meant to be limiting, and other suitable methods will be apparent to the skilled artisan.
  • rAAV Particle Compositions [0512] The present disclosure, among other things, provides a composition comprising a plurality of rAAV particles formed by methods described herein and/or using systems described herein.
  • a composition comprises a pharmaceutical composition Page 143 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) comprising at least one pharmaceutically acceptable component (e.g., a pharmaceutically acceptable carrier, diluent, or excipient).
  • a pharmaceutically acceptable component e.g., a pharmaceutically acceptable carrier, diluent, or excipient.
  • pharmaceutical compositions also contain a pharmaceutically acceptable carrier, excipient, or diluent.
  • excipients include any pharmaceutical agent, e.g., a pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids, such as water, saline, glycerol, sugars, and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, or sulfates; and the salts of organic acids, such as acetates, propionates, malonates, or benzoates.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering substances, may be present in such vehicles.
  • compositions may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, or succinic. Salts tend to be more soluble in aqueous or other protonic solvents than corresponding free base forms.
  • a pharmaceutical composition may be a lyophilized powder.
  • compositions can include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, and isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules, and crystals.
  • Supplementary active compounds can also be incorporated into the compositions.
  • Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art.
  • Page 144 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • compositions suitable for parenteral administration can comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Non-limiting illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils.
  • Aqueous injection suspensions may contain substances that increase the viscosity of a suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions may be prepared as appropriate oil injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents that increase solubility to allow for preparation of highly concentrated solutions.
  • Cosolvents and adjuvants may be added to the formulation.
  • Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.
  • surfactants such as, soya lecithin and oleic acid
  • sorbitan esters such as sorbitan trioleate
  • polyvinylpyrrolidone polyvinylpyrrolidone.
  • compositions e.g., pharmaceutical compositions
  • rAAVs produced with the methods described herein or using systems described herein
  • the route and/or mode of administration can vary depending upon the desired results.
  • dosage regimens can be adjusted to provide the desired response, e.g., a therapeutic response.
  • Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intra-cisterna magna, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intrathecal, intravaginal, transdermal, rectal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin. Mode of administration is left to discretion of a practitioner.
  • a composition may be administered by retinal, subretinal, intravitreal, intracameral or suprachoroidal injection or infusion.
  • Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intra-cisterna magna (ICM), intradermal, intragastric, intramedullary, intramuscular, intranasal, intra-parenchymal (e.g., intra-thalamic), intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, intraspinal, spinal sub-pial, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal administration.
  • bronchial e.g., bronchial instillation
  • buccal enteral
  • interdermal intra-arterial
  • Methods and uses disclosed herein include delivery and administration systemically, regionally or locally, or by any route, for example, by injection or infusion.
  • a composition e.g., a pharmaceutical composition
  • a composition comprising a plurality of rAAV particles formed by methods described herein may be administered by injection or infusion by any route.
  • Delivery of a pharmaceutical composition in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection- enhanced delivery can also be used.
  • compositions may be delivered Page 146 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intra-cisterna magna, intravenously, intra-pleurally, intraarterially, intracoronarily, orally, intrahepatically, via the portal vein, or intramuscularly.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications.
  • a clinician specializing in treatment of patients with certain diseases or disorders may determine the optimal route for administration of vectors described herein.
  • a pharmaceutical composition disclosed herein may also be administered by perfusion, e.g., by limb perfusion.
  • the disclosure provides methods for introducing a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein into a cell, a tissue, or an animal. In some embodiments, such methods comprise contacting a cell, a tissue, or an animal with a composition comprising rAAV particles described herein, such that at least one payload is expressed or present in the cell, tissue, or animal.
  • the disclosure also provides methods for administering a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein to a subject.
  • such methods include administering to a subject (e.g., a mammal), a composition comprising rAAV particles described herein, such that at least one payload is expressed or present in the subject (e.g., in a cell or tissue of a subject).
  • a method includes providing cells of a subject (e.g., a mammal) with a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein, such that at least one payload is expressed or present in the subject.
  • a composition e.g., a pharmaceutical composition
  • a composition comprising rAAV particles described herein can be administered in a sufficient or effective amount to a subject in need thereof.
  • Doses can vary and depend upon a type, onset, progression, severity, frequency, duration, or probability of disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject, and other factors that will be appreciated by a skilled artisan. Dose amount, number, frequency, or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications, or other risk factors of treatment and status of the Page 147 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) subject. A skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
  • a dose to achieve a therapeutic effect will vary based on several factors including, but not limited to: route of administration, level of payload or payload expression required to achieve a therapeutic effect, specific disease treated, any host immune response, and stability of payload or payload expression.
  • route of administration level of payload or payload expression required to achieve a therapeutic effect
  • specific disease treated any host immune response
  • stability of payload or payload expression One skilled in the art can determine a dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors.
  • An effective amount or a sufficient amount can (but need not) be provided in a single administration, may require multiple administrations, and, can (but need not) be, administered alone or in combination with another composition. For example, an amount may be proportionally increased as indicated by need of a subject, type, status, and severity of disease treated or side effects (if any) of treatment.
  • compositions include compositions comprising rAAV particles in an effective amount to achieve an intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using techniques and guidance provided herein. Therapeutic doses can depend on, among other factors, age and general condition of a subject, severity of a disease or disorder, and payload amount or expression in a subject. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on response of an individual patient to rAAV-based treatment.
  • compositions may be delivered to a subject so as to allow production of a payload described herein in vivo by gene- and or cell- based therapies or by ex vivo modification of a patient’s or donor’s cells.
  • a composition e.g., a pharmaceutical composition
  • rAAV particles described herein may be administered to a subject once daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals.
  • a composition comprising rAAV particles described herein may be administered according to a dosing regimen that includes (i) an initial administration that is once Page 148 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals; followed by (ii) a period of no administration of, e.g., 1, 2, 3, 4, 5, 6, 8, or 10 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • a dosing regimen that includes (i) an initial administration that is once Page 148 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals; followed by (ii) a period of no administration of, e.g., 1, 2, 3, 4, 5, 6, 8, or 10 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • a composition comprising rAAV particles described herein may be administered (i) one or more times during an initial time period of up to 2, 4, or 6 weeks or less; followed by (ii) a period of no administration of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • a subject is monitored before and/or following treatment with a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein.
  • AAV capsid reference sequences [0533] SEQ ID NO: 2001: AAV9 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 121 AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE 181 SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI 241 TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR 301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY Q
  • a recombinant adeno-associated virus (rAAV) particle comprising: [0550] (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: [0551] (i) the peptide insertion comprises a sequence of: [0552] (I) RGDX 1 X 2 RX 3 (SEQ ID NO: 1), wherein X 1 and X 2 are independently any amino acid, and X 3 is Y, W, or F; [0553] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0554] (III) RGDHX
  • Embodiment 2 A recombinant adeno-associated virus (rAAV) particle comprising: [0565] (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: [0566] (i) the peptide insertion comprises a sequence provided in Table 1; and [0567] (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and [0568] (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0569] Embodiment 3.
  • Embodiment 4 The rAAV particle of embodiment 1 or 2, wherein the insertion site is located between two non-adjacent amino acids in the variable region of the parental AAV capsid protein.
  • Embodiment 5. The rAAV particle of any one of the preceding embodiments, wherein the insertion of the heterologous peptide replaces a contiguous stretch of amino acids of the parental AAV capsid protein.
  • Embodiment 7 The rAAV particle of any one of the preceding embodiments, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.
  • Embodiment 9 The rAAV particle of embodiment 8, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII of AAV9 comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein.
  • Embodiment 10 The rAAV particle of embodiment 8 or 9, wherein the insertion site is located between amino acids 588 and 589 of VP1 of an AAV9 capsid protein or the corresponding position in the VP1 of another parental AAV capsid protein.
  • Embodiment 11 The rAAV particle of any one of embodiments 8- 10, wherein the insertion site is located between amino acids 588 and 589 of VP2 of an AAV9 capsid protein or the corresponding position in the VP2 of another parental AAV capsid protein.
  • Embodiment 12 The rAAV particle of any one of embodiments 8- 11, wherein the insertion site is located between amino acids 588 and 589 of VP3 of an AAV9 capsid protein or the corresponding position in the VP3 of another parental AAV capsid protein.
  • Embodiment 13 Embodiment 13
  • a VP e.g., VP1, VP2, and/or VP3
  • a VP e.g., VP1, VP2, and/or VP3
  • a VP e.g., VP1, VP2, and/or VP3
  • Embodiment 16 wherein the peptide insertion comprises a sequence provided in Table 2.
  • Embodiment 18 The rAAV particle of embodiment 16 or 17, wherein the peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802).
  • Embodiment 19 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 2.
  • Embodiment 20 The rAAV particle of embodiment 19, wherein the peptide insertion comprises a sequence provided in Table 3.
  • Embodiment 21 Embodiment 21.
  • Embodiment 22 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 3.
  • Embodiment 23 The rAAV particle of embodiment 22, wherein the peptide insertion comprises a sequence provided in Table 4.
  • Embodiment 24 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 4.
  • Embodiment 25 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 4.
  • Embodiment 26 The rAAV particle of embodiment 24 or 25, wherein the peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802).
  • Embodiment 27 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 5. Page 157 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0594] Embodiment 28. The rAAV particle of embodiment 27, wherein the peptide insertion comprises a sequence provided in Table 6. [0595] Embodiment 29.
  • Embodiment 30 The rAAV particle of embodiment 29, wherein the peptide insertion comprises a sequence provided in Table 7.
  • Embodiment 31 The rAAV particle of embodiment 29 or 30, wherein the peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802).
  • Embodiment 32 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 7. [0599] Embodiment 33.
  • Embodiment 34 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 8.
  • Embodiment 35 The rAAV particle of embodiment 34, wherein the peptide insertion comprises a sequence provided in Table 9.
  • Embodiment 36 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 9.
  • Embodiment 37 The rAAV particle of embodiment 36, wherein the peptide insertion comprises a sequence provided in Table 10.
  • Embodiment 38 The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 10. [0605] Embodiment 39. The rAAV particle of embodiment 38, wherein the peptide insertion comprises a sequence provided in Table 11. [0606] Embodiment 40. The rAAV particle of embodiment 38 or 39, wherein the peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551). Page 158 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0607] Embodiment 41.
  • Embodiment 42 The rAAV particle of embodiment 38 or 39, wherein the peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825).
  • Embodiment 42 The rAAV particle of embodiment 38 or 39, wherein the peptide insertion comprises the sequence of RGDYREV (SEQ ID NO: 1829).
  • Embodiment 43 The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein confers increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • Embodiment 44 Embodiment 44.
  • Embodiment 45 The rAAV particle of any one of the preceding embodiments, wherein the muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof.
  • Embodiment 46 Embodiment 46.
  • Embodiment 47. The rAAV particle of embodiment 46, wherein the peptide insertion site is located at or between amino acids 581 and 593 of VP1 of AAV9 or the corresponding position in the capsid protein of another parental AAV capsid protein.
  • Embodiment 48 The rAAV particle of embodiment 46, wherein the peptide insertion site is located between amino acids 588 and 589 of VP1 of AAV9 or the corresponding position in the capsid protein of another parental AAV capsid protein.
  • Embodiment 49 The rAAV particle of any one of embodiments 46-48, wherein the one or more modifications are within about 5 to 10 amino acids upstream or downstream of the location of the peptide insertion site. Page 159 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0616] Embodiment 50. The rAAV particle of embodiment 49, wherein the one or more modifications are within about 5 amino acids upstream or downstream of the location of the peptide insertion site. [0617] Embodiment 51.
  • VR-IV variable region IV
  • Embodiment 52 The rAAV particle of embodiment 51, wherein the AAV9 VP1 VR-IV comprises amino acids 451-475 of VP1 of AAV9.
  • Embodiment 53 Embodiment 53.
  • Embodiment 54 The rAAV particle of any one of embodiments 46-53, wherein the one or more modifications comprises an insertion, deletion, mutation, or a combination thereof.
  • Embodiment 55 The rAAV particle of any one of the preceding embodiments wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region.
  • Embodiment 56 The rAAV particle of embodiment 55, wherein the one or more modifications reduces glycan binding.
  • Embodiment 57 The rAAV particle of embodiment 55 or 56, wherein the glycan is galactose.
  • Embodiment 58 The rAAV particle of any one of embodiments 46-57, wherein the one or more modifications is at or between amino acids: [0625] (a) 271 and 272 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; [0626] (b) 446 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; Page 160 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0627] (c) 470 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; [0628] (d) 501 and 505 (e.g., at any one or all or a combination of
  • Embodiment 59 The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein has at least 90% identity relative to a parental AAV capsid protein.
  • Embodiment 60 The rAAV particle of embodiment 59, wherein percent identity is determined by comparing the sequence of the variant AAV capsid protein without the peptide insertion, with the parental AAV capsid protein.
  • Embodiment 61 Embodiment 61.
  • the rAAV particle of embodiment 60 wherein the variant AAV capsid protein and the parental AAV capsid protein have 100% identity when: [0635] (a) the peptide insertion in the variant AAV capsid protein is not taken into account in the sequence comparison; and [0636] (b) the variant AAV capsid protein does not have one or more modifications other than the peptide insertion. [0637] Embodiment 62.
  • the rAAV particle of embodiment 60 wherein the variant AAV capsid protein and the parental AAV capsid protein have less than 100% identity when: [0638] (a) the peptide insertion in the variant AAV capsid protein is not taken into account in the sequence comparison; and Page 161 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0639] (b) the variant AAV capsid protein comprises one or more modifications other than the peptide insertion. [0640] Embodiment 63. The rAAV particle of any one of the preceding embodiments, wherein the parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001. [0641] Embodiment 64.
  • Embodiment 65 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002.
  • Embodiment 65 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003.
  • Embodiment 66 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2050.
  • Embodiment 67 Embodiment 67.
  • the rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006.
  • Embodiment 70 Embodiment 70.
  • Embodiment 71 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2052.
  • Embodiment 72 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2053.
  • Embodiment 73 Embodiment 73.
  • Embodiment 74 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2054.
  • Embodiment 74 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2055. Page 162 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0652]
  • Embodiment 75 The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2056.
  • Embodiment 76 Embodiment 76.
  • Embodiment 77 The rAAV particle of any one of the preceding embodiments, wherein the payload is a polypeptide.
  • Embodiment 78 Embodiment 78.
  • the rAAV particle of embodiment 77 wherein the polypeptide is or comprises: [0656] (i) a CRISPR-Cas protein or a variant or a fragment thereof; [0657] (ii) a Zinc finger protein, or a variant or a fragment thereof; [0658] (iii) a TAL, or a variant or a fragment thereof; [0659] (iv) a base editor, or a variant or a fragment thereof; [0660] (v) a prime editor, or a variant or a fragment thereof; and/or [0661] (vi) a meganuclease, or a variant or a fragment thereof. [0662] Embodiment 79.
  • a Cas9 protein e.g., a Cas9 protein, a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas
  • Embodiment 81 The rAAV particle of embodiment 80, wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • Embodiment 82 The rAAV particle of embodiment 77, wherein the polypeptide is an enzyme.
  • Embodiment 83 The rAAV particle of embodiment 82, wherein the enzyme is a lysosomal enzyme or an adenosine deaminase enzyme.
  • Embodiment 84 The rAAV particle of embodiment 77, wherein the polypeptide is an antibody.
  • Embodiment 85 The rAAV particle of embodiment 77, wherein the polypeptide is a secreted protein.
  • Embodiment 86 Embodiment 86.
  • Embodiment 87 The rAAV particle of embodiment 86, wherein the RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, circular RNA, a snRNA, or an aptamer.
  • Embodiment 88 The rAAV particle of embodiment 86 or 87, wherein the RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder.
  • Embodiment 89 Embodiment 89.
  • the rAAV particle of embodiment 88 wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • FSHD Facioscapulohumeral muscular dystrophy
  • Embodiment 90 The rAAV particle of any one embodiments 1-76, wherein the payload is a DNA molecule.
  • Embodiment 91 Embodiment 91.
  • Embodiment 92 The rAAV particle of any one of the preceding embodiments, wherein the nucleic acid sequence encoding a payload comprises a promoter.
  • Embodiment 92 The rAAV particle of embodiment 91, wherein the promoter is or comprises a muscle-specific promoter. Page 164 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • Embodiment 93 The rAAV particle of embodiment 92, wherein the muscle- specific promoter is chosen from: MHCK7, CK8, desmin, tMCK, dMCK, or CK6.
  • Embodiment 94 Embodiment 94.
  • Embodiment 95 The rAAV particle of embodiment 94, wherein the dual muscle-liver promoter is SPc5-12.
  • Embodiment 96 A pharmaceutical composition comprising: [0680] (a) a rAAV particle of any one of the preceding embodiments; and [0681] (b) a pharmaceutically acceptable excipient.
  • Embodiment 97 A method of delivering a payload to a muscle cell, comprising administering the pharmaceutical composition of embodiment 96 to the muscle cell.
  • Embodiment 98 Embodiment 98.
  • Embodiment 101 A method of treating a subject having a muscle disorder and/or ameliorating a symptom of a muscle disorder in a subject, the method comprising [0687] administering to the subject the pharmaceutical composition of embodiment 96.
  • Embodiment 102 A method of treating a subject having a muscle disorder and/or ameliorating a symptom of a muscle disorder in a subject, the method comprising [0687] administering to the subject the pharmaceutical composition of embodiment 96.
  • the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy.
  • X-linked myotobular myopathy spinal muscular atrophy
  • Duchenne muscular dystrophy Becker muscular dystrophy
  • a Limb-Girdle muscular dystrophy Emery Dreifuss muscular dystrophy
  • Facioscapulohumeral muscular dystrophy FSHD
  • Charcot-Marie- Tooth disease Nemaline Myopathy
  • Pompe Disease or Myotonic Dystrophy.
  • Embodiment 104 The method of any one of embodiments 101-103, wherein the subject is a human.
  • Embodiment 105 Embodiment 105.
  • Embodiment 106 An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: [0693] (i) the peptide insertion comprises a sequence of: [0694] (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0695] (II) RGDX 3 QX 1 X 2 (SEQ ID NO: 2), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F; [0696] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X
  • Embodiment 107 The isolated nucleic acid of embodiment 106, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.
  • Embodiment 108 The isolated nucleic acid of embodiment 106 or 107, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein.
  • Embodiment 109 Embodiment 109.
  • Embodiment 110 An isolated cell comprising the nucleic acid of any one of embodiments 106-109.
  • Embodiment 111 Embodiment 111.
  • a variant AAV capsid protein wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: [0710] (i) the peptide insertion comprises a sequence of: [0711] (I) RGDX 1 X 2 RX 3 (SEQ ID NO: 1), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F; [0712] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; [0713] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; Page 167 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0714
  • Embodiment 112 The variant AAV capsid protein of embodiment 111, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR- VIII or VR-IX of the parental AAV capsid protein.
  • Embodiment 113 The variant AAV capsid protein of embodiment 111 or 112, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein.
  • Embodiment 114 Embodiment 114.
  • the variant AAV capsid protein of embodiment 113 wherein the parental AAV capsid protein is an AAV9 capsid protein and the VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein.
  • Embodiment 115 Embodiment 115.
  • a composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence of Page 168 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0726] (I) RGDX 1 X 2 RX 3 (SEQ ID NO: 1), wherein X 1 and X 2 are independently any amino acid, and X3 is Y, W, or F; [0727] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X 3 is Y, W, or F; [0728] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0729] (IV) RGDPX 1 X 2 X 3 (SEQ ID NO: 4), wherein X 1 and
  • Embodiment 116 A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence provided in Table 1.
  • Embodiment 117 The composition of embodiment 115 or 116, wherein the targeting moiety is conjugated to the payload.
  • Embodiment 118 The composition of any one of embodiments 115-117, wherein the targeting moiety is inserted into a viral protein. Page 169 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0739] Embodiment 119.
  • the composition of embodiment 118, wherein the viral protein is an AAV capsid protein.
  • Embodiment 120 The composition of any one of embodiments 115-117, wherein the targeting moiety is part of, e.g., incorporated into, a vector.
  • Embodiment 121 The composition of any one of embodiments 115-117, wherein the targeting moiety is not part of a vector.
  • Embodiment 122 The composition of any one of embodiments 115-121, wherein the targeting moiety is a muscle-targeting moiety.
  • Example 1 Identification of RGD-containing capsids with enhanced skeletal muscle tropism
  • This Example describes the identification of RGD-containing capsids obtained by screening an AAV library having RGD-containing variants in non-human primates (NHPs).
  • NIPs non-human primates
  • the plasmid backbone for Round 1 library consists of an AAV2 inverted terminal (ITR2)-flanked sequence containing a CBh promoter, AAV2 P40 promoter, a partial AAV9 VP1 coding sequence, and SV40 polyadenylation signal.
  • ITR2 AAV2 inverted terminal
  • Oligonucleotides encoding RGDxxxx and xRGDxxx peptide insertions were synthesized by Integrated DNA Technologies, where x represents standard trinucleotide 19 mix without cysteine.
  • Oligonucleotides were pooled and used as PCR primers to amplify DNA fragments encoding the AAV9 VP1 C-terminus with RGD peptides inserted between amino acid residues 588 and 589 (AAV9 VP1 numbering), which were cloned into AgeI-linearized plasmid Page 170 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) backbone via Gibson assembly. Assembled products were electroporated into NEB 10-beta cells for DNA library production. [0747] AAV Library Production.
  • Virus production was performed by transfecting HEK293T cells with an Ad helper plasmid, a plasmid supplying Rep2, and a limited amount of DNA library at ⁇ 2500 copies per cell using PEI Max.
  • Virus was harvested 3 days post- transfection, and purified through iodixanol density gradient. Virus preparations were buffer exchanged into phosphate-buffered saline (pH 7.4) with 0.001% pluronic F-68, and subsequently subjected to digital droplet PCR (ddPCR)-based titration, purity analysis by SYPRO-Ruby staining of PAGE gels, and endotoxin measurement. [0748] In vivo library screening.
  • In vivo library screening was performed on adult/juvenile cynomolgus macaques sero-negative for AAV9 neutralizing antibodies.
  • two animals were injected intravenously, each with 1E14 genome copies (GC) of AAV library. Necropsies were performed 21 days post-injection, and major organs collected for analysis.
  • Trizol-based RNA isolation was performed from skeletal muscle and heart. To enrich capsid mRNA, hybridization-based mRNA capture was performed using biotinylated antisense oligos and streptavidin paramagnetic beads. Captured RNA was reverse transcribed, and cDNA used for PCR amplification for NGS analysis as well as subsequent round library cloning.
  • Round 2 DNA library construction A synthetic oligo pool consisting of 24,000 oligos, with two oligos encoding each peptide insertion for the top 12,000 enriched Round 1 variants, was synthesized by Twist Biosciences. Round 1 muscle amplicons as well as this oligo pool were used to clone Round 2 DNA library. Recovered Round 1 muscle amplicons were inserted into the AgeI-digested plasmid library backbone by Gibson assembly whereas the synthetic oligo pool underwent low-cycle high-fidelity PCR amplification for subsequent insertion into a modified plasmid backbone with BsaI sites flanking the insertion site by Golden Gate assembly. [0751] NGS and bioinformatics analysis.
  • a custom AAV amplicon-sequencing pipeline was developed to process the NGS raw data. Briefly, paired-end reads were merged, followed by Page 171 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sequential trimming on constant regions allowing maximum 10% error rate. Variable and flanking regions were quality filtered with minimum Phred score of 20. Sequences matching RGDxxxx or xRGDxxx trimer nucleotide sequences are retained for further analysis. Variant fraction in a given AAV or tissue amplicon library is normalized to reads per million (rpm) plus a pseudo count of 0.1.
  • Table 12 VR-VIII peptide insertion sequences for top muscle-targeting capsids with >5 fold enhanced skeletal muscle transduction compared to AAV9.
  • Peptide SEQ ID NO Fold change over AAV9 Page 172 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • Peptide SEQ ID NO Fold change over AAV9 1 7 C Page 173 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • a Page 174 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01)
  • Peptide SEQ ID NO Fold change over AAV9 9 3 A Page 175 of 248 11821383v1
  • RGD tripeptide predominantly resides at the very N-terminus of peptide positions (1951 out of 1991).
  • RGDxxRW SEQ ID NO: 2070
  • RGDYQxx SEQ ID NO: 2071
  • RGDHxxW SEQ ID NO: 2072
  • RGDPxxW SEQ ID NO: 2073
  • RGDYxxV SEQ ID NO: 2074
  • RGDxQxW SEQ ID NO: 2075
  • RGDYxSx SEQ ID NO: 2076
  • LRGDxQ[W/F] SEQ ID NO: 2077.
  • RGDxxRW SEQ ID NO: 2070
  • FDR ⁇ 0.1 WT AAV9
  • FIG. 1 A pool of RGD-containing capsid candidates with enhanced skeletal muscle tropism was identified through a combinatorial approach based on rational design and directed evolution. Several distinct peptide motifs emerged among top candidates, suggesting that a comprehensive functional interrogation was achieved within the RGD peptide space.
  • RGD-containing capsids disclosed herein can be used to target muscle cells for delivery of a payload to muscle cells.
  • an AAV capsid protein having an RGD-peptide has increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • an AAV capsid protein having an RGD-peptide can be used to deliver a payload to a muscle cell, e.g., to treat a muscle disorder, or a glycogen or sugar storage disorder.
  • Example 2 Identification of RGD-containing capsids with enhanced skeletal muscle tropism
  • This Example describes the identification of RGD-containing capsids obtained by screening an AAV library having RGD-containing variants in non-human primates (NHPs). Page 225 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0760] Materials and Methods [0761] Round 3 DNA library construction.
  • the Round 3 library includes 73 capsid variants selected from the Round 2 library.
  • RGDPQRW SEQ ID NO: 802
  • RGDYGLH SEQ ID NO: 1574
  • wtAAV8 wtAAV9
  • five previously identified myotropic capsids having SNSRGDYNSL (SEQ ID NO: 2017), STVRGDYTSV (SEQ ID NO: 2019), QERRGDYTSM (SEQ ID NO: 2014), ASTRGDHGVL (SEQ ID NO: 2048), or ENRRGDFNNT (SEQ ID NO: 2049) peptide insertions
  • SNSRGDYNSL SEQ ID NO: 2017
  • STVRGDYTSV SEQ ID NO: 2019
  • QERRGDYTSM SEQ ID NO: 2014
  • ASTRGDHGVL SEQ ID NO: 2048
  • ENRRGDFNNT SEQ ID NO: 2049
  • the sequence encoding the capsid gene was inserted into a RepCap plasmid backbone for AAV production by adherent triple transfection.
  • the plasmid containing the expression cassette included a ubiquitous CAG promoter-driven H2B-EGFP transgene and a bGH polyadenylation signal.
  • a unique 16 bp barcode was inserted into the 3’ UTR (FIG.2).
  • Each capsid variant was produced independently in a 2-layer Cellstack and packaged with three unique barcodes.
  • AAV library and single capsid production. Capsid variants in the Round 3 library were individually produced by adherent triple transfection in HEK293T cells.
  • Producer cells were transfected with an Ad helper plasmid, a plasmid supplying the ITR-flanked barcoded transgene cassette, and the RepCap plasmid supplying the capsid coding sequence.
  • Ad helper plasmid a plasmid supplying the ITR-flanked barcoded transgene cassette
  • RepCap plasmid supplying the capsid coding sequence.
  • Three days following transfection, AAV vectors were harvested and pooled for purification through a discontinuous iodixanol density gradient. The AAV library was then buffer exchanged and concentrated into phosphate-buffered saline (pH 7.4) with 0.001% Pluronic F-68.
  • Genome titer was performed using digital droplet PCR (ddPCR), purity was determined by SDS-PAGE, and endotoxin measurements were obtained using LAL method.
  • Peptides in category E have a 2-fold or more enhanced skeletal muscle transduction compared to AAV9
  • peptides in category F have a more than 3-fold enhanced skeletal muscle transduction compared to AAV9
  • peptides in category G have a more than 4-fold enhanced skeletal muscle transduction compared to AAV9
  • peptides in category H have a more than 5-fold enhanced skeletal muscle transduction compared to AAV9.
  • Table 13 Skeletal muscle transduction of Round 3 RGD capsid variants relative to AAV9.
  • RGDYLNT SEQ ID NO: 1696
  • RGDYESR SEQ ID NO: 1556
  • RGDYERI SEQ ID NO: 1551
  • RGDPSPW SEQ ID NO: 833
  • RGDSERW SEQ ID NO: 1088
  • RGDYLSV SEQ ID NO: 1713
  • RGDYREI SEQ ID NO: 1825
  • RGDPQRW SEQ ID Page 228 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) NO: 802)
  • RGDYREV SEQ ID NO: 1829
  • RGDPIRW SEQ ID NO: 772
  • capsid variants RGDYREI SEQ ID NO: 1825
  • RGDYERI SEQ ID NO: 1551
  • RGDYREV capsid variants
  • Capsid variants RGDYREI SEQ ID NO: 1825
  • RGDYERI SEQ ID NO: 1551
  • RGDYREV SEQ ID NO: 1829
  • RGD Round 3 pooled screening data FOGs.7A-7J
  • a pool of RGD-containing capsid candidates with enhanced skeletal muscle tropism was identified through a combinatorial approach based on rational design and directed evolution. Several distinct peptide motifs emerged among top candidates, suggesting that a comprehensive functional interrogation was achieved within the RGD peptide space.
  • RGD-containing capsids disclosed herein can be used to target muscle cells for delivery of a payload to muscle cells.
  • Example 3 Characterization of RGD-containing capsids with enhanced skeletal muscle tropism. [0774] This Example describes the characterization of RGD-containing capsids obtained by screening an AAV library having RGD-containing variants in non-human primates (NHPs), as described in Examples 1 and 2. [0775] Materials and Methods [0776] Vector production.
  • AAV particles having a wildtype AAV9 capsid and AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid packaged with a CAGG-driven FLAG- tagged mCherry transgene were produced through suspension transient transfection of HEK293T cells. Crude lysates were harvested 3 days post-transfection and purified using POROS CaptureSelect AAV9 affinity resin. AAV preparations were then concentrated and buffer Page 229 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) exchanged into phosphate-buffered saline (pH 7.4) with 0.001% Poloxamer 188 by tangential flow filtration (TFF).
  • TMF tangential flow filtration
  • Genome titer quantification was performed using digital droplet PCR (ddPCR). Purity was assessed by SDS-PAGE followed by SYPRO Ruby staining, and endotoxin was measured using a limulus amebocyte lysate (LAL) assay.
  • ddPCR digital droplet PCR
  • LAL limulus amebocyte lysate
  • AAV vectors (also referred to herein as AAV particles) were administered through a single intravenous bolus injection at 5x10 13 vg/kg. Animals were euthanized 21 days post-injection, and tissues were collected for analysis. [0778] Mice studies. Male C57Bl/6 mice were obtained from Charles River Laboratories.
  • Mouse tissues were harvested 4 weeks post AAV injection and were processed for FFPE or snap-frozen in liquid nitrogen for mRNA analysis.
  • mice were housed on a 12-h light/dark cycle and given food and water ad libitum.
  • IV intravenously
  • 5E12 vg/kg of AAV capsids packaged with a CBA-SV40 intron- mCherry-GT2A-Fluc2-WPRE-hGHpA reporter at 8 weeks old, followed by bioluminescence imaging (BLI) sessions using an IVIS SpectrumCT In Vivo Imaging System (PerkinElmer) at day 14 post dose.
  • mice were injected intraperitoneally (IP) with 150 mg/kg of d-luciferin at 0 minutes, placed in an induction chamber containing 2.5% isoflurane at 2 minutes, and positioned in nose cones on the IVIS imaging platform at 6 minutes. An image was captured at 10 minutes for analysis. Total radiance was measured from same size of region of interest (ROI) across animals, using Living Image software (version 4.7.2, PerkinElmer). Page 230 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0780] Vector genome biodistribution and transgene mRNA quantification. NHP tissues were homogenized in Qiagen Buffer RLT Plus using Genogrinder.
  • Tissue homogenates were aliquoted for DNA extraction using Qiagen DNeasy kit, and RNA extraction using TRIzol LS reagent following manufacturer’s protocol.
  • Vector genome biodistribution was performed via qPCR using primer-probe sets against the WPRE element in the vector genome and the endogenous RPP30 gene.
  • Transgene mRNA quantification was performed via RT-qPCR, using primer-probe sets against the WPRE element, as well as the endogenous GAPDH as control.
  • Transgene mRNA level was normalized against the endogenous GAPDH mRNA level per sample for cross-sample comparison. [0781] Immunohistochemistry studies.
  • Tissues were drop-fixed in 10% neutral-buffer formalin for 2 days, washed in PBS, and paraffin blocked.
  • immunohistochemical (IHC) staining of mCherry staining was conducted on the Leica Bond RXm platform using standard chromogenic methods.
  • HIER antigen retrieval
  • slides were heated in a pH9 EDTA-based buffer for 25 minutes at 94C, followed by a 30-minute antibody incubation (1:6000, Abcam ab167453).
  • Antibody binding was detected using an HRP-conjugated secondary polymer (Biocare, MACH 2 goat anti-rabbit, Ref# RHRP520MM, ready to use at room temperature), followed by chromogenic visualization with diaminobenzidine (DAB).
  • human immortalized myoblasts obtained from Institut de Myologie were used. Cells were grown in growth medium (PromoCell cat # C-39360 with 15% HI FBS (Hyclone cat# SH30070.01HI)).3D myobundles were established using CuriBio’s Mantarray casting platform.
  • RNA isolation and qRT-PCR analysis Trizol-based RNA isolation was performed for skeletal muscles (diaphragm, gastrocnemius, triceps, tibialis anterior, soleus), heart, liver, lung, kidney, and brain.
  • qRT-PCR was performed using TaqMan Assays (Applied Biosystems) in a QuantStudio 12k Flex Real-Time PCR System (Thermo Fisher Scientific). To quantify relative transcript levels, 2 ⁇ Ct was used to compare samples to GAPDH as a housekeeping gene and then to the AAV9-CAG-mCherry group.
  • mCherry fw 5'- TTGGACATCACCTCCCACAAC-3' (SEQ ID NO: 2060); mCherry rev: 5'- CCTCGGCGCGTTCGTA-3’(SEQ ID NO: 2061); mCherry probe: 5'-6FAM- ACTACACCATCGTGGAAC-MGB/NFQ-3' (SEQ ID NO: 2062); mouse GAPDH endogenous control FAM/MGB probe (Mm99999915_g1; Applied Biosystems), NHP GAPDH FAM/MGB probe (Mf04392546_g1; Applied Biosystems).
  • SEQ ID NO: 2062 is an oligonucleotide probe used in TaqMan real-time PCR for mCherry transgene mRNA detection. It has a 6- carboxyfluorescein (6-FAM) conjugated at the 5’-end, and a nonfluorescent quencher-minor groove binder (NFQ-MGB) conjugated at the 3’-end.
  • 6-FAM 6- carboxyfluorescein
  • NFQ-MGB nonfluorescent quencher-minor groove binder
  • RGDYREI SEQ ID NO: 1825
  • RGDYERI capsid variants substantially outperformed AAV9 based on mCherry fluorescence (FIGS.8A-8C and FIGS.9A-D).
  • capsid variant RGDYREV SEQ ID NO: 1829
  • capsid variant RGDYREV packaged with a dual reporter containing mCherry and luciferase, was tested on immortalized human myoblasts differentiated to myotubes.
  • Capsid variant RGDYREV SEQ ID NO: 1829
  • also outperformed AAV9 based on mCherry fluorescence and luciferase activity FIGGS.10B-10C).
  • capsids variants were assessed in contractile human muscle tissues (myobundles). These engineered muscle tissues can respond to electrical and chemical signals to contract, and are more mature than 2D cultures resembling human muscle tissues better.
  • Human myobundles treated with capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) showed enhanced expression of mCherry fluorescent reporter as shown by immunofluorescence analysis of myobundle cross-sections 7 days post-treatment compared to AAV9 (FIGS.11A- 11F).
  • Top capsid variants showed enhanced transduction in mouse muscles following intravenous administration.
  • AAV particles having capsid variants RGDYREI SEQ ID NO: 1825
  • RGDYERI SEQ ID NO: 1551
  • Transduction levels were evaluated by observing reporter gene (mCherry) expression: red coloration of the muscle indicated expression of mCherry with darker red associated with higher mCherry expression.
  • the data shows that AAV particles having capsid variants RGDYREI and RGDYERI transduced mouse quadriceps muscles more efficiently than AAV9 (FIGS.12A-12C).
  • mCherry expression was analyzed at 4 weeks post-treatment by qPCR in skeletal muscle tissues, heart, lung, kidney, brain and liver of C57Bl/6 mice treated with Page 234 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) AAVs containing CAG promoter driven mCherry. Two doses of virus were used for AAV9 (low dose and high dose) and one low dose was used for RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551).
  • mCherry expression was comparable or higher in skeletal muscles of mice treated with RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsids at 5X lower dose compared to AAV9 (high dose).
  • Biodistribution analysis showed that AAV particles having capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) delivered vector genomes to skeletal muscles (Gastrocnemius, Tibialis anterior and diaphragm), with no significant enhancement over AAV9 (FIGS.15A-15F).
  • vector genome distribution may not be directly correlated with AAV transduction (e.g., as measured by payload expression).
  • AAV transduction e.g., as measured by payload expression.
  • FIG. 1 To evaluate muscle transduction for AAV particle having capsid variant RGDYREV (SEQ ID NO: 1829) in vivo, B6 albino mice were i.v.
  • mice injected with AAV particles having a wildtype AAV9 capsid or capsid variant RGDYREV showed higher BLI signal in their limbs compared to mice injected with AAV particles having a wildtype AAV9 capsid (FIGS.16A-16D).
  • AAV capsid variant RGDYREI (SEQ ID NO: 1825) was selected for further validation in NHP.
  • AAV particles having a wildtype AAV9 capsid and AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid were packaged with a CAGG-driven FLAG-tagged mCherry transgene, and the AAV particles were intravenously dosed at 5x10E13 vg/kg to three adult cynomolgus macaques.
  • Dexamethasone in combination with Tacrolimus was orally administered daily to test animals to dampen immune responses.
  • Page 235 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0791]
  • AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid were well tolerated with minimal safety or pathology findings.
  • Vector genome biodistribution and transgene mRNA expression were analyzed in muscle and liver by qPCR and RT-qPCR, respectively (FIG.17 and FIGS.18A-18B).
  • AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid showed similar vector genome biodistribution to that of AAV particles having a wildtype AAV9 capsid in all tested muscles, which is consistent with observations in mice. Although there was no increase in vector genome biodistribution to muscle, AAV particles displaying a RGDYREI (SEQ ID NO: 1825) peptide mediated about 3- fold to about 36- fold enhanced transgene mRNA expression over AAV9 in various muscle tissues including rectus femoris, biceps, triceps, gastrocnemius, and tibialis anterior in NHPs #1 and #3 (FIGS.18A-18B).
  • NHP #2 may have had neutralizing antibodies against AAV9 which could have affected transduction with AAV9 particles displaying RGD-containing peptides.
  • RGD- containing capsids e.g., capsid variants RGDYREI (SEQ ID NO: 1825), RGDYERI (SEQ ID NO: 1551), and RGDYREV (SEQ ID NO: 1829) have enhanced targeting to a muscle cell as compared to a WT AAV9 capsid protein.
  • an AAV capsid protein having an RGD-peptide disclosed herein has increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.
  • an AAV capsid protein having an RGD-peptide disclosed herein can be used to deliver a payload to a muscle cell, e.g., to treat a muscle disorder, or a glycogen or sugar storage disorder.
  • Future experiments to further characterize variant AAV capsid proteins disclosed herein may include evaluation of anti-AAV neutralizing antibodies (e.g., titers of anti-AAV neutralizing antibodies such as titers of anti-AAV9 neutralizing antibodies) in subjects administered AAV particles comprising a variant AAV capsid protein disclosed herein (e.g., Page 236 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) prior to or after administration of AAV particles).
  • such studies may be useful in evaluating vector distribution and/or payload expression, e.g., in samples from subject administered AAV particles comprising a variant AAV capsid protein disclosed herein. Page 237 of 248 11821383v1

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Abstract

The present disclosure pertains to muscle-targeting moieties comprising RGD- containing peptides which peptides can be inserted in the capsid of a recombinant adeno- associated virus (rAAV) vector. Also disclosed herein are compositions comprising muscle- targeting moieties disclosed herein and methods of making and using the same.

Description

Attorney Docket No.: 2011256-1804 (P1811WO01) RGD-CONTAINING PEPTIDES FOR DELIVERING PAYLOADS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63/446,649, filed on February 17, 2023 and U.S. Provisional Patent Application No.63/547,663 filed on November 7, 2023, the entire contents of each of which are hereby incorporated by reference in their entirety. SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said sequence listing, created on February 14, 2024 is named 2011256-1804.xml and is 1,834,072 bytes in size. BACKGROUND [0003] Targeted delivery of payloads (e.g., using recombinant adeno-associated viruses) to cells or tissues for treating and/or preventing diseases remains a challenge. SUMMARY [0004] The present disclosure identifies certain challenges with existing targeted delivery of payloads. For example, the present disclosure identifies that a lack of targeting moieties that can specifically deliver payloads to a target cell or tissue with decreased non-specific delivery to other cells or tissues is a key challenge. Another challenge identified by the present disclosure is the poor potency associated with delivery vectors, e.g., recombinant adeno-associated virus vectors. In some embodiments, improving the potency of delivery vectors could be beneficial in obtaining clinically relevant outcomes. [0005] Among other things, the present disclosure provides technologies that can address certain limitations identified in existing targeted delivery of payloads. The technologies provided herein are particularly useful for specifically delivering payloads to a target cell or tissue. In some embodiments, by specifically delivering payloads to a target cell or tissue, technologies provided here can also increase the potency of a payload. The technologies provided herein are useful for delivering payloads to a target cell or tissue, e.g., muscle cells or tissue. Page 1 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0006] The present disclosure encompasses -targeting moieties comprising a peptide comprising an RGD-motif. Targeting moieties disclosed herein include muscle-targeting moieties. Also disclosed herein are recombinant adeno-associated virus (rAAV) particles comprising a variant AAV capsid comprising a targeting moiety, e.g., a muscle-targeting moiety disclosed herein. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety in a variant AAV capsid is also referred to as a “peptide insertion.” In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, e.g., in a variant AAV capsid, provides muscle-cell tropism. In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein binds to and/or recognizes a target on a cell, e.g., a muscle cell. Also disclosed herein are compositions comprising rAAV particles disclosed herein, and uses of the same. [0007] Disclosed herein is a recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2 X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; or (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. Page 2 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0008] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F ; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments X3 is W or F. In some embodiments X3 is Y. In some embodiments X3 is W. In some embodiments X3 is F. [0009] In some embodiments, a peptide insertion comprises a sequence provided in Table 2. In some embodiments, a peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0010] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F ; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments X3 is W or F. In some embodiments X3 is Y. In some embodiments X3 is W. In some embodiments X3 is F. [0011] In some embodiments, a peptide insertion consists of a sequence provided in Table 2. In some embodiments, a peptide insertion consists of the sequence of RGDPQRW (SEQ ID NO: 802). [0012] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. Page 3 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0013] In some embodiments, a peptide insertion comprises a sequence provided in Table 3. In some embodiments, a peptide insertion does not comprise the sequence of RGDYQAV (SEQ ID NO: 1764). In some embodiments, a peptide insertion does not comprise the sequence of RGDYQTL (SEQ ID NO: 1814). In some embodiments, a peptide insertion does not comprise RGDYQEL (SEQ ID NO: 2008). [0014] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0015] In some embodiments, a peptide insertion consists of a sequence provided in Table 3. [0016] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0017] In some embodiments, a peptide insertion comprises a sequence provided in Table 4. In some embodiments, a peptide insertion does not comprise the sequence of RGDHASW (SEQ ID NO: 191). In some embodiments, a peptide insertion does not comprise the sequence of RGDHSGW (SEQ ID NO: 322). In some embodiments, a peptide insertion does not comprise the sequence of RGDHSTW (SEQ ID NO: 333). In some embodiments, a peptide insertion does not comprise the sequence of RGDHTQW (SEQ ID NO: 349). In some Page 4 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, a peptide insertion does not comprise the sequence of RGDHQNF (SEQ ID NO: 283). [0018] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consist of the sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0019] In some embodiments, a peptide insertion consists of a sequence provided in Table 4. [0020] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0021] In some embodiments, a peptide insertion comprises a sequence provided in Table 5. In some embodiments, a peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0022] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consist of the sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid Page 5 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0023] In some embodiments, a peptide insertion consists of a sequence provided in Table 5. In some embodiments, a peptide insertion consists of the sequence of RGDPQRW (SEQ ID NO: 802). [0024] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0025] In some embodiments, a peptide insertion comprises a sequence provided in Table 6. [0026] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0027] In some embodiments, a peptide insertion consists of a sequence provided in Table 6. [0028] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV Page 6 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0029] In some embodiments, a peptide insertion comprises a sequence provided in Table 7. In some embodiments, a peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0030] In some embodiments, a peptide insertion does not comprise the sequence of RGDVQNF (SEQ ID NO: 2011). In some embodiments, a peptide insertion does not comprise the sequence of RGDHQNF (SEQ ID NO: 283). [0031] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0032] In some embodiments, a peptide insertion consists of a sequence provided in Table 7. In some embodiments, a peptide insertion consists of the sequence of RGDPQRW (SEQ ID NO: 802). [0033] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0034] In some embodiments, a peptide insertion comprises a sequence provided in Table 8. In some embodiments, a peptide insertion does not comprise the sequence of Page 7 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) RGDYVSV (SEQ ID NO: 1948). In some embodiments, a peptide insertion does not comprise the sequence of RGDYSSV (SEQ ID NO: 1882). In some embodiments, a peptide insertion does not comprise the sequence of RGDYHSF (SEQ ID NO: 1623). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTSM (SEQ ID NO: 1906). In some embodiments, a peptide insertion does not comprise the sequence of RGDYASL (SEQ ID NO: 2015). In some embodiments, a peptide insertion does not comprise the sequence of RGDYNSL (SEQ ID NO: 2016). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTSV (SEQ ID NO: 2018). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTST (SEQ ID NO: 2021). In some embodiments, a peptide insertion does not comprise the sequence of RGDYTSL (SEQ ID NO: 2023). [0035] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0036] In some embodiments, a peptide insertion consists of a sequence provided in Table 8. In some embodiments, a peptide insertion does not consists of the sequence of RGDYVSV (SEQ ID NO: 1948). [0037] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. Page 8 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0038] In some embodiments, a peptide insertion comprises a sequence provided in Table 9. [0039] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) a peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0040] In some embodiments, a peptide insertion consists of a sequence provided in Table 9. [0041] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. [0042] In some embodiments, a peptide insertion comprises a sequence provided in Table 10. [0043] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. Page 9 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0044] In some embodiments, a peptide insertion consist of a sequence provided in Table 10. [0045] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0046] In some embodiments, a peptide insertion comprises a sequence provided in Table 11. In some embodiments, a peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide comprises the sequence of RGDYREV (SEQ ID NO: 1829). [0047] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) a peptide insertion consists of the sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0048] In some embodiments, a peptide insertion consists of a sequence provided in Table 11. In some embodiments, a peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide consists of the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide consists of the sequence of RGDYREV (SEQ ID NO: 1829). [0049] This disclosure further provides a rAAV particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence provided in Table 1; and (ii) the peptide insertion site is in a variable region of a Page 10 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0050] Also provided herein is a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) the peptide insertion comprises a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; or (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0051] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion site is located between two adjacent amino acids in the variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0052] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion site is located between two non-adjacent amino acids in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0053] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0054] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion is in VR-VIII of a parental AAV capsid Page 11 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein (e.g., an AAV9 capsid protein). In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2, or VP3 of an AAV9 capsid protein or the corresponding positions in a capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein. [0055] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein. [0056] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion of the heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0057] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion of the heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0058] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding position in the capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein. [0059] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. [0060] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion Page 12 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) comprising any one sequence provided in any one of Tables 1-11, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. [0061] In some embodiments, disclosed herein is a targeting moiety, e.g., a muscle- targeting moiety, conjugated to a payload. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, comprises a peptide sequence provided in any one of Tables 1-11. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is part of (e.g., incorporated into) a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, encapsidates a payload, e.g., as described herein. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is tethered to a payload, e.g., as described herein. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1825. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1551. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1829. [0062] Also disclosed herein is an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein. [0063] Further disclosed herein is an isolated cell transduced with an rAAV particle disclosed herein. Also disclosed herein is a cell comprising an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein. [0064] This disclosure provides a composition comprising a targeting moiety, e.g., a muscle-targeting moiety,, and a payload, wherein the targeting moiety, e.g., a muscle-targeting moiety, comprises a peptide comprising a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; or Page 13 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I. [0065] Also provided herein is a composition comprising a targeting moiety, e.g., a muscle-targeting moiety, and a payload, wherein the targeting moiety, e.g., a muscle-targeting moiety, comprises a peptide comprising a sequence provided in any one of Tables 1-11. In some embodiments, a peptide insertion in a targeting moiety, e.g., a muscle-targeting moiety, comprises a sequence of SEQ ID NO: 1825. In some embodiments, a peptide insertion in a targeting moiety, e.g., a muscle-targeting moiety, comprises a sequence of SEQ ID NO: 1551. In some embodiments, a peptide comprises the sequence of SEQ ID NO: 1829. [0066] In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is conjugated to the payload. [0067] In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is inserted into a viral protein, e.g., an AAV capsid protein. [0068] In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is part of, e.g., incorporated into, a vector. [0069] In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is not part of a vector. [0070] This disclosure further provides a pharmaceutical composition comprising: (a) a rAAV particle disclosed herein; and (b) a pharmaceutically acceptable excipient. [0071] Also provided herein is a method of delivering a payload to a muscle cell, comprising administering a pharmaceutical composition disclosed herein to a muscle cell. [0072] In some embodiments, a muscle cell is in vitro. [0073] In some embodiments, a muscle cell is in vivo. [0074] In some embodiments, a muscle cell is from a subject that has, or has been determined to have, a muscle disorder. Page 14 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0075] This disclosure provides a method of treating a subject having a muscle disorder and/or ameliorating a symptom of a muscle disorder in a subject, the method comprising administering to the subject a pharmaceutical composition disclosed herein. [0076] In some embodiments, a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0077] In some embodiments, a pharmaceutical composition is administered via a route of administration chosen from: intramuscular, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, intra-cisterna magna, or limb perfusion. [0078] In some embodiments, a subject is a human. [0079] In some embodiments of a rAAV particle, a variant AAV capsid, a composition, or a method disclosed herein a variant AAV capsid protein confers increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0080] In some embodiments of a rAAV particle, a variant AAV capsid, a composition, or a method disclosed herein, a variant AAV capsid protein confers at least 5-fold, at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0081] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein confers about 5-fold, about 10-fold, about 15-fold, about 20 fold, about 25-fold, about 30-fold, about 40-fold, or about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. Page 15 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0082] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein confers about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30 fold, about 5-fold to about 25-fold, about 5-fold to about 20-fold, about 5-fold to about 15-fold, about 5-fold to about 10- fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, 20-fold to about 50-fold, 25- fold to about 50-fold, 30-fold to about 50-fold, or 40-fold to about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0083] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof. [0084] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a peptide insertion site is located at or between amino acids 581 and 593 of VP1, VP2 or VP3 of AAV9 or the corresponding position in the capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein. [0085] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein. In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site. In some embodiments, one or more modifications are within about 10 amino acids upstream or downstream of the location of a peptide insertion site. In some embodiments, one or more modifications are within about 5 amino acids upstream or downstream of the location of a peptide insertion site. [0086] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications are located in a variable Page 16 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) region of parental AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, one or more modifications are located in an AAV9 capsid protein variable region, e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX, or any combination thereof. [0087] In some embodiments, one or more modifications are located in: VR-VIII of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein, VR-IV of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein, or VR-V of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein, or any combination thereof. [0088] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a VR-IV comprises amino acids 451-475 of VP1, VP2 or VP3 of an AAV9 capsid protein. [0089] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, VR-V comprises amino acids 488-506 of VP1, VP2 or VP3 of an AAV9 capsid protein. [0090] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications comprises an insertion, deletion, mutation, or a combination thereof. [0091] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. [0092] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose. [0093] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another Page 17 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f). In some embodiments, a parental AAV capsid protein is an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein. [0094] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant capsid has 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% identity relative to a parental AAV capsid protein. In some embodiments, percent identity is determined by comparing the sequence of the variant capsid without the peptide insertion, with a parental AAV capsid protein (e.g., an AAV9 capsid protein). [0095] In some embodiments, a variant capsid protein and a parental AAV capsid protein have 100% identity when: (a) the peptide insertion in the variant capsid protein is not taken into account in the sequence comparison; and (b) the variant capsid protein does not have one or more modifications other than the peptide insertion. [0096] In some embodiments, a variant capsid protein and a parental AAV capsid protein have less than 100% identity when: (a) the peptide insertion in the variant capsid protein is not taken into account in the sequence comparison; and (b) the variant capsid protein comprises one or more modifications other than the peptide insertion. [0097] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001. Page 18 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0098] In some embodiments, a parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002. [0099] In some embodiments, a parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003. [0100] In some embodiments, a parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2050. [0101] In some embodiments, a parental AAV capsid protein is an AAV4 capsid protein of SEQ ID NO: 2051. [0102] In some embodiments, a parental AAV capsid protein is an AAV5 capsid protein of SEQ ID NO: 2004. [0103] In some embodiments, a parental AAV capsid protein is an AAV6 capsid protein of SEQ ID NO: 2005. [0104] In some embodiments, a parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2052. [0105] In some embodiments, a parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006. [0106] In some embodiments, a parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2053. [0107] In some embodiments, a parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2054. [0108] In some embodiments, a parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2055. [0109] In some embodiments, a parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2056. [0110] In some embodiments, a parental AAV capsid protein is an AAVrh74 capsid protein of SEQ ID NO: 2057. Page 19 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0111] In some embodiments of a rAAV particle, a variant AAV capsid, a composition, or a method disclosed herein, a payload is a polypeptide that is encoded by a nucleic acid sequence within the rAAV particle. [0112] In some embodiments, a polypeptide is or comprises a CRISPR-Cas protein. In some embodiments, a CRISPR-Cas protein is chosen from: a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment thereof. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the CRISPR-Cas protein. In some embodiments a CRISPR-Cas protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain. In some embodiments a CRISPR-Cas protein is a nuclease. In some embodiment a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleic acid molecule. In some embodiment a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule. [0113] In some embodiments, a polypeptide is or comprises a Zinc finger protein, or a variant or fragment thereof. In some embodiments, a Zinc finger protein is chosen from: a Zinc finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeted zinc-finger DNA-binding unit optionally fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or combination of any of the foregoing. In some embodiments a Zinc finger protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain. [0114] In some embodiments, a polypeptide is or comprises a Transcription Activator- Like Effector (TAL) protein, or a variant or fragment thereof. In some embodiments, a TAL comprises: a TAL effector DNA binding domain (e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.), a Transcription Activator-Like Effector Nuclease (TALEN), e.g., a TAL effector DNA binding domain fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or combination of any of the foregoing. In some embodiments a TAL protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain. Page 20 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0115] In some embodiments, a polypeptide is or comprises a base editor, or a variant or fragment thereof. In some embodiments, a base editor comprises a deaminase, an adenosine deaminase enzyme (ABE), a cytosine deaminase enzyme (CBE), an APOBEC1, an APOBEC3A, an APOBEC3G, an evoAPOBEC, a BE4-YE1, a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine deaminases (TadA*), an E. coli tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA binding domain monomer, a base editing enzyme that is RNA guided, a DNA glyosylase inhibitor, one or more DNA glycosylase inhibitor domains, a 5-methylcytosine deaminase, a cytidine deaminase domain, an adenine deaminase domain, an adenosine base editor (ABE), a Target-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax., a circularly permuted base editor, an adenosine deaminase enzyme (ADAR), a RNA editing for programmable adenosine to inosine replacement (REPAIR), a leveraging endogenous ADAR for programmable editing of RNA (LEAPER) or a variant or fragment or combination of any of the foregoing. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the base editor. [0116] In some embodiments, a polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising the same. In some embodiments, a prime editor and/or system comprising the same comprises: a reverse transcriptase, a prime editing enzyme, an editing enzyme that includes a reverse transcriptase domain, an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Murine Leukemia Virus (MLV) Reverse Transcriptase, a HIV- 1 reverse transcriptase, a bacterial reverse transcriptase, a reverse transcriptase associated with a DNA binding domain and/or protein, a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase), a prime editing 1 system (PE1), a prime editing 2 system (PE2), a prime editing 3 system (PE3), a prime editing 3b system (PE3b) or a variant or fragment or combination of any of the foregoing. In some embodiments, the payload also comprises a prime editing gRNA (pegRNA) or an extended sgRNA that interacts with the prime editor. [0117] In some embodiments, a polypeptide is or comprises a meganuclease, or a variant or fragment thereof. In some embodiments, a meganuclease is chosen from: a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His- Page 21 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Cyst box family meganuclease, or HNH family endonuclease, an I-SeeI, an I-CeuI, a PI-PspI, a PI-SceI, an I-SceIV, an I-CsmI, an I-PanI, an I-SceII, an I-PpoI, an I-SceIII, an I-CreI, an I-TevI, an I-TevII an I-TevIII or a variant or fragment or combination of any of the foregoing. [0118] In some embodiments, a polypeptide is associated with a muscle disorder, or a glycogen or sugar storage disorder. In some embodiments, a muscle disorder is chosen from: X- linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0119] In some embodiments, a polypeptide is an enzyme. In some embodiments, an enzyme is a lysosomal enzyme or an adenosine deaminase enzyme. [0120] In some embodiments, a polypeptide is an antibody. [0121] In some embodiments, a polypeptide is a secreted protein. [0122] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a payload is an RNA molecule. In some embodiments, an RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, circular RNA, a snRNA, or an aptamer. In some embodiments, an RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder. In some embodiments, a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0123] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a payload is a DNA molecule, e.g., a donor DNA molecule that is integrated into a host genome via homologous recombination. In some embodiments, a DNA molecule comprises a nucleic acid sequence of up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc. In some embodiments of a rAAV particle, Page 22 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) a variant AAV capsid protein, a composition, or a method disclosed herein, a nucleotide sequence encoding a payload comprises a promoter. [0124] In some embodiments, a promoter is or comprises a muscle-specific promoter. In some embodiments, a muscle-specific promoter is chosen from: MHCK7, CK8, desmin, tMCK, dMCK, or CK6, a variant or fragment of any of the foregoing. [0125] In some embodiments, a promoter is or comprises a dual muscle-liver promoter. In some embodiments, a dual muscle-liver promoter is SPc5-12 or a variant or fragment thereof. [0126] As would be understood by one with ordinary skill in the art, a peptide sequence disclosed herein, e.g., a peptide comprising an RGD motif as disclosed in any one of Tables 1- 11, can be used as a targeting moiety, e.g., a muscle-targeting moiety, to deliver a payload. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, can be conjugated to a payload. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is inserted in a viral protein, e.g., an AAV capsid protein. [0127] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWING [0128] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee. [0129] The Figures described below, which together make up the Drawing, are for illustration purposes only, not for limitation. [0130] FIG.1 is a graph showing enhanced skeletal muscle transduction in cynomolgus macaque with RGDxxRW variants. [0131] FIG.2 is a schematic of the transgene cassette used for the Round 3 library. Gene expression is initiated by a ubiquitous CAG promoter driving transcription of a histone-2B Page 23 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (H2B) enhanced green fluorescent protein (EGFP) coding sequence with a unique barcode (BC) in the 3’ UTR. Each capsid variant in the Round 3 library is packaged with three unique 16 bp barcodes. [0132] FIG.3 is a graph showing average capsid enrichment in skeletal muscle tissue following Round 3 library screening in cynomolgus macaque (n = 2). Enrichment values were calculated from the average calculated log2 fold-change (log2FC) versus AAV9 in all analyzed skeletal muscle tissue (gastrocnemius, biceps brachii, quadriceps, soleus, diaphragm, pectoralis major, and rectus abdominis) in each of the two animals. [0133] FIG.4 is a graph showing average capsid enrichment in heart tissue following Round 3 library screening in cynomolgus macaque (n = 2). Enrichment values were calculated from the average log2 fold-change (log2FC) versus AAV9 in heart tissue in both animals. [0134] FIG.5 is a graph showing that top-performing Round 3 capsids in cynomolgus macaque also achieved enhanced skeletal muscle transduction in C57BL/6J mice. The log2 fold- change (log2FC) enrichment values represent the average of all analyzed muscle tissue in the two species. Error bars represent standard error of the mean. [0135] FIG.6 is a graph showing the productivity of select capsid variants before and after purification (n = 1 per capsid). Pre-purification is represented by the measured genome copies (GC) in the harvested lysate following harvest tangential flow filtration (hTFF). Post- purification is represented by the measured genome titer in the final formulation following iodixanol discontinuous gradient ultracentrifugation and formulation TFF (fTFF). [0136] FIGS.7A-7J are graphs showing transduction enhancement observed across multiple muscle tissue regions in cynomolgus macaque. Dotted line represents AAV9 transduction level. [0137] FIGS.8A-8C show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. AAV9 and two RGD capsid variants (with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated primary myotubes from healthy human donors. Images were taken at 1 Page 24 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) day and 5 days post-transduction. FIG.8A is in color, FIG.8B is in greyscale, and FIG.8C is in black and white. [0138] FIGS.9A-9D show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. AAV9, and two capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551), with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits, were packaged with a CAG-mCherry reporter (FIG.9A). Three independent primary myoblast cell lines (hSKMDC_con1, hSKMDC_con2, hSKMDC_con3) were differentiated to myotubes (as described in Example 3). On day 6 of differentiation, cells were transduced with the corresponding AAV particles at the indicated doses (1E4 vg/cell or 1E5 vg/cell). Quantification of mCherry fluorescence was performed 5 days after transduction with AAVs (FIGs.9B-9D). [0139] FIGS.10A-10C show that AAV particles displaying RGD-containing peptides efficiently transduced immortalized human myotubes (ImmSkMDC_AB1190) in culture. AAV9 and capsid variant RGDYREV (SEQ ID NO: 1829; with the specified peptide inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits) were packaged with a CBA- mCherry-Fluc2 dual reporter. Quantification of mCherry fluorescence (FIG.10B) and luciferase activity (FIG.10C) was performed 5 days after transduction with the AAV particles at the indicated doses (1E4 vg/cell, 3E4 vg/cell or 1E5 vg/cell). [0140] FIGS.11A-11F show that AAV particles displaying RGD-containing peptides transduced 3D myobundles more efficiently than AAV particles having a wildtype AAV9 capsid. AAV9 and capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551), with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits, were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated myobundles generated from immortalized myoblasts from healthy human donors. Myobundles were transduced with AAV particles at 7 days post-differentiation at a multiplicity of infection (MOI) of 1E5 vg/cell, and images were taken at 7 days post-viral transduction. Each channel was imaged under identical exposure times across the samples. FIGS.11A, 11C, and 11E show images taken with 4x magnification (scale bar = 250 μm). FIGS.11B, 11D, and 11F show images taken with 10x magnification (scale bar = 250μm). Page 25 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) FIGS.11A-11B are in color, FIGS.11C-11D are in greyscale, and FIGS.11E-11F are in black and white. [0141] FIGS.12A-12C provide a visualization of mCherry expression in transduced mouse quadriceps. AAV9 and two RGD capsid variants (with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits) were packaged with a CAG-mCherry reporter and the corresponding AAV particles were administered via intravenous injection. Images were taken 4 weeks post-transduction. Grid = 10mm. FIG.12A is in color, FIG.12B is in greyscale, and FIG.12C is in black and white. [0142] FIGS.13A-13C are a series of images showing mCherry expression in transduced striated muscle via IHC of tissues from mice treated with AAV particles displaying RGD-containing peptides compared to mice treated with AAV particles having a wild type AAV9 capsid at 4 weeks post-injection. AAV9 and RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variants, with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits, were packaged with a CAG- mCherry reporter. Tissues from animals administered a low dose (2E13 vg/kg) of AAV particles having RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) demonstrated mCherry expression comparable to tissues from animals administered a high dose (1E14 vg/kg) AAV particles having a wild type AAV9 capsid. Tissues were stained for mCherry with a hematoxylin counterstain. FIG.13A is in color, FIG.13B is in greyscale, and FIG.13C is in black and white. [0143] FIGS.14A-14J show that administration of a low dose of AAV particles displaying a RGDYREI (SEQ ID NO: 1825) peptide or a RGDYERI (SEQ ID NO: 1551) peptide have similar or enhanced mCherry mRNA expression compared to administration of a high dose of AAV particles having a wildtype AAV9 capsid in samples from mice administered the AAV particles. The specified peptides were inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. Non-muscle targets (lung, kidney, brain, and liver) showed decreased transduction with RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551). Samples were normalized to GAPDH and compared to low dose AAV9 expression. Page 26 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0144] FIGS.15A-15F show the quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles having a wildtype AAV9 capsid, or AAV particles displaying a RGDYREI (SEQ ID NO: 1825) peptide or a RGDYERI (SEQ ID NO: 1551) peptide with a CAG-mCherry reporter. The specified peptides were inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. Vector genome values were normalized to the RPP30 gene with a standard curve determined from a pUC57 plasmid control. FIG.15A: tibialis anterior; FIG.15B: gastrocnemius muscle; FIG.15C: diaphragm; FIG.15D: brain; FIG. 15E: kidney; FIG.15F: liver. [0145] FIGS.16A-16D show that AAV particles displaying a RGDYREV (SEQ ID NO: 1829) peptide have higher hindlimb transduction compared to AAV particles having a wildtype AAV9 capsid in mice. RGDYREV was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. FIG.16A shows representative images of bioluminescence imaging 14 days after IV injection of AAV particles having capsids containing a dual reporter (CBA- mCherry-Fluc2) at 5E12 vg/kg. Red circles represent regions of interest (ROIs) for measurement. FIGS.16B-16D show average radiance for each ROI throughout time. Data are presented as mean ± SEM (n=5 per group). Two-way ANOVA with Sidak’s multiple comparison’s test. ****p<0.001. [0146] FIG.17 shows qPCR quantification of vector genome biodistribution in NHPs dosed with AAV particles having a wild type AAV9 capsid or AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. Y-axis denotes vector genome copy number per diploid genome (vg/dg). [0147] FIGS.18A-18B are graphs showing RT-qPCR quantification of mCherry mRNA levels in NHPs dosed with AAV particles having a wildtype AAV9 capsid or AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. FIG.18A shows quantification from tissues collected in both AAV9 and RGDYREI capsid groups, where y-axis denotes relative mCherry mRNA levels compared to those from AAV9 transduction in the same tissues. FIG.18B shows quantification from tissues collected solely from the RGDYREI Page 27 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) capsid group, where y-axis denotes relative mCherry levels normalized to that from AAV9 transduction in gastrocnemius. [0148] FIGS.19A-19C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. Animals administered AAV particles displaying a RGDYREI peptide expressed mCherry in the tongue, upper esophageal, and the heart of NHP #1 and NHP #3 (top and bottom panels, respectively). Immunohistochemistry images show mCherry expression was moderate in the liver relative to muscle tissues in all NHPs. NHPs #1 and #2 were male, and NHP #3 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500μm. FIG.19A is in color, FIG.19B is in greyscale, and FIG.19C is in black and white. [0149] FIGS.20A-20C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. Animals administered AAV particles displaying a RGDYREI peptide expressed mCherry in the triceps brachili, medial gastrocnemius, lateral gastrocnemius, soleus, and the vastus lateralis of NHP #1 and NHP #3. NHPs #1 and #2 were male, and NHP #3 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500μm. FIG.20A is in color, FIG.20B is in greyscale, and FIG.20C is in black and white. [0150] FIGS.21A-21C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles displaying a RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between AAV9 VP1 positions 588 and 589 in in all viral protein subunits. Animals administered AAV particles displaying a RGDYREI peptide expressed mCherry in the rectus femoris, tibialis anterior, biceps brachii, pectoral, and the diaphragm of NHP #1 and NHP #3. NHPs #1 and #2 were male, and NHP #3 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500μm. FIG.21A is in color, FIG.21B is in greyscale, and FIG.22C is in black and white. Page 28 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0151] FIGS.22A-22C are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs dosed with AAV particles having a wildtype AAV9 capsid packaged with a CAG-mCherry reporter. NHPs #4 and #5 were male, and #6 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500μm. FIG.22A is in color, FIG.22B is in greyscale, and FIG.22C is in black and white. DEFINITIONS [0152] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included. [0153] 5’ and 3’: The terms “5’” and “3’” are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence. Thus, 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment, while 5’ indicates a segment of the nucleic acid that is upstream of another segment. For example, 3’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence. Similarly, 5’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence. Unless indicated otherwise, the directionality of a nucleic acid will be in the 5’ to 3’ direction of translation. [0154] About or approximately: As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). [0155] Adeno-associated virus (AAV): As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in whole or in part, of the family Parvoviridae and the Page 29 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) genus Dependoparvovirus. AAV is a small replication-defective, nonenveloped virus. AAV includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotypes 4, AAV serotypes 5, AAV serotypes 6, AAV serotypes 7, AAV serotypes 8, AAV serotypes 9, AAV serotypes 10, AAV serotypes 11, AAV serotypes 12, AAV serotype 13, AAVrh74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, non-human primate AAV, e.g., from rhesus monkeys, and any variant of any of the foregoing. Wild-type AAV is replication deficient and requires co- infection of cells by a helper virus, e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate. [0156] Ad2 helper: As used herein, the term “Ad2 helper” refers to the Adenovirus serotype 2 (Ad2) helper virus (e.g., wildtype or recombinantly engineered Ad2 helper virus) and various Ad2 helper genes and/or Ad2 helper polypeptides, including, but not limited, to E1a, E1b, E2a, E4Orf6, VA RNA, and any variant or fragment of any of the foregoing. In some embodiments, an Ad2 helper vector (e.g., plasmid) encodes Ad2 helper polypeptides (e.g., one, two, three, or four of E1 (e.g., E1a and/or E1b), E2A, E4, or VA RNA) necessary to generate functional rAAV particles. In certain embodiments, the Ad2 helper vector is transfected into an E1 complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad2 helper vector and Ad2 helper virus genes can be derived from the Adenovirus 2 genome (Genbank Accession No. J01917.1). [0157] Ad5 helper: As used herein, the term “Ad5 helper” refers to the Adenovirus serotype 5 (Ad5) helper virus (e.g., wildtype or recombinantly engineered Ad5 helper virus) and various Ad5 helper genes and/or Ad5 helper polypeptides, including, but not limited, to E1a, E1b, E2a, E4Orf6, and/or VA RNA. In some embodiments, an Ad5 helper vector (e.g., plasmid) comprises Ad5 helper genes (e.g., one, two, three, or four of E1 (e.g., E1a and/or E1b), E2A, E4, or VA RNA) necessary to generation functional rAAV particles. In certain embodiments, the Ad5 helper vector is transfected into an E1 complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad5 helper vector and Ad5 helper genes can be derived from the Adenovirus 5 genome (Genbank Accession No. AY601635). [0158] Administration: As used herein, the term “administration” refers to the administration of a composition comprising rAAV particles as described herein to a subject. Page 30 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Administration may be by any appropriate route. For example, in some embodiments, administration may be local or systemic administration (e.g., to a mammal, e.g., to a human, e.g., a patient). A composition of the disclosure may be administered by injection or infusion by any route. For example, a composition may be administered by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-cisterna magna, or intrathecal injection or infusion. Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal. [0159] Bioreactor: The term “bioreactor,” as used herein, refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). The bioreactor can be of any size and/or any shape so long as it is useful for culturing a cell culture (e.g., a mammalian cell culture). [0160] Cap polypeptide: As used herein, the term “Cap polypeptide” refers to the structural proteins that form a functional AAV capsid, which can in turn package DNA and infect or transduce a target cell. In some embodiments, a Cap polypeptide comprises a variant AAV capsid as disclosed herein. In some embodiments, Cap polypeptides will comprise all of the AAV capsid subunits, but less than all of the capsid subunits may be present as long as a functional capsid is produced. In some embodiments, the nucleic acid sequence encoding Cap polypeptides will be present on a single vector (e.g., plasmid). In some embodiments, the Cap polypeptide comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAVrh74 Cap polypeptide, or a variant of any of the foregoing. AAV capsid genes and proteins have been described in, e.g., Knipe et al., Fields Virology, Volume 1, (6th ed., Lippincott-Raven Publishers), which is hereby incorporated by reference in its entirety. [0161] Cell Density: As used herein, the term “cell density” refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area. For example, cell density may be represented as viable cells (vc)/cm2 of culture medium or vc/mL. Page 31 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0162] Culture: As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and/or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium. [0163] Fragment: As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole. [0164] Gene: As used herein, the term “gene” refers to a DNA sequence that codes for a product (e.g., an RNA product and/or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or effect one or more aspects of gene expression (e.g., inducible expression, etc.). [0165] Gene therapy: As used herein, the term “gene therapy” refers to insertion or deletion of specific genomic DNA sequences to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells). Target cells may be from a mammal and/or may be cells in a mammalian subject. Mammals include but are not limited to humans, dogs, Page 32 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) cats, cows, sheep, pigs, llamas, etc. In some embodiments, heterologous DNA is transferred to target cells. The heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. Additionally or alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product, or it may encode a product, such as a peptide or RNA that in some manner mediates or modulates, directly or indirectly, expression of a therapeutic product. Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression. Such an inhibitor or repressor or other modulator can be a polypeptide, peptide, or nucleic acid (e.g., DNA or RNA). Gene therapy may include in vivo or ex vivo techniques. In some embodiments, viral and non-viral based gene transfer methods can be used to introduce a nucleic acid encoding a polypeptide of interest or to introduce a therapeutic nucleic acid into mammalian cells or target tissues. Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as poloxamers or liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808- 813 (1992); Miller, Nature 357:455-460 (1992); Feuerbach et al., Kidney International 49:1791- 1794 (1996); Urnov et al., Nature Reviews Genetics 11, 636–646 (2010); and Collins et al., Proceedings Biological Sciences / The Royal Society, 282(1821):pii 20143003 (2015), each of which is hereby incorporated by reference in its entirety. [0166] Host Cell: As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used Page 33 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). [0167] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Page 34 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0168] Improve, increase, inhibit, or reduce: As used herein the terms “improve”, “increase,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample, e.g., of a culture medium) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. [0169] Medium: As used herein, the terms “medium,” “culture medium,” and “growth medium” refer to a solution comprising nutrients to nourish cells (e.g., growing cells, e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for survival and/or minimal growth. The solution can also comprise components that enhance survival and/or growth above the minimal rate, including hormones and growth factors. The solution can be formulated to a pH and concentration of one or more salts that are optimal for cellular survival and/or proliferation. For example, the medium can also be a “defined medium” or “chemically defined medium,” e.g., a serum-free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. One of skill in the art understands a defined medium can comprise recombinant polypeptides, for example, but not limited to, hormones, cytokines, interleukins, and/or other signaling molecules. [0170] Muscle targeting moiety: The phrase “muscle targeting moiety” as used herein refers to a peptide containing an RGD-motif which is effective in targeting a muscle cell or muscle tissue. In some embodiments, a muscle-targeting moiety can target a muscle cell or tissue by: (i) contacting a muscle cell or muscle tissue (e.g., binding to one or more receptors expressed on a muscle cell or tissue); (ii) contacting a cell in contact with a muscle cell or tissue (e.g., binding to one or more receptors expressed on a cell in contact with a muscle cell or tissue); (iii) delivering a payload to a muscle cell or tissue; or (iv) any combination of (i)-(iii). In some embodiments, delivering a payload to a muscle cell or muscle tissue comprises transducing a Page 35 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) muscle cell or muscle tissue. In some embodiments, delivering a payload to a muscle cell or muscle tissue results in expression of (e.g., detectable expression of) a payload in a muscle cell or muscle tissue. In some embodiments, a muscle cell comprises a skeletal muscle cell, a cardiac muscle cell, a smooth muscle cell, or a muscle stem cell, e.g., a muscle satellite cell. In some embodiments, a muscle targeting moiety can be conjugated to a payload. In some embodiments, a muscle targeting moiety can be incorporated into a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a muscle targeting moiety can be inserted in an AAV capsid to form a variant AAV capsid as disclosed herein. [0171] Nucleic acid: The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and single- stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and/or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo- deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and/or modified nucleobases; nucleic acids derived from sugars and/or modified sugars; and nucleic acids derived from phosphate bridges and/or modified phosphorus-atom bridges (also referred to herein as “internucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. In accordance with the methods and compositions described herein, in some embodiments, an RNA comprises a short hairpin RNA (shRNA), small interfering RNA Page 36 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (siRNA), mRNA, snRNA, CRISPR/Cas guide RNA, microRNA (miRNA), and/or a precursor thereof. [0172] Payload: As used herein, the term “payload” refers to a nucleic acid sequence of interest (e.g., comprising a sequence that encodes a target payload, such as a target polypeptide or RNA) that is desired to be introduced into a cell, tissue, organ, organism, and/or system comprising cells; or a polypeptide. A target payload can be a heterologous protein with a therapeutic purpose, e.g., an enzyme or antibody. The target payload can be a heterologous nucleic acid with a therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR/Cas guide RNA, or a precursor thereof. One of skill in the art will recognize that the target payload can be selected from any heterologous protein or nucleic acid of interest. As used herein, “encode” or “encodes” means directs the expression of or processed into. For example, as used herein, a nucleic acid encodes a polypeptide sequence if it directs the expression of that polypeptide sequence. As another example, as used herein, a nucleic acid precursor (e.g., a pri- miRNA or pre-miRNA) encodes a further processed version of the nucleic acid (e.g., mature miRNA) if it is processed into the further processed version. [0173] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition comprising rAAV particles that is suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen. In some embodiments, a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to achieve a desired therapeutic effect when administered to a subject or population in need thereof (e.g., by a statistically significant probability). A pharmaceutical composition may be specially formulated for administration in solid or liquid form. In some embodiments, a pharmaceutical composition is formulated for administration by parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection. In some embodiments, a pharmaceutical composition is formulated as a sterile solution or suspension, e.g., in a sustained- release formulation. Pharmaceutical compositions of the disclosure may be formulated for administration by injection or infusion (e.g., subcutaneous, intramuscular, intravenous or Page 37 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) epidural injection or infusion). For example, compositions may be formulated for administration by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-cisterna magna, or intrathecal injection or infusion. In some embodiments, a pharmaceutical composition is intended and suitable for administration to a human subject. In some embodiments, a pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free). Formulations of the pharmaceutical compositions may include, but are not limited to, formulations for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; topical application, such as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces. [0174] Polypeptide: The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to Page 38 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. [0175] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and/or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and/or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and/or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and/or otherwise generating a nucleic acid that encodes and/or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc). [0176] Recombinant AAV (rAAV) particle: A “recombinant AAV particle”, or “rAAV particle,” as used herein, refers to an infectious, replication-defective viral particle comprising an AAV protein shell encapsulating a payload that is flanked on both sides by ITRs. An AAV particle is produced in a suitable host cell (e.g., a HEK293 cell). For example, the host cell is transfected with at least one vector encoding one or more helper polypeptides (e.g., Ad2 helper polypeptides), at least one Rep polypeptide, at least one Cap polypeptide, and at least one payload (e.g., for polypeptide expression or a therapeutic nucleic acid), such that the host cell is capable of producing the Rep and Cap polypeptides necessary for packing the rAAV particle. rAAV particles may be used for subsequent gene delivery. [0177] Rep polypeptide: The term “Rep polypeptide”, as used herein, refers to the AAV non-structural proteins that mediate AAV replication for the production of AAV particles. The Page 39 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) AAV replication genes and proteins have been described in, e.g., Knipe et al., FIELDS VIROLOGY, Volume 1, (6th ed., Lippincott-Raven Publishers), which is hereby incorporated by reference in its entirety. [0178] RGD motif: The phrase “RGD motif” as used herein refers to a peptide comprising the amino acids R, G, and D in consecutive order. Peptides comprising an RGD- motif are provided in Tables 1-11. [0179] Seeding: The term “seeding” as used herein refers to the process of providing a cell culture to a vessel (e.g., a bioreactor or culture flask). For example, the process of providing a cell culture may include propagation of the cells in another bioreactor or vessel before providing to the bioreactor or other vessel. The cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term “seeding” refers to providing any number of cells, including a single cell. [0180] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a neurological disease or disorder or a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g,. clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered. [0181] Titer: As used herein, the term “titer” refers to the quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or Page 40 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume. [0182] Transfection: As used herein, the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells (e.g., mammalian cells). For example, transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and/or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and/or transfection based on cationic polymers (e.g., DEAE-dextran or polyethylenimine). In some embodiments, viral-based transfection is also referred to herein as transduction. [0183] Treating: As used herein, the term “treating” refers to providing treatment, e.g., providing any type of medical or surgical management of a subject. The treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition. “Prevent” refers to causing a disease, disorder, condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering an agent to the subject following the development of one or more symptoms or manifestations indicative of a condition, disease, or disorder, e.g., in order to reverse, alleviate, reduce the severity of, and/or inhibit or prevent the progression of the condition and/or to reverse, alleviate, reduce the severity of, and/or inhibit or one or more symptoms or manifestations of the condition. A composition comprising rAAV particles of the disclosure can be administered to a subject who has developed a disorder or is at increased risk of developing such a disorder relative to a member of the general population. A composition of the disclosure can be administered prophylactically or before development of any symptom or manifestation of the condition. Typically, in this case, the subject will be at risk of developing the condition. [0184] Variant: As used herein in the context of molecules, e.g., nucleic acids, or proteins, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In Page 41 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, Page 42 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature. [0185] Vector: As used herein, the term “vector” refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into a cell. By way of non-limiting example, one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be packaged into a viral capsid and can be transferred into another cell and/or organism. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.” [0186] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's Page 43 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference in its entirety. [0187] VP: As used herein, the term “VP” refers to an AAV VP1 capsid protein, an AAV VP2 capsid protein, an AAV VP3 capsid protein, or variants or fragments or combinations of any of the foregoing. The term “capsid protein” is used interchangeably herein with VP. The numbering used herein in describing exemplary locations of peptide insertions in VP1, VP2 or VP3 are used relative to AAV VP1 numbering. For example VP1, VP2 and VP3 of the AAV9 capsid protein correspond to amino acids 1 to 736 of VP1, amino acids 138 to 736 of VP1 and amino acids 203 to 736 of VP1, respectively. Thus, reference to a peptide insertion between positions 588 and 589 in an AAV capsid variant refers to positions 588 and 589 in VP1, VP2 or VP3 relative to VP1 numbering. Those with knowledge in the pertinent field would be able to readily ascertain the corresponding position in VP2 and VP3, e.g., by comparing the sequences of VP1, VP2 and VP3 of the parental AAV capsid proteins using methods known in the field such as sequence alignment. In some embodiments, a VP capsid protein is a VP1 capsid protein. In some embodiments, a VP capsid protein is a VP2 capsid protein. In some embodiments, a VP capsid protein is a VP3 capsid protein. In some embodiments, a VP protein comprises a peptide insertion disclosed herein. [0188] Variant AAV capsid protein: As used herein, the term “variant AAV capsid protein” refers to a VP capsid protein (e.g., a VP1, VP2, or VP3) comprising a peptide insertion relative to a corresponding parental AAV capsid protein (e.g., a parental VP1, VP2, or VP3). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0189] The present disclosure provides, inter alia, improved recombinant adeno- associated virus (rAAV) particles that can be used for targeting cells or tissue, e.g., muscle cells or tissue. Safe and efficient therapeutic payload delivery to muscle, e.g., skeletal muscle and/or cardiac muscle, remains a major challenge in gene therapy. Recombinant adeno-associated Page 44 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) viruses (rAAVs) have emerged as some of the most promising vectors for in vivo gene therapy, and are currently under clinical evaluation for a number of disorders including muscular disorders. However, naturally occurring AAV capsids sub-optimally target skeletal muscle, and require extremely high doses to achieve minimum effective transgene expression. This poses daunting manufacturing challenges as well as safety concerns. [0190] The present disclosure is based, in part, on the discovery that AAV muscle tropism can be obtained by inserting a short peptide onto an AAV capsid to direct said AAV capsid to a muscle cell. In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein bind to and/or recognize a target on a muscle cell. Without wishing to be bound by any particular theory, in some embodiments, rAAV particles comprising a variant capsid comprising a peptide insertion disclosed herein can enhance vector attachment, internalization, and/or payload expression in muscle cells. AAV9 [0191] Adeno-associated viruses (AAVs) are small, nonenveloped, single-stranded DNA (ssDNA) viruses that belong to the Parvoviridae family. At least twelve distinct AAV serotypes have been identified from human and nonhuman primate sources (see DiMattia MA et al., (2012) J. Virology 86(12):6947, the entire contents of which is hereby incorporated by reference). AAV9 is one of the human AAV serotypes that has enhanced transduction efficiency in cardiac and skeletal muscle, liver tissue, pancreatic tissue, and the eye compared to other serotypes (DiMattia 2012). [0192] The AAV wild-type genome contains at least three genes, rep, cap and X (Buning and Srivastava, (2019) Molecular Therapy: Methods & Clinical Development vol.12 pages 248- 265). The cap gene encodes for viral proteins VP1, VP2, and VP3, and assembly-activating protein (AAP). All three VP proteins are capsid monomers. Transcription of the cap gene results in two messenger RNA: a messenger RNA which encodes VP1 and a messenger RNA which encodes VP2 and VP3 (as described in Warrington KH et al., (2004) Journal of Virology volume 78(12) pages 6595-6609). VP1, VP2, and VP3 are present at ratios of 1:1:10, respectively. The Page 45 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) VP3 region is observed in all capsid structures of AAV serotypes that have been studied (DiMattia 2012). [0193] VPs comprise beta strands, alpha helical regions, and structurally variable regions (VRs) in the surface loops which connect the beta strands. Without wishing to be bound by any particular theory, it is believed that differences in sequence and/or conformations of VRs contribute to the variability in cellular tropism, differences in tissue transduction efficiently, and/or antigenic reactivity among different AAV serotypes. In some embodiments, differences in VR sequence and/or structure among different AAV serotypes allow for differential recognition of cell surface glycans and/or tissue specific protein or lipid receptor interaction for internalization. [0194] Wild type AAV9 (WT AAV9) has nine variable regions VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX (DiMattia 2012, see also Table 3 therein). AAV9 VR-I encompasses amino acid positions 262-269. AAV VR-II encompasses amino acid positions 327-332 and has a role, e.g., in genome packaging. AAV9 VR-III encompasses amino acid positions 382-386. AAV9 VR-IV encompasses amino acid positions 452-460 and has a role, e.g., in liver transduction and/or a delayed blood clearance phenotype. AAV9 VR-V encompasses amino acid positions 488-505 and has a role, e.g., in LamR receptor binding, liver and/or muscle-specific transduction, and/or a delayed blood clearance phenotype. AAV9 VR-VI encompasses amino acid positions 527-539 and has a role, e.g., in LamR receptor binding, and/or a delayed blood clearance phenotype. AAV9 VR-VII encompasses amino acid positions 545-558 and has a role, e.g., in liver transduction and/or delayed blood clearance phenotype. AAV9 VR- VIII encompasses amino acid positions 581-593 and has a role, e.g., in LamR receptor binding and/or transduction. AAV9 VR-IX encompasses amino acid positions 704-714 and has a role, e.g., in heart tropism, melanoma tropism and/or altered tropism. [0195] In some embodiments, a rAAV particle disclosed herein is a recombinant AAV (rAAV) particle. In some embodiments, a rAAV particle comprises a variant AAV9 capsid protein comprising a peptide insertion disclosed herein. In some embodiments, a peptide insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of a parental AAV capsid protein. Page 46 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0196] In some embodiments, a parental AAV capsid protein comprises the sequence of a wildtype AAV capsid protein, or a sequence having at least 95% identity to the sequence of a wildtype AAV capsid protein, or a sequence having no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein. [0197] In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to the sequence of a wildtype AAV capsid protein and one or more mutations, e.g., as disclosed herein. [0198] In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0199] In some embodiments, a parental AAV capsid protein is other than an AAV9 capsid protein and comprises one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) at a position of VP1, VP2 or VP3 corresponding to position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein. Page 47 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0200] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose. [0201] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f). [0202] In some embodiments, a parental AAV capsid protein is chosen from: an AAV9 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, or an AAVrh74 capsid protein. [0203] In some embodiments, a parental AAV capsid protein comprises: an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises: the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. Page 48 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0204] In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. [0205] In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. Exemplary mutations including liver de-targeting mutations are disclosed in Pulicherla N. et al., (2011) Molecular Therapy volume 19, pages 1070-1078, the entire contents of which are hereby incorporated by reference. [0206] In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de- targeting mutation) comprises a mutation at position 503, e.g., a W503R mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 595, e.g., a W595C mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 457, e.g., a N457H mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 574, e.g., a T574S mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 592, e.g., a Q592L mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 498, e.g., a N498Y or an N498I mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 602, e.g., a L602F mutation. In some embodiments, a mutation that alters a Page 49 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 468, e.g., a P468T mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 500, e.g., a E500D mutation. [0207] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose. [0208] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f). [0209] In some embodiments, a parental AAV capsid protein comprises an AAV1 capsid protein. In some embodiments, an AAV1 capsid protein sequence is provided in SEQ ID NO: 2002. [0210] In some embodiments, a parental AAV capsid protein comprises an AAV2 capsid protein. In some embodiments, an AAV2 capsid protein sequence is provided in SEQ ID NO: 2003. [0211] In some embodiments, a parental AAV capsid protein comprises an AAV3B capsid protein. In some embodiments, an AAV3B capsid protein sequence is provided in SEQ ID NO: 2050. Page 50 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0212] In some embodiments, a parental AAV capsid protein comprises an AAV4 capsid protein. In some embodiments, an AAV4 capsid protein sequence is provided in SEQ ID NO: 2051. [0213] In some embodiments, a parental AAV capsid protein comprises an AAV5 capsid protein. In some embodiments, an AAV5 capsid protein sequence is provided in SEQ ID NO: 2004. [0214] In some embodiments, a parental AAV capsid protein comprises an AAV6 capsid protein. In some embodiments, an AAV6 capsid protein sequence is provided in SEQ ID NO: 2005. [0215] In some embodiments, a parental AAV capsid protein comprises an AAV7 capsid protein. In some embodiments, an AAV7 capsid protein sequence is provided in SEQ ID NO: 2052. [0216] In some embodiments, a parental AAV capsid protein comprises an AAV8 capsid protein. In some embodiments, an AAV8 capsid protein sequence is provided in SEQ ID NO: 2006. [0217] In some embodiments, a parental AAV capsid protein comprises an AAV10 capsid protein. In some embodiments, an AAV10 capsid protein sequence is provided in SEQ ID NO: 2053. [0218] In some embodiments, a parental AAV capsid protein comprises an AAV11 capsid protein. In some embodiments, an AAV11 capsid protein sequence is provided in SEQ ID NO: 2054. [0219] In some embodiments, a parental AAV capsid protein comprises an AAV12 capsid protein. In some embodiments, an AAV12 capsid protein sequence is provided in SEQ ID NO: 2055. [0220] In some embodiments, a parental AAV capsid protein comprises an AAV13 capsid protein. In some embodiments, an AAV13 capsid protein sequence is provided in SEQ ID NO: 2056. Page 51 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0221] In some embodiments, a parental AAV capsid protein comprises an AAVrh74 capsid protein. In some embodiments, an AAVrh74 capsid protein sequence is provided in SEQ ID NO: 2057. [0222] In some embodiments, a peptide insertion is in VR-I of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0223] In some embodiments, a peptide insertion is in VR-II of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0224] In some embodiments, a peptide insertion is in VR-III of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0225] In some embodiments, a peptide insertion is in VR-IV of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0226] In some embodiments, a peptide insertion is in VR-V of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0227] In some embodiments, a peptide insertion is in VR-VI of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0228] In some embodiments, a peptide insertion is in VR-VII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0229] In some embodiments, a peptide insertion is in VR-VIII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. Page 52 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0230] In some embodiments, a peptide insertion is in VR-IX of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74. [0231] In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding positions in the capsid proteins of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein. [0232] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein. [0233] In some embodiments, a peptide insertion is in a VP (e.g., VP1, VP2, and/or VP3) of a parental AAV capsid protein. [0234] In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of a parental AAV capsid protein (e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74). [0235] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein. [0236] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein. [0237] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein. [0238] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein. [0239] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein. Page 53 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0240] In some embodiments, a peptide insertion site is located between two adjacent amino acids in VR-VIII of a parental AAV capsid protein. [0241] In some embodiments, a peptide insertion site is located between two non- adjacent amino acids in VR-VIII of a parental AAV capsid protein. [0242] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein. [0243] In some embodiments, insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein. [0244] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid protein, e.g., as compared to a WT AAV9 capsid protein. In some embodiments, an AAV9 WT capsid protein sequence is provided in SEQ ID NO: 2001. In some embodiments, a variant AAV9 capsid protein has 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% identity relative to a WT AAV9 capsid protein. [0245] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV1 capsid protein, e.g., as compared to a WT AAV1 capsid protein. In some embodiments, an AAV1 WT capsid protein is provided in SEQ ID NO: 2002. In some embodiments, a variant AAV1 capsid protein has 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% identity relative to a WT AAV1 capsid. [0246] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV2 capsid protein, e.g., as compared to a WT AAV2 capsid protein. In some embodiments, an AAV2 WT capsid protein is provided in SEQ ID NO: 2003. In some embodiments, a variant AAV2 capsid protein has 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% identity relative to a WT AAV2 capsid protein. [0247] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV3B capsid protein, e.g., as compared to a WT AAV3B capsid protein. In some Page 54 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, an AAV3B WT capsid protein is provided in SEQ ID NO: 2050. In some embodiments, a variant AAV3B capsid protein has 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% identity relative to a WT AAV3B capsid protein. [0248] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV5 capsid protein, e.g., as compared to a WT AAV5 capsid protein. In some embodiments, an AAV5 WT capsid protein is provided in SEQ ID NO: 2004. In some embodiments, a variant AAV5 capsid protein has 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% identity relative to a WT AAV5 capsid protein. [0249] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV6 capsid protein, e.g., as compared to a WT AAV6 capsid protein. In some embodiments, an AAV6 WT capsid protein is provided in SEQ ID NO: 2005. In some embodiments, a variant AAV6 capsid protein has 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% identity relative to a WT AAV6 capsid protein. [0250] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV8 capsid protein, e.g., as compared to a WT AAV8 capsid protein. In some embodiments, an AAV8 WT capsid protein is provided in SEQ ID NO: 2006. In some embodiments, a variant AAV8 capsid protein has 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% identity relative to a WT AAV8 capsid protein. [0251] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV4 capsid protein, e.g., as compared to a WT AAV4 capsid protein. In some embodiments, an AAV4 WT capsid protein is provided in SEQ ID NO: 2051. In some embodiments, a variant AAV4 capsid protein has 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% identity relative to a WT AAV4 capsid protein. Page 55 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0252] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV7 capsid protein, e.g., as compared to a WT AAV7 capsid protein. In some embodiments, an AAV7 WT capsid protein is provided in SEQ ID NO: 2052. In some embodiments, a variant AAV7 capsid protein has 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% identity relative to a WT AAV7 capsid protein. [0253] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV10 capsid protein, e.g., as compared to a WT AAV10 capsid protein. In some embodiments, an AAV10 WT capsid protein is provided in SEQ ID NO: 2053. In some embodiments, a variant AAV10 capsid protein has 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% identity relative to a WT AAV10 capsid protein. [0254] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV11 capsid protein, e.g., as compared to a WT AAV11 capsid protein. In some embodiments, an AAV11 WT capsid protein is provided in SEQ ID NO: 2054. In some embodiments, a variant AAV11 capsid protein has 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% identity relative to a WT AAV11 capsid protein. [0255] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV12 capsid protein, e.g., as compared to a WT AAV12 capsid protein. In some embodiments, an AAV12 WT capsid protein is provided in SEQ ID NO: 2055. In some embodiments, a variant AAV12 capsid protein has 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% identity relative to a WT AAV12 capsid protein. [0256] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV13 capsid protein, e.g., as compared to a WT AAV13 capsid protein. In some embodiments, an AAV13 WT capsid protein is provided in SEQ ID NO: 2056. In some embodiments, a variant AAV13 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least Page 56 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV13 capsid protein. [0257] In some embodiments, a rAAV particle disclosed herein comprises a variant AAVrh74 capsid protein, e.g., as compared to a WT AAVrh74 capsid protein. In some embodiments, an AAVrh74 WT capsid protein is provided in SEQ ID NO: 2057. In some embodiments, a variant AAVrh74 capsid protein has 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% identity relative to a WT AAVrh74 capsid protein. [0258] Additional modifications to an AAV9 capsid protein (not including peptide insertions disclosed herein) are possible including, for example, variants disclosed in International Patent Application WO 2003/052052 filed on November 12, 2002, the entire contents of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in WO 2003/052052. [0259] Several other reports disclose modifications to an AAV9 capsid protein, including: Pulicherla N. et al., (2011) Mol Ther.19(6): pp.1070–1078; Wang D. et al., (2018) Mol Ther Methods Clin Dev. (9): pp.234-246; Adachi K. et al., (2014) Nat. Comm. (5): art. 3075; or Bell CL. Et al., (2012) J Virol.86(13): pp.7326–7333, the entire contents each of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in any of the reports referenced herein. AAV9 capsid variants with peptide insertion [0260] Among other things, disclosed herein, are AAV9 capsid protein variants having one or more peptide insertions, e.g., as disclosed herein. [0261] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein. In some embodiments, a peptide Page 57 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of AAV9. [0262] In some embodiments, a peptide insertion is in VR-I of an AAV9 capsid protein. [0263] In some embodiments, a peptide insertion is in VR-II of an AAV9 capsid protein. [0264] In some embodiments, a peptide insertion is in VR-III of an AAV9 capsid protein. [0265] In some embodiments, a peptide insertion is in VR-IV of an AAV9 capsid protein. [0266] In some embodiments, a peptide insertion is in VR-V of an AAV9 capsid protein. [0267] In some embodiments, a peptide insertion is in VR-VI of an AAV9 capsid protein. [0268] In some embodiments, a peptide insertion is in VR-VII of an AAV9 capsid protein. [0269] In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. [0270] In some embodiments, a peptide insertion is in VR-IX of an AAV9 capsid protein. [0271] In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580-585, amino acids 585-590, amino acids 590-595, or amino acids 595-601 of VP1, VP2 or VP3 of AAV9. In some embodiments, a peptide insertion site is located between amino acids 580-581, between amino acids 581-582, between amino acids 582-583, between amino acids 583-584, between amino acids 584-585, between amino acids 585-586, between amino acids 586-587, between amino acids 587-588, between amino acids 588-589, between amino acids 589-590, between amino acids 590-591, between amino acids 591-592, between amino acids 592-593, between amino acids 593-594, between amino acids 594-595, between amino acids 595-596, Page 58 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) between amino acids 596-597, between amino acids 597-598, between amino acids 598-599, between amino acids 599-600, or between amino acids 600-601 of VP1, VP2 or VP3 of an AAV9 capsid protein. [0272] In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein. [0273] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP3 of an AAV9 capsid protein. [0274] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein. [0275] In some embodiments, a peptide insertion site is located between two adjacent amino acids in VR-VIII of an AAV9 capsid protein [0276] In some embodiments, a peptide insertion site is located between two non- adjacent amino acids in VR-VIII of an AAV9 capsid protein. [0277] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein. [0278] In some embodiments, insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein. [0279] In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising a peptide insertion disclosed herein, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a peptide Page 59 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) insertion comprises a sequence provided in any one of Tables 1-11. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 1, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 2, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 3, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 4, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 5, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 6, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 7, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 8, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 9, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 10, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising any one of the peptides provided in Table 11, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of an AAV9 capsid protein. Page 60 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0280] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV capsid protein comprising a consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) one or more sequences of a VP (e.g., VP1, VP2, or VP3) of an AAV9 capsid protein. [0281] In some embodiments, a peptide insertion does not comprise an additional sequence N-terminal of a peptide sequence provided in any one of Tables 1-11. [0282] In some embodiments, a peptide insertion does not comprise an additional sequence C-terminal of a peptide sequence provided in any one of Tables 1-11. [0283] In some embodiments, a peptide insertion does not comprise an additional sequence N-terminal and C-terminal of a peptide sequence provided in any one of Tables 1-11. [0284] All of the peptides provided in Tables 1-11 have a more than 5-fold enhanced skeletal muscle transduction compared to AAV9. The fold change data is presented in categories A, B, C and D. Peptides in category A have a more than 5 fold enhanced skeletal muscle transduction compared to AAV9, peptides in category B have a more than 10 fold enhanced skeletal muscle transduction compared to AAV9, peptides in category C have a more than 15 fold enhanced skeletal muscle transduction compared to AAV9, and peptides in category D have a more than 20 fold enhanced skeletal muscle transduction compared to AAV9. [0285] Table 1: Exemplary peptide insertions. Peptide SEQ ID NO Fold change over AAV9 Page 61 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 R DHA 173 C Page 62 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 R DRD L 978 B Page 63 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 R DYI 1667 B Page 64 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0286] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein does not comprise RGDRDAL (SEQ ID NO: 975). [0287] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein is not YGVRGDRDAL (SEQ ID NO: 2025). [0288] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1X2R[W/F] (SEQ ID NO: 2026), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 2. [0289] Table 2: Exemplary peptides encompassed by SEQ ID NO: 1. Peptide SEQ ID NO Fold change over AAV9 Page 65 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 862 D [0290] ein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]QX1X2 (SEQ ID Page 66 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) NO: 2027), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 3. [0291] Table 3: Exemplary peptides encompassed by SEQ ID NO: 2. Peptide SEQ ID NO Fold change over AAV9 Page 67 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 R DY H 1795 A Page 68 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 R D M 1452 A [0292] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDYQAV (SEQ ID NO: 1764). [0293] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDYQAV (SEQ ID NO: 1764). [0294] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYQTL (SEQ ID NO: 1814). [0295] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are Page 69 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not SKDRGDYQTL (SEQ ID NO: 2007). [0296] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYQEL (SEQ ID NO: 2008). [0297] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not LSRRGDYQEL (SEQ ID NO: 2009). [0298] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYQAV (SEQ ID NO: 1764), SKDRGDYQTL (SEQ ID NO: 2007) or LSRRGDYQEL (SEQ ID NO: 2009). [0299] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2[W/F] (SEQ ID NO: 2028), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 4. [0300] Table 4: Exemplary peptides encompassed by SEQ ID NO: 3. Peptide SEQ ID NO Fold change over AAV9 Page 70 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 Page 71 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 [0301] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are Page 72 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHASW (SEQ ID NO: 191). [0302] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHASW (SEQ ID NO: 191). [0303] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHSGW (SEQ ID NO: 322). [0304] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHSGW (SEQ ID NO: 322). [0305] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHSTW (SEQ ID NO: 333). [0306] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHSTW(SEQ ID NO: 333). [0307] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHTQW (SEQ ID NO: 349). [0308] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are Page 73 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHTQW (SEQ ID NO: 349). [0309] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDHQNF (SEQ ID NO: 283). [0310] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not ANTRGDHQNF (SEQ ID NO: 2010). [0311] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion is not RGDHASW (SEQ ID NO: 191), RGDHSGW (SEQ ID NO: 322), RGDHSTW (SEQ ID NO: 333), RGDHTQW (SEQ ID NO: 349) or ANTRGDHQNF (SEQ ID NO: 2010). [0312] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPX1X2[W/F] (SEQ ID NO: 2029), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 5. [0313] Table 5: Exemplary peptides encompassed by SEQ ID NO: 4. Peptide SEQ ID NO Fold change over AAV9 Page 74 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 Page 75 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 [0314] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]X1X2V (SEQ ID NO: 2030), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 6. [0315] Table 6: Exemplary peptides encompassed by SEQ ID NO: 5. Peptide SEQ ID NO Fold change over AAV9 Page 76 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYERV 1554 B Page 77 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYSIV 1856 A [0316] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2[W/F] (SEQ ID NO: 2031), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 7. [0317] Table 7: Exemplary peptides encompassed by SEQ ID NO: 6. Page 78 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDAQRW 61 A Page 79 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDFQSY 125 A Page 80 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0318] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDVQNF (SEQ ID NO: 2011). [0319] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not NGVRGDVQNF (SEQ ID NO: 2012). [0320] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDHQNF (SEQ ID NO: 283). [0321] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not ANTRGDHQNF (SEQ ID NO: 2010). [0322] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not NGVRGDVQNF (SEQ ID NO: 2012) or ANTRGDHQNF(SEQ ID NO: 2010). [0323] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]X1SX2 (SEQ ID NO: 2032), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 8. [0324] Table 8: Exemplary peptides encompassed by SEQ ID NO: 7. Page 81 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYASH 1518 A Page 82 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYQSV 1808 B [0325] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are Page 83 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYVSV (SEQ ID NO: 1948). [0326] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYVSV (SEQ ID NO: 1948). [0327] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYSSV (SEQ ID NO: 1882). [0328] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYSSV (SEQ ID NO: 1882). [0329] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYHSF (SEQ ID NO: 1623). [0330] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYHSF (SEQ ID NO: 1623). [0331] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTSM (SEQ ID NO: 1906). [0332] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are Page 84 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not STVRGDYTSM (SEQ ID NO: 2013). [0333] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not QERRGDYTSM (SEQ ID NO: 2014). [0334] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYASL (SEQ ID NO: 2015). [0335] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYASL (SEQ ID NO: 2015). [0336] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYNSL (SEQ ID NO: 2016). [0337] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not SNSRGDYNSL (SEQ ID NO: 2017). [0338] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTSV (SEQ ID NO: 2018). [0339] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are Page 85 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not STVRGDYTSV (SEQ ID NO: 2019). [0340] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not SNSRGDYTSV (SEQ ID NO: 2020). [0341] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTST (SEQ ID NO: 2021). [0342] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not SAMRGDYTST (SEQ ID NO: 2022). [0343] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion does not comprise RGDYTSL (SEQ ID NO: 2023). [0344] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not TSQRGDYVSL (SEQ ID NO: 2024). [0345] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F with the proviso that the peptide insertion is not RGDYSSV (SEQ ID NO: 1882), RGDYASL (SEQ ID NO: 2015), RGDYVSV (SEQ ID NO: 1948), RGDYHSF (SEQ ID NO: 1623), STVRGDYTSM (SEQ ID NO: 2013), QERRGDYTSM (SEQ ID NO: 2014), SNSRGDYNSL (SEQ ID NO: 2017), STVRGDYTSV Page 86 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (SEQ ID NO: 2019), SNSRGDYTSV (SEQ ID NO: 2020), SAMRGDYTST (SEQ ID NO: 2022), or TSQRGDYVSL (SEQ ID NO: 2024). [0346] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of LRGDX1X2[W/F] (SEQ ID NO: 2033), wherein X1 and X2 are independently any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 9. [0347] Table 9: Exemplary peptides encompassed by SEQ ID NO: 8. Peptide SEQ ID NO Fold change over AAV9 LRGDFHF 14 A [0348] I otein disclosed herein comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W/F]GLX (SEQ ID NO: 2034) wherein X is any amino acid. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYGLX (SEQ ID NO: 2035), wherein X is any amino acid. In some embodiments, a peptide insertion comprises a sequence provided in Table 10. [0349] Table 10: Exemplary peptides encompassed by SEQ ID NO: 9. Peptide SEQ ID NO Fold change over AAV9 Page 87 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0350] In some embodiments, a peptide insertion in an AAV capsid protein comprises any one of the peptides disclosed in Table 11. [0351] Table 11: Additional exemplary peptides. Peptide SEQ ID NO Fold change over AAV9 [0352] In rotein disclosed herein comprises a sequence of RGDPIRW (SEQ ID NO: 772). [0353] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPQRW (SEQ ID NO: 802). [0354] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPSPW (SEQ ID NO: 833). [0355] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDSERW (SEQ ID NO: 1088). [0356] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYESR (SEQ ID NO: 1556). [0357] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYLNT (SEQ ID NO: 1696). [0358] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYLSV (SEQ ID NO: 1713). Page 88 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0359] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I. [0360] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2063), wherein X1 is E, X2 is E or R, and X3 is V or I. [0361] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2068), wherein X1 is R, X2 is E or R, and X3 is V or I. [0362] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2064), wherein X1 is E or R, X2 is E, and X3 is V or I. [0363] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2067), wherein X1 is E or R, X2 is R, and X3 is V or I. [0364] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2066), wherein X1 is E or R, X2 is E or R, and X3 is V. [0365] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2065), wherein X1 is E or R, X2 is E or R, and X3 is I. [0366] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYERI (SEQ ID NO: 1551). [0367] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2047 as shown below: Page 89 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0368] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYERIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPH TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL. [0369] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I with the proviso that the peptide insertion is not NATRGDYREI (SEQ ID NO: 2069). [0370] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I with the proviso that the peptide insertion is not RRGDYREIPL (SEQ ID NO: 2059). [0371] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYREI (SEQ ID NO: 1825). [0372] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2046 as shown below: [0373] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL Page 90 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYREIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPH TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL [0374] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include a T residue immediately upstream (or 5’) of the peptide insertion. [0375] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include an N residue at the third position immediately upstream (or 5’) of the peptide insertion. [0376] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include a R residue immediately upstream (or 5’) of the peptide insertion. [0377] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include a P residue immediately downstream (or 3’) of the peptide insertion. [0378] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include an L residue at the second position immediately downstream (or 3’) of the peptide insertion. [0379] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYREV (SEQ ID NO: 1829). In some embodiments, a Page 91 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYREV (SEQ ID NO: 1829). [0380] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2058 as shown below: [0381] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYREVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL [0382] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid protein comprising a peptide insertion disclosed in any one of Tables 1-11 and one or more modifications to an amino acid sequence flanking the peptide insertion site. In some embodiments, one or more modifications are within about 10 amino acids, e.g., within about 5 amino acids, upstream or downstream of the location of the peptide insertion site. [0383] In some embodiments, one or more modifications are located in a variable region IV (VR-IV) of a VP1, VP2, or VP3 of an AAV9 capsid protein, or a variable region V (VR-V) of VP1, VP2, or VP3 of an AAV9 capsid protein, or both. [0384] In some embodiments, VR-IV of a VP1, VP2, or VP3 of an AAV9 capsid protein comprises amino acids 451-475 of a VP1, VP2, or VP3 of an AAV9 capsid protein. [0385] In some embodiments, VR-V of a VP1, VP2, or VP3 of an AAV9 capsid protein comprises amino acids 488-506 of a VP1, VP2, or VP3 of an AAV9 capsid protein. Page 92 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0386] In some embodiments, one or more modifications comprises an insertion, deletion, mutation, or a combination thereof. [0387] In some embodiments, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose. [0388] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f). Exemplary capsid variants with peptide insertions in VR-VIII [0389] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises any one of the peptide insertions disclosed herein; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. [0390] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 1; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is Page 93 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0391] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) a peptide Page 94 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0392] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some Page 95 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 2; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. Page 96 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0393] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile Page 97 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0394] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 3; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 Page 98 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0395] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver Page 99 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0396] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 4; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, Page 100 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0397] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a Page 101 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0398] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 5; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein Page 102 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0399] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: Page 103 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0400] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 6; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% Page 104 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0401] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the Page 105 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0402] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 7; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as Page 106 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0403] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some Page 107 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0404] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 8; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% Page 108 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0405] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR- VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as Page 109 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0406] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 9; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity Page 110 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0407] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; (2) a peptide insertion site is in a VR- VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some Page 111 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0408] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 10; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid Page 112 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0409] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDYX1X2I (SEQ ID NO: 10), wherein X1 is E or R, and X2 is E or R; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, Page 113 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de- targeting mutation) comprises a W503R mutation. [0410] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a peptide insertion provided in Table 11; (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid Page 114 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0411] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2065), wherein X1 is E or R, X2 is E or R, and X3 is I; (2) a peptide insertion site is in a VR- VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an Page 115 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0412] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDYERI (SEQ ID NO: 1551); (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental Page 116 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. [0413] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some Page 117 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, (1) a peptide insertion comprises a sequence of RGDYREI (SEQ ID NO: 1825); (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion consists of the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. Page 118 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0414] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) a peptide insertion comprises a sequence of RGDYREV (SEQ ID NO: 1829); (2) a peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion consists of the sequence of RGDYREV (SEQ ID NO: 1829). In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a Page 119 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. Characterization of AAV9 capsid variants [0415] AAV capsid variants disclosed herein have enhanced muscle-tropism. In some embodiments, an AAV capsid variant disclosed herein is characterized in that when administered to a cell or tissue or subject, an AAV capsid variant confers increased infectivity and/or transduction of a muscle cell compared to infectivity and/or transduction of a muscle cell by a control AAV particle comprising a corresponding parental AAV capsid protein. [0416] In some embodiments, a variant AAV capsid protein confers at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0417] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein confers about 5-fold, about 10-fold, about 15-fold, about 20 fold, about 25-fold, about 30-fold, about 40-fold, or about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0418] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein confers about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30 fold, about 5-fold to about 25-fold, about 5-fold to about 20-fold, about 5-fold to about 15-fold, about 5-fold to about 10- fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, 20-fold to about 50-fold, 25- fold to about 50-fold, 30-fold to about 50-fold, or 40-fold to about 50-fold increased infectivity and/or transduction of a muscle cell compared the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0419] In some embodiments, a variant AAV capsid is an AAV9 variant capsid. Page 120 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0420] In some embodiments, a rAAV particle comprising an AAV capsid variant disclosed herein and a heterologous nucleic acid comprising a nucleotide sequence encoding a payload is characterized in that when administered to a cell or tissue or subject, delivery of a payload is enhanced to a muscle cell as compared to delivery of a similar payload with an otherwise similar AAV particle without an AAV capsid variant disclosed herein. [0421] In some embodiments, a muscle cell is chosen from: a skeletal muscle cell, a cardia muscle cell, a smooth muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof. Payloads for use in AAV particles comprising an AAV9 capsid variant [0422] A rAAV particle comprising an AAV capsid variant disclosed herein can also comprise a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0423] In some embodiments, a payload is a polypeptide. In some embodiments, a payload polypeptide is chosen from: a CRISPR-Cas protein, a Zinc finger protein, a TAL, a base editor, a prime editor, a meganuclease, or any combination thereof. [0424] In some embodiments, a polypeptide is or comprises a CRISPR-Cas protein. In some embodiments, a CRISPR-Cas protein is chosen from: a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment thereof. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the CRISPR-Cas protein. In some embodiments a CRISPR-Cas protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain. In some embodiments a CRISPR-Cas protein is a nuclease. In some embodiment a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleic acid molecule. In some embodiment a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule. [0425] In some embodiments, a polypeptide is or comprises a Zinc finger protein, or a variant or fragment thereof. In some embodiments, a Zinc finger protein is chosen from : a Zinc Page 121 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeted zinc-finger DNA-binding unit with a nuclease domain (e.g., Fok1 nuclease domain) fusion protein, or a variant or fragment or combination of any of the foregoing. In some embodiments a Zinc finger protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain. [0426] In some embodiments, a polypeptide is or comprises a Transcription Activator- Like Effector (TAL) protein, or a variant or fragment thereof. In some embodiments, a TAL comprises: a TAL effector DNA binding domain (e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.), a Transcription Activator-Like Effector Nuclease (TALEN), e.g., a TAL effector DNA binding domain fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or combination of any of the foregoing. In some embodiments a TAL protein is fused with one or more domains, e.g., an activator domain and/or a repressor domain. [0427] In some embodiments, a polypeptide is or comprises a base editor, or a variant or fragment thereof. In some embodiments, a base editor comprises a deaminase, an adenosine deaminase enzyme (ABE), a cytosine deaminase enzyme (CBE), an APOBEC1, an APOBEC3A, an APOBEC3G, an evoAPOBEC, a BE4-YE1, a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine deaminases (TadA*), an E. coli tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA binding domain monomer, a base editing enzyme that is RNA guided, a DNA glyosylase inhibitor, one or more DNA glycosylase inhibitor domains, a 5-methylcytosine deaminase, a cytidine deaminase domain, an adenine deaminase domain, an adenosine base editor (ABE), a Target-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax., a circularly permuted base editor, an adenosine deaminase enzyme (ADAR), a RNA editing for programmable adenosine to inosine replacement (REPAIR), a leveraging endogenous ADAR for programmable editing of RNA (LEAPER) or a variant or fragment or combination of any of the foregoing. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA/tracrRNA that interacts with the base editor. [0428] In some embodiments, a polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising the same. In some embodiments, a prime editor and/or system comprising the same comprises: a reverse transcriptase, a prime editing enzyme, Page 122 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) an editing enzyme that includes a reverse transcriptase domain, an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Murine Leukemia Virus (MLV) Reverse Transcriptase, a HIV- 1 reverse transcriptase, a bacterial reverse transcriptase, a reverse transcriptase associated with a DNA binding domain and/or protein, a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase), a prime editing 1 system (PE1), a prime editing 2 system (PE2), a prime editing 3 system (PE3), a prime editing 3b system (PE3b) or a variant or fragment or combination of any of the foregoing. In some embodiments, the payload also comprises a prime editing gRNA (pegRNA) or an extended sgRNA that interacts with the prime editor. [0429] In some embodiments, a polypeptide is or comprises a meganuclease, or a variant or fragment thereof. In some embodiments, a meganuclease is chosen from: a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His- Cyst box family meganuclease, or HNH family endonuclease, an I-SeeI, an I-CeuI, a PI-PspI, a PI-SceI, an I-SceIV, an I-CsmI, an I-PanI, an I-SceII, an I-PpoI, an I-SceIII, an I-CreI, an I-TevI, an I-TevII an I-TevIII or a variant or fragment or combination of any of the foregoing. [0430] In some embodiments, a polypeptide is associated with a muscle disorder, or a glycogen or sugar storage disorder. [0431] In some embodiments, a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0432] In some embodiments, a polypeptide is an enzyme. In some embodiments, an enzyme is a lysosomal enzyme or an adenosine deaminase enzyme. [0433] In some embodiments, a polypeptide is an antibody. [0434] In some embodiments, a polypeptide is a secreted protein. Page 123 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0435] In some embodiments, a payload is an RNA molecule. In some embodiments, an RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, a snRNA, circular RNA or an aptamer, or combinations thereof. [0436] In some embodiments, an RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder. [0437] In some embodiments, a muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchene muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0438] In some embodiments, a payload is a DNA molecule. In some embodiments, a DNA molecule comprises a nucleic acid sequence of up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc. [0439] In some embodiments, a nucleotide sequence encoding a payload comprises a promoter. In some embodiments, a promoter is a muscle-specific promoter. In some embodiments, a muscle-specific promoter is chosen from: MHCK7, CK8, desmin, tMCK, dMCK, or CK6. [0440] In some embodiments, a promoter is a dual muscle-liver promoter. In some embodiments, a dual muscle-liver promoter is SPc5-12. Uses of AAV particles comprising an AAV9 capsid variant [0441] The present disclosure, among other things, provides methods of delivering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles as described herein to a cell or tissue, e.g., a muscle cell or tissue. The present disclosure also provides methods of treating a subject with a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles produced using a method or system described herein. Page 124 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0442] In some embodiments, disclosed herein is a method for treating a muscle disorder in a subject comprising administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein. [0443] In some embodiments, also disclosed herein is a method for amelioration a symptom of a muscle disorder in a subject comprising administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein. [0444] In some embodiments, a muscle disorder is chosen from: (a) a muscular dystrophy, e.g., X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker Muscular dystrophy, a Limb-Girdle muscular dystrophy, an Emery Dreifuss muscular dystrophy, a myotonic dystrophy, or Facioscapulohumeral muscular dystrophy (FSHD); (b) a neuro-muscular disease, e.g., Charcot-Marie-Tooth disease, a Myotonic Dystrophy, Nemaline Myopathy, or Facioscapulohumeral muscular dystrophy (FSHD); (c) a sugar or glycogen storage disease, e.g., MPS type III disease or Pompe disease; (d) an expanded repeat disease, e.g., a Myotonic Dystrophy, or Facioscapulohumeral muscular dystrophy (FSHD), or (e) a combination of any one of or all of (a)-(d). [0445] In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles as described herein is administered to a subject suffering from or at risk of a disease, disorder, or condition. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles as described herein is administered in combination with one or more additional therapeutics agents to a subject. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles as described herein is contacted with an organ, tissue, or cells ex vivo. The organ, tissue, or cells can be introduced into a subject and can be protected from damage that would otherwise be caused by the recipient’s immune system. [0446] In some embodiments, methods and kits of the present invention may be used for the evaluation and/or monitoring of gene therapy. In some embodiments, gene therapy comprises administration of a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein. In some embodiments, samples for evaluating and/or monitoring gene therapy may be obtained prior to the initiation of gene therapy. In some Page 125 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, samples are obtained after a first gene therapy treatment or dose. In some embodiments, samples are obtained after the conclusion of gene therapy. In some embodiments, samples are obtained at specific time points, intervals, or any other metric of time before, during, or after gene therapy is performed. Method of transfecting host cells using AAV particles comprising an AAV9 capsid variant [0447] The present disclosure, among other things, provides methods for transfection of a host cell comprising: combining nucleic acids with a transfection reagent and introducing the mix to host cells under conditions that lead to transfection of the host cells with the nucleic acids. [0448] In some embodiments, nucleic acids used in a method disclosed herein comprise one or more vectors. In some embodiments, nucleic acids disclosed herein comprise one or more vectors encoding: (i) at least one payload flanked by an AAV inverted terminal repeat (ITR) on either side of the at least one payload, (ii) at least one AAV Rep polypeptide, (iii) at least one AAV Cap polypeptide, and/or (iv) at least one Adenoviral helper polypeptide. [0449] A host cell (e.g., a mammalian host cell, e.g., a HEK293) can be transfected with: at least one helper polypeptide (e.g., at least one Ad2 helper polypeptides), at least one Rep polypeptide or a fragment thereof, at least one Cap polypeptide or a fragment thereof, and at least one payload (e.g., for polypeptide expression or an inhibitory or guide nucleic acid). [0450] In some embodiments, a transfection method disclosed herein is or comprises transient transfection. In some embodiments, a transient transfection method is a suspension transient transfection (sTT). In some embodiments, a transient transfection method is an adherent transient transfection. [0451] In some embodiments, the disclosure provides transfected host cells comprising two, three, or four vectors as described herein. [0452] In some embodiments, the method comprises transfecting a host cell with three vectors. In some embodiments, the three vectors comprise: (i) a first vector encoding at least one payload flanked by an AAV ITR on either side of the at least one payload, (ii) a second vector encoding at least one AAV Rep polypeptide and at least one AAV Cap polypeptide, and (iii) a third vector encoding at least one Adenoviral helper polypeptide. Page 126 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0453] In some embodiments, the method comprises transfecting a host cell with two vectors. In some embodiments, the two vectors comprises (i) a first vector encoding at least one AAV Cap polypeptide and at least one payload flanked by an AAV ITR on either side of the at least one payload; and (ii) a second vector encoding at least one Adenoviral helper polypeptide and at least one AAV Rep polypeptide. [0454] Transfection methods disclosed herein comprise transfection of nucleic acids (e.g., comprising one or more vector) with any transfection reagent known to a skilled person for introducing nucleic acid molecules into host cells (e.g., mammalian cells, such as HEK293). In some embodiments, a transfection reagent comprises a lipid, a polymer, or a combination thereof. In some embodiments, a transfection reagent is a reagent that can form a complex with the nucleic acids. [0455] In some embodiments, a transfection reagent comprise a polymer, a lipid, or both a polymer and a lipid. In some embodiments, a transfection reagent is or comprises a polymer. In some embodiments, a transfection reagent is or comprises lipid. In some embodiments, a transfection reagent comprises a polymer and a lipid. [0456] In some embodiments, a transfection reagent is or comprises a polymer, e.g., a cationic polymer. In some embodiments, a transfection reagent comprises polyethyleneimine (PEI), FectoVIR, TransIT-VirusGEN, or a combination thereof. In some embodiments, a transfection reagent is or comprises polyethyleneimine (PEI). [0457] In some embodiments, host cells are transfected with PEI. In some embodiments, host cells are transfected with a weight (wt.) ratio of DNA to transfection reagent (e.g., PEI) of about 1:1 to about 1:2, about 1:1 to about 1:5, or about 1:1 to about 1:10, e.g., about 1:0.05, about 1:1, about 1:1.25, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In some embodiments, a wt. ratio of DNA to transfection reagent is dependent on cell culture density (e.g., of adherent or suspension host cells). [0458] In some embodiments, a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is used in a method of Page 127 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) transfection disclosed herein. In some embodiments, a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is about 1:1:1. In some embodiments, a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is not about 1:1:1. [0459] In some embodiments, a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is about 1:0.5:1, about 1:1:2, about 1:1:3, about 1:1:4, about 1:1:5, about 1:1:6, about 1:1:7, about 1:1:8, about 1:1:9, about 1:1:10, about 5:10:1, about 1:0.5:2, about 1:0.5:10, about 1:0.5:5, about 0.5:5:1, about 1:10:20, about 1:2:1, about 1:3:1, about 1:4:1, about 1:5:1, about 1:6:1, about 1:7:1, about 1:8:1, about 1:9:1, about 1:10:1, about 10:1:1, about 9:1:1, about 8:1:1, about 7:1:1, about 6:1:1, about 6:1:1, about 4:1:1, about 3:1:1, about 2:1:1, or about 1:0.5:5. [0460] In some embodiments, a vector mass ratio of: (i) a first vector encoding at last one payload to (ii) a second vector encoding at least one Rep polypeptide and/or at least one Cap polypeptide to (iii) a third vector encoding at least one helper polypeptide is about 1:0.5:1 to about 1:0.5:10; about 1:1:1 to about 1:1:10; about 0.5:1:1 to about 5:1:1; about 1:1:1 to about 1:10:1; or about 1:1:1 to about 10:1:1. Host Cells [0461] The present disclosure, among other things, provides host cells for transfection with at least one vector as described herein for production of rAAV particles. A host cell includes a progeny cell of an original cell transfected with at least one vector described herein. A progeny cell of a parental cell may not be substantially identical in morphology or genomic content as a parent cell due to natural, accidental, or deliberate mutation. [0462] Components for a host cell to produce rAAV particles may be provided in trans on at least one vector. A stable host cell may comprise at least one polypeptide to produce rAAV particles using methods known to those of skill in the art. In some embodiments, a stable host cell comprises at least one polypeptide under control of an inducible promoter. In other Page 128 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, a stable host cell comprises at least one polypeptide under control of a constitutive promoter. For example, a stable host cell (e.g., a HEK293 cell) may comprise a nucleic acid encoding an E1 helper polypeptide under the control of a constitutive promoter. Other stable host cells may be generated by one of skill in the art using routine methods. [0463] Exemplary host cells include prokaryotes or eukaryotes (single-cell or multiple- cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp), mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, or Trichoplusia ni), non-human animal cells, human cells, or cell fusions, such as hybridomas or quadromas. In some embodiments the host cell is a mammalian cell. In some embodiments, the host cell is a human, monkey, ape, hamster, rat, or mouse cell. [0464] In some embodiments, the host cell is selected from a kidney cell (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, or BHK), CHO cell (e.g., CHO Kl, DXB-11 CHO, or Veggie-CHO), COS cell (e.g., COS-7), retinal cell, Vero cell, CV1 cell, HepG2 cell, WI38 cell, MRC 5 cell, Colo205 cell, HB 8065 cell, HL-60 cell (e.g., BHK21), Jurkat cell, Daudi cell, A431 cell (epidermal), CV-1 cell, U937 cell, 3T3 cell, L cell, C127 cell, SP2/0 cell, NS-0 cell, MMT 060562 cell, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, or a cell line derived from an aforementioned cell. [0465] In some embodiments, the host cell comprises a kidney cell (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, or BHK). In certain embodiments, the host cell comprises a HEK293 cell. In some embodiments, the host cell (e.g., a HEK 293 cell) comprises or expresses an E1 polypeptide. In some embodiments, the host cell does not comprise or express an E1 polypeptide. In some embodiments, the host cell comprises a CHO cell (e.g., CHO-K, DXB-11 CHO, or Veggie-CHO). In certain embodiments, the host cell comprises a CHO-K cell. [0466] In some embodiments, host cells are or comprise suspension cells. In some embodiments, at least 10% +/- 15%, at least 15 +/- 15%, at least 20 +/- 15%, at least 25 +/- 15%, at least 30 +/- 15%, at least 35 +/- 15%, at least 40 +/- 15%, at least 45 +/- 15%, at least 50 +/- 15%, at least 55 +/- 15%, at least 60 +/- 15%, at least 65 +/- 15%, at least 70 +/- 15%, at least 75 +/- 15%, at least 80 +/- 15%, at least 85 +/- 15%, at least 90 +/- 15%, at least 95 +/- 15%, at least 99 +/- 15%, or more host cells in culture are suspended. Page 129 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0467] In some embodiments, prior to transfection, host cells (e.g., adherent or suspended host cells) are seeded at a certain density. In some embodiments, prior to transfection, host cells (e.g., adherent host cells) are seeded at a density of at least about 1.0 x 104 viable cells (vc)/cm2, e.g., at a density of about 1.0 x 104 vc/cm2 to about 2.0 x 104 vc/cm2, e.g., about 1.0 x 104 vc/cm2, about 1.1 x 104 vc/cm2, about 1.2 x 104 vc/cm2, about 1.3 x 104 vc/cm2, about 1.4 x 104 vc/cm2, about 1.5 x 104 vc/cm2, about 1.6 x 104 vc/cm2, about 1.7 x 104 vc/cm2, about 1.8 x 104 vc/cm2, about 1.9 x 104 vc/cm2, or about 2.0 x 104 vc/cm2. In some embodiments, prior to transfection, host cells (e.g., suspended host cells) are seeded at a density of at least 1.0 x 106 vc/cm2 +/- 15%, e.g., at a density of 1.0 x 106 vc/cm2 +/- 15% to 2.0 x 106 vc/cm2 +/- 15%, e.g., 1.0 x 106 vc/cm2 +/- 15%, 1.1 x 106 vc/cm2 +/- 15%, 1.2 x 106 vc/cm2 +/- 15%, 1.3 x 106 vc/cm2 +/- 15%, 1.4 x 106 vc/cm2 +/- 15%, 1.5 x 106 vc/cm2 +/- 15%, 1.6 x 106 vc/cm2 +/- 15%, 1.7 x 106 vc/cm2 +/- 15%, 1.8 x 106 vc/cm2 +/- 15%, 1.9 x 106 vc/cm2 +/- 15%, or 2.0 x 106 vc/cm2 +/- 15%. Vectors [0468] Many forms of vectors can be used in methods of producing rAAV particles described herein. Non-limiting examples of vectors include plasmids, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, and viral vectors (e.g., vectors suitable for gene therapy). A vector genetic element may be delivered by any suitable method known in the art, e.g., 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.). [0469] In some embodiments, a vector encodes at least one helper polypeptide. In some embodiments, a vector encodes at least one Rep polypeptide and/or at least one Cap polypeptide. In some embodiments, a vector encodes at least one payload (e.g., for expression of polypeptide or as an inhibitory or guide nucleic acid). In some embodiments, a vector encodes at least one helper polypeptide and at least one Rep polypeptide. In some embodiments, a vector encodes at least one Cap polypeptide and at least one payload. [0470] A vector can include conventional control elements operably linked to a nucleic acid encoding any polypeptide or payload described herein, in a manner that permits transcription, translation and/or expression in a cell transfected with a vector described herein. Page 130 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters that are native, constitutive, inducible, and/or tissue- specific, are known in the art and may be included in a vector described herein. [0471] Examples of constitutive promoters include, but are not limited to, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter (optionally with CMV enhancer), an SV40 promoter, and a dihydrofolate reductase promoter. [0472] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors, such as temperature, or the presence of a specific physiological state (e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only). Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include a zinc-inducible sheep metallothionine (MT) promoter, a dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, a T7 polymerase promoter system, an ecdysone insect promoter, a tetracycline-repressible system, a tetracycline-inducible system, a RU486-inducible system, and a rapamycin-inducible system. Still other types of inducible promoters that may be useful are regulated by a specific physiological state, such as temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only. [0473] In another embodiment, a native promoter or fragment thereof for a nucleic acid encoding any polypeptide or payload described herein may be used. In some embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, may also be used to mimic native expression. Vector encoding Helper Polypeptides Page 131 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0474] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one helper polypeptide. AAV is a helper-dependent DNA parvovirus, which belongs to the genus Dependovirus. Production of recombination AAV requires co-infection with a related virus (e.g., adenovirus, herpes, or vaccinia virus) or a helper vector encoding helper polypeptides, such as structural proteins and proteins for viral genome replication. [0475] A helper vector can comprise nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication, which may include, but are not limited to, activation of gene transcription, stage specific mRNA splicing, DNA replication, synthesis of at least one Cap polypeptide, and/or capsid assembly. Viral-based helper polypeptides can be derived from any known helper viruses such as adenovirus, herpesvirus, vaccinia virus, or a combination thereof. Thus, a helper vector (e.g., a plasmid) for culturing of the host cell can comprise sufficient helper polypeptides to permit packaging of the recombinant AAV vector into the AAV capsid polypeptides. [0476] In some embodiments, a helper vector comprises an Ad2 helper vector. In certain embodiments, a nucleic acid sequence of an Ad2 helper vector is derived from an Adenovirus 2 genome (Genbank Accession No. J01917.1). In some embodiments, a helper vector comprises an Ad5 helper vector. In certain embodiments, a nucleic acid sequence of an Ad5 helper vector is derived from an Adenovirus 5 genome (Genbank Accession No. AY601635). [0477] Helper polypeptides can comprise at least one, two, three, or four of E1, E2A, E4, or VA RNA. In some embodiments, E1 comprises E1a and/or E1b. In some embodiments, one or both of E2A and VA RNA increase stability and/or efficiency of AAV mRNA translation, such as for cap gene transcripts. In some embodiments, E4 facilitates DNA replication. In some embodiments, E1a comprises a transactivator (e.g., regulating activity of at least one Ad gene, AAV rep gene, and/or AAV cap gene). In some embodiments, E1b comprises a viral mRNA transport. Helper polypeptides are described in further detail in Coura and Nardi, A role for adeno-associated viral vectors in gene therapy, Genetics and Molecular Biology, 31(1): 1-11 (2008), which is hereby incorporated by reference in its entirety. [0478] In some embodiments, a helper vector comprises a selection marker. Exemplary selection markers include, but are not limited to, antibiotic resistance genes. In some embodiments, an antibiotic resistance gene is not a gene encoding penicillin. In some Page 132 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) embodiments, an antibiotic resistance gene is not a gene encoding a penicillin-derivative. In some embodiments, an antibiotic resistance gene comprises an antibiotic resistance gene chosen from kanamycin, puromycin, neomycin, hygromycin, blasticidin, gentamycin, Gr18, or zeocin. In certain embodiments, an antibiotic resistance gene comprises an antibiotic resistance gene for kanamycin. [0479] In some embodiments, nucleic acids encoding helper polypeptides are oriented in the same direction (e.g., 5’ to 3’) on a helper vector. In some embodiments, nucleic acids encoding helper polypeptides are transcribed in the same direction from a helper vector. In certain embodiments, helper polypeptides comprise VA RNA and E4 oriented in the same direction on a helper vector. In certain embodiments, helper polypeptides comprise E4 and E2A oriented in the same direction on a helper vector. In certain embodiments, helper polypeptides comprise VA RNA, E4, and E2A oriented from 5’ to 3’ in direction on a helper vector. In some embodiments, a helper vector does not comprise a nucleic acid sequence encoding a Fiber protein or a fragment thereof (e.g., does not comprise a nucleic acid sequence of Genbank Accession No. AP_000226.1 or a fragment thereof). Vector encoding Rep and/or Cap Polypeptides [0480] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one Rep polypeptide and/or at least one Cap polypeptide (e.g., a variant Cap disclosed herein). Production of rAAV particles can include culturing of a host cell with at least one Rep polypeptide and at least one Cap polypeptide (e.g., a variant Cap disclosed herein). Rep proteins (e.g., one, two, three, or four Rep78, Rep68, Rep52, and Rep40) are involved in viral DNA replication, resolution of replicative intermediates, and generation of single-stranded genomes. In some embodiments, a vector comprises a nucleic acid sequence encoding one, two, three, or four of Rep78, Rep68, Rep52, or Rep40, or a variant of any of the foregoing. [0481] In some embodiments, a Rep polypeptide comprises a nucleic acid sequence derived from an AAV2 serotype. For example, a nucleic acid sequence encoding a Rep polypeptide may be derived from the AAV2 genome (as found in Accession No. NC_001401). In some embodiments, a Rep polypeptide comprises an AAV2 Rep polypeptide operably linked to a p5 and/or p19 promotor (as found in Accession No. NC_001401). In some embodiments, a Page 133 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Rep polypeptide comprises an amino acid sequence of YP_680422.1 or a fragment thereof. In some embodiments, a promoter is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In certain embodiments, a promoter operably linked to a nucleic acid sequence encoding at least one Rep polypeptide comprises a p5 and/or p19 promoter. In some embodiments, a wildtype promoter of AAV2 or a variant thereof is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In some embodiments, a promoter (e.g., a p5 promoter) regulating expression of at least one Rep polypeptide is located in a different location on a vector than a wildtype promoter of AAV2 or a variant thereof. In certain embodiments, a promoter (e.g., a p5 promoter) is located 3’ of a nucleic acid encoding at least one Rep polypeptide. In certain embodiments, a promoter (e.g., a p5 promoter) is located 5’ of a nucleic acid encoding at least one Rep polypeptide. [0482] Cap polypeptides (e.g., VP1, VP2, and VP3) are structural proteins comprising a Capsid. In some embodiments, a vector comprises a nucleic acid sequence encoding one, two, or three of VP1, VP2, and VP3, e.g., comprising a variant AAV capsid protein disclosed herein. In some embodiments, a vector comprises a nucleic acid sequence encoding at least one Cap polypeptide (e.g., a variant AAV capsid polypeptide disclosed herein) and at least one Rep polypeptide. In other embodiments, a vector encodes at least one Cap polypeptide (e.g., a variant AAV capsid polypeptide disclosed herein) and a separate vector encodes at least one Rep polypeptide. [0483] For example, a nucleic acid sequence encoding a Cap polypeptide comprising an AAV variant capsid disclosed herein may further comprise a nucleic acid sequence derived from a known AAV genome sequence including, but not limited to: AAV9/hu14 provided as SEQ ID NO: 123 in U.S. Patent 7,906,111; AAV1 Accession No. NC_002077 or AF063497; AAV2 Accession No. NC_001401; AAV5 Accession No. Y18065 or AF085716; Accession No. AAV6 NC_001862; or AAV8 Accession No NC_006261.1. In certain embodiments, a nucleic acid sequence encoding a Cap polypeptide (e.g., a variant AAV capsid polypeptide disclosed herein) is derived from an AAV genome sequence or a variant thereof as described in US Patent No. 7,906,111, which is hereby incorporated by reference in its entirety. In certain embodiments, a nucleic acid sequence encoding a Cap polypeptide (e.g., a variant AAV capsid polypeptide disclosed herein) is derived from an AAV genome sequence or a variant thereof as described in Page 134 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) International Publication No. WO 2018/160582, which is hereby incorporated by reference in its entirety. [0484] In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV2 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV2 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV5 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV8 serotype, or a variant thereof. In certain embodiments, a Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises a nucleic acid sequence derived from an AAV9 serotype, or a variant thereof. [0485] In some embodiments, a promoter is operably linked to a nucleic acid sequence encoding at least one Cap polypeptide comprising an AAV variant capsid disclosed herein. In some embodiments, a wildtype promoter of AAV2, AAV5, AAV8, AAV9, is operably linked to a nucleic acid sequence encoding at least one Rep polypeptide. In certain embodiments, a p40 promoter is operably linked to a nucleic acid sequence encoding at least one Cap polypeptide comprising an AAV variant capsid disclosed herein further comprises. Vector encoding Payload [0486] The present disclosure, among other things, provides vectors (e.g., plasmids) encoding at least one payload. A payload sequence is generally a sequence of interest that is desired to be introduced into a cell, tissue, organ, or organism. [0487] In some embodiments, a payload is flanked by inverted terminal repeats (ITRs). The AAV sequences of a rAAV vector typically comprise cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses,” ed., P. Tijsser, CRC Press, pp.155-168 (1990), which is hereby incorporated by reference in its entirety). ITR sequences are typically about 145 bp in length. In some embodiments, one or both of a 5’ITR or a 3’ ITR nucleic acid sequence are modified relative to a known ITR nucleic acid sequence. Page 135 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Modification of ITR nucleic acid sequences is within one of skill in the art (See, e.g., Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520532 (1996), each of which is hereby incorporated by reference in its entirety). AAV ITR sequences may be obtained from any known AAV, including mammalian AAV types. [0488] In some embodiments, a payload is a heterologous protein with a therapeutic purpose, e.g., an enzyme, cytokine, antibody, receptor, fusion protein, or chimeric polypeptide. In some embodiments, a payload is linked to a secretion signal sequence for secretion of an expressed polypeptide from a host cell. In some embodiments, a payload is a heterologous nucleic acid with a therapeutic purpose, e.g., a miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR/Cas guide RNA, or a precursor thereof. One of skill in the art will recognize that a payload can be selected from any heterologous protein or nucleic acid of interest. In some embodiments, a payload sequence comprises one or more aptamer-binding domains or polypeptide-binding domains (e.g., transcription factor binding domains). A vector will also typically include other regulatory elements (e.g., promoters, introns, and/or enhancers) to regulate expression or amount of a payload in a cell or tissue. [0489] In accordance with various embodiments, a payload sequence can be of any length, e.g., between 2 and 5,100 nucleotides in length or any integer value there between. In some embodiments, a nucleic acid sequence encoding a payload comprises at least 20 nucleotides, at least 50 nucleotides, at least 75 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, at least 550 nucleotides, at least 600 nucleotides, at least 650 nucleotides, at least 700 nucleotides, at least 750 nucleotides, at least 800 nucleotides, at least 850 nucleotides, at least 900 nucleotides, at least 950 nucleotides, at least 1000 nucleotides, at least 1100 nucleotides, at least 1200 nucleotides, at least 1300 nucleotides, at least 1400 nucleotides, at least 1500 nucleotides, at least 1600 nucleotides, at least 1700 nucleotides, at least 1800 nucleotides, at least 2000 nucleotides, at least 2500 nucleotides, at least 3000 nucleotides, at least 4000 nucleotides, at least 5000 nucleotides.. In some embodiments, a nucleic acid sequence encoding a payload comprises between about 50 and about 5,100 nucleotides in length, between about 100 and about Page 136 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 5,100 nucleotides in length, between about 500 and about 5,100 nucleotides in length, between about 1,000 and about 5,100 nucleotides in length, and/or between about 2,000 and about 5,100 nucleotides in length. Culture vessels and culturing parameters [0490] The present disclosure, among other things, provides methods for culturing of a host cell with at least one vector described herein for production of rAAV particles. A wide variety of growth media (e.g., mammalian growth media) may be used in accordance with the present invention. In certain embodiments, cells may be grown in one of a variety of chemically defined media, wherein the components of the media are both known and controlled. In certain embodiments, cells may be grown in a complex medium, in which not all components of the medium are known and/or controlled. [0491] A culture of host cells can be prepared in any medium suitable for a particular cell type being cultured. In some embodiments, a host cell medium comprises, e.g., inorganic salts, carbohydrates (e.g., sugars, such as glucose, galactose, maltose, or fructose), amino acids, vitamins (e.g., B group vitamins (e.g., B12), vitamin A, vitamin E, riboflavin, thiamine, or biotin), fatty acids (e.g., cholesterol or steroids), proteins (e.g., albumin, transferrin, fibronectin, or fetuin), serum (e.g., albumins, growth factors, or growth inhibitors, such as, fetal bovine serum, newborn calf serum, or horse serum), trace elements (e.g., zinc, copper, selenium, or tricarboxylic acid intermediates), hydrolysates (e.g., derived from plant or animal sources), or combinations thereof. [0492] Commercially available media can be used for culturing host cells described herein. Exemplary media can include, but is not limited to, Dulbecco's Modified Eagle's Medium ([DMEM], Sigma), FreeStyle™ F17 Expression Medium (ThermoFisher), DMEM/F12 medium (Invitrogen), CD OptiCHO™ medium (Invitrogen), CD EfficientFeed™ media (Invitrogen), Cell Boost (HyClone™) media (GE Life Sciences), BalanCD™ CHO Feed (Irvine Scientific), BD Recharge™ (Becton Dickinson), Cellvento Feed™ (EMD Millipore), Ex-cell CHOZN Feed™ (Sigma-Aldrich), CHO Feed Bioreactor Supplement (Sigma-Aldrich), SheffCHO™ (Kerry), Zap-CHO™ (Invitria), ActiCHO™ (PAA/GE Healthcare), Minimal Essential Medium (Sigma), or RPMI-1640 (Sigma). Media can be supplemented as necessary Page 137 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) with hormones and/or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, or phosphate), buffers (e.g., HEPES), nucleosides (e.g., adenosine or thymidine), antibiotics (e.g., kanamycin, puromycin, neomycin, hygromycin, blasticidin, gentamycin, Gr18, or zeocin), trace elements, lipids (e.g., linoleic or other fatty acids), or glucose or an equivalent energy source. In some embodiments, the media for culturing host cells comprises glutamine or a glutamine dipeptide. In some embodiments, the media for culturing host cells comprises a surfactant. In some embodiments, the nutrient media is serum-free media, a protein-free media, or a chemically defined media. Any other necessary supplements can also be included at appropriate concentrations that would be known to those skilled in the art. [0493] After culturing of host cells as described herein, a plurality of rAAV particles are recovered. In some embodiments, rAAV particles are recovered by lysing host cells and recovering rAAV particles from lysate, e.g., after centrifugation. In some embodiments, rAAV particles are recovered from culture supernatant. In some embodiments, a lysis solution for host cells comprises chemical reagents, e.g., detergents (e.g., sodium dodecyl sulfate (SDS), ethyl trimethyl ammonium bromide, Triton X-100, bile salts, such as cholate, or zwitterionic detergents, such as CHAPS). In some embodiments, a lysis solution for host cells comprises a salt (e.g., NaCl) and a high pH (e.g., a pH of greater than about 7). In some embodiments, rAAV particles are purified using purification methods, such as chromatography (e.g., affinity chromatography or ion-exchange chromatography (e.g., cation exchange chromatography)) or filtration (e.g., UF/DF filtration)). [0494] In some embodiments, a plurality of rAAV particles are produced in a large-scale preparation. In some embodiments, a large-scale preparation of host cells (e.g., suspension host cells) is at least 3 liters +/- 15% of culture media, 10 liters +/- 15% of culture media, e.g., between 50 liters +/- 15% to 1000 liters +/- 15% of culture media or between 50 liters +/- 15% to 2000 liters+/- 15% of culture media, e.g., at least 20 liters +/- 15%, 30 liters +/- 15%, 40 liters +/- 15%, 50 liters +/- 15%, 55 liters +/- 15%, 60 liters +/- 15%, 65 liters +/- 15%, 70 liters +/- 15%, 75 liters +/- 15%, 80 liters +/- 15%, 85 liters +/- 15%, 90 liters +/- 15%, 95 liters +/- 15%, 100 liters +/- 15%, 200 liters +/- 15%, 300 liters +/- 15%, 400 liters +/- 15%, 500 liters +/- 15%, 600 liters +/- 15%, 700 liters +/- 15%, 800 liters +/- 15%, 900 liters +/- 15%, 1,000 liters +/- 15%, Page 138 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 1,250 liters +/- 15%, 1,500 liters +/- 15%, 1,750 liters +/- 15%, 2,000 liters +/- 15%, or more of culture media. [0495] In some embodiments, a large-scale preparation of host cells (e.g., adherent host cells) is at least 5 m2 +/- 15% of culture media, e.g., between 5 m2 +/- 15% to 500 m2 +/- 15% of culture media, e.g., at least 5 m2 +/- 15%, 10 m2 +/- 15%, 15 m2 +/- 15%, 20 m2 +/- 15%, 25 m2 +/- 15%, 20 m2 +/- 15%, 35 m2 +/- 15%, 40 m2 +/- 15%, 45 m2 +/- 15%, 50 m2 +/- 15%, 55 m2 +/- 15%, 60 m2 +/- 15%, 65 m2 +/- 15%, 75 m2 +/- 15%, 80 m2 +/- 15%, 85 m2 +/- 15%, 90 m2 +/- 15%, 95 m2 +/- 15%, 100 m2 +/- 15%, 150 m2 +/- 15%, 175 m2 +/- 15%, 200 m2 +/- 15%, 225 m2 +/- 15%, 250 m2 +/- 15%, 275 m2 +/- 15%, 300 m2 +/- 15%, 325 m2 +/- 15%, 330 m2 +/- 15%, 340 m2 +/- 15%, 350 m2 +/- 15%, 375 m2 +/- 15%, 400 m2 +/- 15%, 425 m2 +/- 15%, 450 m2 +/- 15%, 475 m2 +/- 15%, 500 m2 +/- 15%, 600 m2 +/- 15%, 700 m2 +/- 15%, 800 m2 +/- 15%, 900 m2 +/- 15%, 1000 m2 +/- 15%, 1100 m2 +/- 15%, 1200 m2 +/- 15%, 1300 m2 +/- 15%, 1400 m2 +/- 15%, 1500 m2 +/- 15%, 1600 m2 +/- 15%, 1700 m2 +/- 15%, 1800 m2 +/- 15%, 1900 m2 +/- 15%, 2000 m2 +/- 15%, 2500 m2 +/- 15%, 3000 m2 +/- 15%, 3500 m2 +/- 15%, 4000 m2 +/- 15%, 4500 m2 +/- 15%, 5000 m2 +/- 15%, 5500 m2 +/- 15%, 6000 m2 +/- 15%, 6500 m2 +/- 15%, 7000 m2 +/- 15%, 7500 m2 +/- 15%, 8000 m2 +/- 15%, 8500 m2 +/- 15%, 9000 m2 +/- 15%, 9500 m2 +/- 15%, 10,000 m2 +/- 15%, 11,000 m2 +/- 15%, 12,000 m2 +/- 15%, 13,000 m2 +/- 15%, 14,000 m2 +/- 15%, 15,000 m2 +/- 15%, or more of culture media. [0496] A host cell can be cultured in a cell culture vessel or a bioreactor. In some embodiments, a cell culture vessel is suitable for/used for culturing adherent cells. In other embodiments, a cell culture vessel is suitable for/used for culturing suspension cells. Exemplary cell culture vessels include 35mm, 60mm, 100mm, or 150mm dishes, multi-well plates (e.g., 6- well, 12-well, 24-well, 48-well, or 96 well plates), or flasks (e.g., T-flasks, e.g., T-25, T-75, or T- 160 flasks), or shaker flasks. [0497] In some embodiments, a host cell is cultured in a bioreactor. In some embodiments, a bioreactor is suitable for/used for culturing adherent cells. In some embodiments, a bioreactor is suitable for/used for culturing suspension cells. A bioreactor can be, e.g., a continuous flow batch bioreactor, a perfusion bioreactor, a batch process bioreactor, or a fed batch bioreactor. An exemplary bioreactor is a fixed bed bioreactor, e.g., an iCELLis bioreactor (used for culturing adherent cells). A bioreactor can be maintained under conditions Page 139 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sufficient to produce rAAV particles. Culture conditions can be modulated to optimize yield, purity, or structure of rAAV particles. [0498] In some embodiments, a bioreactor comprises a plurality of host cells. In some embodiments, host cells in a bioreactor comprise viable cells (vc). [0499] In some embodiments, a bioreactor comprises at least about 1 x 106, about 1 x 107, about 1 x 108, about 1 x 109, about 1 x 1010, about 1 x 1011, about 1 x 1012, about 1 x 1013, or about 1 x 1014 host cells (e.g., viable host cells). In some embodiments, a bioreactor comprises between 1 x 106 to 1 x 1014 host cells; between 1 x 106 to 0.5 x 1014 host cells; between 1 x 106 to 1 x 1013 host cells; between 1 x 106 to 0.5 x 1013 host cells; between 1 x 106 to 1 x 1012 host cells; between 1 x 106 to 0.5 x 1012 host cells; between 1 x 106 to 1 x 1011 host cells; between 1 x 106 to 0.5 x 1011 host cells; between 1 x 106 to 1 x 1010 host cells; between 1 x 106 to 0.5 x 1010 host cells; between 1 x 106 to 1 x 109 host cells; between 1 x 106 to 0.5 x 109 host cells; between 1 x 106 to 1 x 108 host cells; between 1 x 106 to 0.5 x 108 host cells; between 1 x 106 to 1 x 107 host cells; between 1 x 106 to 0.5 x 107 host cells; between 0.5 x 107 to 1 x 1014 host cells; between 1 x 108 to 1 x 1014 host cells; between 0.5 x 109 to 1 x 1014 host cells; between 1 x 109 to 1 x 1014 host cells; between 0.5 x 1010 to 1 x 1014 host cells; between 1 x 1010 to 1 x 1014 host cells; between 0.5 x 1011 to 1 x 1014 host cells; between 1 x 1011 to 1 x 1014 host cells; between 0.5 x 1012 to 1 x 1014 host cells; between 1 x 1012 to 1 x 1014 host cells; between 0.5 x 1013 to 1 x 1014 host cells; between 1 x 1013 to 1 x 1014 host cells; or between 0.5 x 1013 to 1 x 1014 host cells. [0500] In some embodiments, a bioreactor comprises about 0.5 million host cells/mL, about 1 million host cells/mL, about 1.5 million host cells/mL, about 2 million host cells/mL, about 2.5 million host cells/mL, about 3 million host cells/mL, about 3.5 million host cells/mL, about 4 million host cells/mL, about 4.5 million host cells/mL, about 5 million host cells/mL, about 5.5 million host cells/mL, about 6 million host cells/mL, about 7 million host cells/mL, about 8 million host cells/mL, about 9 million host cells/mL, about 10 million host cells/mL. In some embodiments, host cells in a bioreactor comprise viable cells (vc). [0501] In some embodiments, a bioreactor comprises at least about 1 liter, about 2 liters, about 3 liters, about 10 liters, about 20 liters, about 30 liters, about 40 liters, about 50 liters, about 55 liters, about 60 liters, about 65 liters, about 70 liters, about 75 liters, about 80 liters, about 85 liters, about 90 liters, about 95 liters, about 100 liters, about 200 liters, about 300 liters, Page 140 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) about 400 liters, about 500 liters, about 600 liters, about 700 liters, about 800 liters, about 900 liters, about 1000 liters, about 1500 liters, about 2000 liters or about 3000 liters of culture media. [0502] In an embodiment, a bioreactor is maintained under conditions that promote growth of a host cell, e.g., at a temperature (e.g., 37°C) and gas concentration (e.g., 5% - 10% CO2) that is permissive for growth of the host cell. For example, a bioreactor can perform one or more of the following: feeding of nutrients and/or carbon sources, injection of suitable gas (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), cleaning, and/or sterilization. Exemplary bioreactor units may contain multiple reactors within a unit, e.g., a unit can comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100, or more bioreactors. Any suitable bioreactor diameter and/or shape can be used. In some embodiments, suitable reactors can be round, e.g., cylindrical. In some embodiments, suitable reactors can be square, e.g., rectangular. rAAV Particle Production [0503] The present disclosure, among other things, rAAV particles produced using methods described herein. Generally, rAAV particles produced using methods described herein may be of any AAV serotype. AAV serotypes generally have different tropisms to infect and/or transduce different tissues. In some embodiments, an AAV serotype is selected based on a tropism. [0504] In some embodiments, a rAAV particle may comprise or be based on a serotype selected from any of the following serotypes, and variants thereof, including, but not limited to: AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 and AAVrh74. [0505] In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV1 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV2 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV3B serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV4 serotype Page 141 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV5 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV6 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV7 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV8 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV9 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV10 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV11 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV12 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAV13 serotype or a variant thereof. In certain embodiments, a rAAV particle comprising an AAV variant capsid disclosed herein comprises an AAVrh74 serotype or a variant thereof. [0506] In some embodiment, a plurality of rAAV particles are produced with methods described herein at a higher titer, e.g., such there is improved rAAV particle production. In some embodiments, the improved production comprises a higher yield of the plurality of rAAV particles relative to a plurality of rAAV particles produced with a helper vector comprising a nucleic acid sequence of an antibiotic resistance gene other than KanR (e.g., an Ampicillin resistance gene). In some embodiments, a high titer is relative to AAV particles produced from a reference helper vector (e.g., an Ad5 vector, e.g., an Ad5 vector described herein), e.g., under otherwise identical conditions. [0507] In some embodiments, a high titer is greater than 7.0 x 109 vg/mL +/- 15%, e.g., when cultured in suspension. In some embodiments, a high titer is greater than about 7.0 x 109 vg/mL, e.g., greater than about 7.5 x 109 vg/mL, 8.0 x 109 vg/mL, 8.5 x 109 vg/mL, 9.0 x 109 vg/mL, 1.0 x 1010 vg/mL, 1.5 x 1010 vg/mL, 2.0 x 1010 vg/mL, 2.5 x 1010 vg/mL, 3.0 x 1010 vg/mL, 3.5 x 1010 vg/mL, 4.0 x 1010 vg/mL, 4.5 x 1010 vg/mL, 5.0 x 1010 vg/mL, 5.5 x 1010 vg/mL, 6.0 x 1010 vg/mL, 6.5 x 1010 vg/mL, 7.0 x 1010 vg/mL, 7.5 x 1010 vg/mL, 8.0 x 1010 Page 142 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) vg/mL, 8.5 x 1010 vg/mL, 9.0 x 1010 vg/mL, 9.5 x 1010 vg/mL, 1.0 x 1011 vg/mL, 1.5 x 1011 vg/mL, 2.0 x 1011 vg/mL, or higher, e.g., when cultured in suspension. [0508] In some embodiments, a high titer of rAAV particles is at least about 7.0 x 109 vg/cm2, about 7.5 x 109 vg/cm2, about 8.0 x 109 vg/cm2, about 8.5 x 109 vg/cm2, about 9.0 x 109 vg/cm2, about 9.5 x 109 vg/cm2, about 1.0 x 1010 vg/cm2, about 1.5 x 1010 vg/cm2, or higher, e.g., when cultured in a bioreactor, e.g., a fixed bed bioreactor. [0509] In some embodiments, a high titer of rAAV particles is greater than 5.0 x 1013 vg/m2 +/- 15%, e.g., greater than 6.0 x 1013 vg/m2 +/- 15, 7.0 x 1013 vg/m2 +/- 15, 8.0 x 1013 vg/m2 +/- 15, 9.0 x 1013 vg/m2 +/- 15, 1.0 x 1014 vg/m2 +/- 15, 2.0 x 1014 vg/m2 +/- 15, 3.0 x 1014 vg/m2 +/- 15, 4.0 x 1014 vg/m2 +/- 15, 5.0 x 1014 vg/m2 +/- 15, 6.0 x 1014 vg/m2 +/- 15, 7.0 x 1014 vg/m2 +/- 15, 8.0 x 1014 vg/m2 +/- 15, 9.0 x 1014 vg/m2 +/- 15, or more. [0510] In some embodiments, a plurality of rAAV particles described herein is harvested after at least 3 days of culturing. In some embodiments, a plurality of rAAV particles described herein is harvested after at least about 3 days to about 10 days of culturing, e.g., about 3 days to about 7 days, about 3 days to about 5 days, about 4 days to about 9 days, about 4 days to about 8 days, or about 4 days to about 6 days of culturing, e.g., after at least about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, or longer after culturing. In some embodiments, a plurality of rAAV particles produced with methods described herein is substantially free of one or both of a helper adenovirus or a herpes virus. In some embodiments, a plurality of rAAV particles is substantially free of one or both of a helper adenovirus or a herpes virus, e.g., a purity of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more free of one or both of a helper adenovirus or a herpes virus [0511] The foregoing methods for producing recombinant vectors are not meant to be limiting, and other suitable methods will be apparent to the skilled artisan. rAAV Particle Compositions [0512] The present disclosure, among other things, provides a composition comprising a plurality of rAAV particles formed by methods described herein and/or using systems described herein. In some embodiments, a composition comprises a pharmaceutical composition Page 143 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) comprising at least one pharmaceutically acceptable component (e.g., a pharmaceutically acceptable carrier, diluent, or excipient). Such pharmaceutical compositions are useful for, among other things, administration to a subject in vivo or ex vivo. [0513] In some embodiments, pharmaceutical compositions also contain a pharmaceutically acceptable carrier, excipient, or diluent. Such excipients include any pharmaceutical agent, e.g., a pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids, such as water, saline, glycerol, sugars, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, or sulfates; and the salts of organic acids, such as acetates, propionates, malonates, or benzoates. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such vehicles. [0514] Pharmaceutical compositions may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, or succinic. Salts tend to be more soluble in aqueous or other protonic solvents than corresponding free base forms. In some embodiments, a pharmaceutical composition may be a lyophilized powder. [0515] Pharmaceutical compositions can include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, and isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules, and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral, and antifungal agents) can also be incorporated into the compositions. [0516] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, Page 144 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. [0517] Compositions suitable for parenteral administration can comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils. Aqueous injection suspensions may contain substances that increase the viscosity of a suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions may be prepared as appropriate oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase solubility to allow for preparation of highly concentrated solutions. [0518] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone. [0519] After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. Such labeling can include amount, frequency, and method of administration. [0520] Pharmaceutical compositions and delivery systems appropriate for the compositions, methods and uses of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy.21st Edition. Philadelphia, PA. Lippincott Williams & Wilkins, 2005). Page 145 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Administration [0521] The present disclosure, among other things, provides methods of administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles formed by methods described herein and/or produced using systems described herein. Compositions (e.g., pharmaceutical compositions) comprising rAAVs produced with the methods described herein or using systems described herein can be used to treat a muscle disorder, e.g., subjects suffering from or susceptible to a muscle disorder described herein. The route and/or mode of administration can vary depending upon the desired results. One with skill in the art (e.g., a physician), is aware that dosage regimens can be adjusted to provide the desired response, e.g., a therapeutic response. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intra-cisterna magna, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intracerebral, intrathecal, intravaginal, transdermal, rectal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin. Mode of administration is left to discretion of a practitioner. [0522] For example, a composition may be administered by retinal, subretinal, intravitreal, intracameral or suprachoroidal injection or infusion. Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intra-cisterna magna (ICM), intradermal, intragastric, intramedullary, intramuscular, intranasal, intra-parenchymal (e.g., intra-thalamic), intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, intraspinal, spinal sub-pial, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal administration. [0523] Methods and uses disclosed herein include delivery and administration systemically, regionally or locally, or by any route, for example, by injection or infusion. A composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles formed by methods described herein may be administered by injection or infusion by any route. [0524] Delivery of a pharmaceutical composition in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection- enhanced delivery can also be used. For example, compositions may be delivered Page 146 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intra-cisterna magna, intravenously, intra-pleurally, intraarterially, intracoronarily, orally, intrahepatically, via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. A clinician specializing in treatment of patients with certain diseases or disorders may determine the optimal route for administration of vectors described herein. [0525] Additionally, a pharmaceutical composition disclosed herein may also be administered by perfusion, e.g., by limb perfusion. [0526] The disclosure provides methods for introducing a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein into a cell, a tissue, or an animal. In some embodiments, such methods comprise contacting a cell, a tissue, or an animal with a composition comprising rAAV particles described herein, such that at least one payload is expressed or present in the cell, tissue, or animal. [0527] The disclosure also provides methods for administering a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein to a subject. In some embodiments, such methods include administering to a subject (e.g., a mammal), a composition comprising rAAV particles described herein, such that at least one payload is expressed or present in the subject (e.g., in a cell or tissue of a subject). In some embodiments, a method includes providing cells of a subject (e.g., a mammal) with a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein, such that at least one payload is expressed or present in the subject. [0528] A composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein can be administered in a sufficient or effective amount to a subject in need thereof. Doses can vary and depend upon a type, onset, progression, severity, frequency, duration, or probability of disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject, and other factors that will be appreciated by a skilled artisan. Dose amount, number, frequency, or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications, or other risk factors of treatment and status of the Page 147 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) subject. A skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit. [0529] A dose to achieve a therapeutic effect will vary based on several factors including, but not limited to: route of administration, level of payload or payload expression required to achieve a therapeutic effect, specific disease treated, any host immune response, and stability of payload or payload expression. One skilled in the art can determine a dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors. [0530] An effective amount or a sufficient amount can (but need not) be provided in a single administration, may require multiple administrations, and, can (but need not) be, administered alone or in combination with another composition. For example, an amount may be proportionally increased as indicated by need of a subject, type, status, and severity of disease treated or side effects (if any) of treatment. Amounts considered effective also include amounts that result in a reduction of use of another treatment, therapeutic regimen, or protocol. [0531] Accordingly, pharmaceutical compositions include compositions comprising rAAV particles in an effective amount to achieve an intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using techniques and guidance provided herein. Therapeutic doses can depend on, among other factors, age and general condition of a subject, severity of a disease or disorder, and payload amount or expression in a subject. Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on response of an individual patient to rAAV-based treatment. Pharmaceutical compositions may be delivered to a subject so as to allow production of a payload described herein in vivo by gene- and or cell- based therapies or by ex vivo modification of a patient’s or donor’s cells. [0532] In some embodiments, a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein may be administered to a subject once daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein may be administered according to a dosing regimen that includes (i) an initial administration that is once Page 148 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals; followed by (ii) a period of no administration of, e.g., 1, 2, 3, 4, 5, 6, 8, or 10 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein may be administered (i) one or more times during an initial time period of up to 2, 4, or 6 weeks or less; followed by (ii) a period of no administration of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, a subject is monitored before and/or following treatment with a composition (e.g., a pharmaceutical composition) comprising rAAV particles described herein. AAV capsid reference sequences [0533] SEQ ID NO: 2001: AAV9 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 121 AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE 181 SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI 241 TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR 301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH 361 EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV 421 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP 481 GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS 541 LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG 601 ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT 661 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV 721 YSEPRPIGTR YLTRNL [0534] SEQ ID NO: 2002: AAV1 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVEEGAKTAP GKKRPVEQSP QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE 181 SVPDPQPLGE PPATPAAVGP TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI 241 TTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQ 361 GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEEVP 421 FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP 481 GPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD EDKFFPMSGV 541 MIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG 601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPA 661 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGL 721 YTEPRPIGTR YLTRPL [0535] SEQ ID NO: 2003: AAV2 VP1 capsid reference sequence 1 MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD Page 149 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 61 KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVEEPVKTAP GKKRPVEHSP VEPDSSSGTG KAGQQPARKR LNFGQTGDAD 181 SVPDPQPLGQ PPAAPSGLGT NTMATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI 241 TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG 361 CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFT FSYTFEDVPF 421 HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT PSGTTTQSRL QFSQAGASDI RDQSRNWLPG 481 PCYRQQRVSK TSADNNNSEY SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL 541 IFGKQGSEKT NVDIEKVMIT DEEEIRTTNP VATEQYGSVS TNLQRGNRQA ATADVNTQGV 601 LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN TPVPANPSTT 661 FSAAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNPEIQY TSNYNKSVNV DFTVDTNGVY 721 SEPRPIGTRY LTRNL [0536] SEQ ID NO: 2050: AAV3B VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWALKPGVP QPKANQQHQD NRRGLVLPGY KYLGPGNGLD 61 KGEPVNEADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRILEPLG LVEEAAKTAP GKKRPVDQSP QEPDSSSGVG KSGKQPARKR LNFGQTGDSE 181 SVPDPQPLGE PPAAPTSLGS NTMASGGGAP MADNNEGADG VGNSSGNWHC DSQWLGDRVI 241 TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGFRPKK LSFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG 361 CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFQ FSYTFEDVPF 421 HSSYAHSQSL DRLMNPLIDQ YLYYLNRTQG TTSGTTNQSR LLFSQAGPQS MSLQARNWLP 481 GPCYRQQRLS KTANDNNNSN FPWTAASKYH LNGRDSLVNP GPAMASHKDD EEKFFPMHGN 541 LIFGKEGTTA SNAELDNVMI TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTRTVNDQG 601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPT 661 TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VDFTVDTNGV 721 YSEPRPIGTR YLTRNL [0537] SEQ ID NO: 2004: AAV5 VP1 capsid reference sequence 1 MSFVDHPPDW LEEVGEGLRE FLGLEAGPPK PKPNQQHQDQ ARGLVLPGYN YLGPGNGLDR 61 GEPVNRADEV AREHDISYNE QLEAGDNPYL KYNHADAEFQ EKLADDTSFG GNLGKAVFQA 121 KKRVLEPFGL VEEGAKTAPT GKRIDDHFPK RKKARTEEDS KPSTSSDAEA GPSGSQQLQI 181 PAQPASSLGA DTMSAGGGGP LGDNNQGADG VGNASGDWHC DSTWMGDRVV TKSTRTWVLP 241 SYNNHQYREI KSGSVDGSNA NAYFGYSTPW GYFDFNRFHS HWSPRDWQRL INNYWGFRPR 301 SLRVKIFNIQ VKEVTVQDST TTIANNLTST VQVFTDDDYQ LPYVVGNGTE GCLPAFPPQV 361 FTLPQYGYAT LNRDNTENPT ERSSFFCLEY FPSKMLRTGN NFEFTYNFEE VPFHSSFAPS 421 QNLFKLANPL VDQYLYRFVS TNNTGGVQFN KNLAGRYANT YKNWFPGPMG RTQGWNLGSG 481 VNRASVSAFA TTNRMELEGA SYQVPPQPNG MTNNLQGSNT YALENTMIFN SQPANPGTTA 541 TYLEGNMLIT SESETQPVNR VAYNVGGQMA TNNQSSTTAP ATGTYNLQEI VPGSVWMERD 601 VYLQGPIWAK IPETGAHFHP SPAMGGFGLK HPPPMMLIKN TPVPGNITSF SDVPVSSFIT 661 QYSTGQVTVE MEWELKKENS KRWNPEIQYT NNYNDPQFVD FAPDSTGEYR TTRPIGTRYL 721 TRPL [0538] SEQ ID NO: 2005: AAV6 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPFG LVEEGAKTAP GKKRPVEQSP QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE 181 SVPDPQPLGE PPATPAAVGP TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI 241 TTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQ 361 GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEDVP 421 FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP 481 GPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGV Page 150 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 541 MIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG 601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA 661 EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGL 721 YTEPRPIGTR YLTRPL [0539] SEQ ID NO: 2006: AAV8 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPARK RLNFGQTGDS 181 ESVPDPQPLG EPPAAPSGVG PNTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWAL PTYNNHLYKQ ISNGTSGGAT NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA 361 HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFQFTYTFED 421 VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQTTGGTANT QTLGFSQGGP NTMANQAKNW 481 LPGPCYRQQR VSTTTGQNNN SNFAWTAGTK YHLNGRNSLA NPGIAMATHK DDEERFFPSN 541 GILIFGKQNA ARDNADYSDV MLTSEEEIKT TNPVATEEYG IVADNLQQQN TAPQIGTVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP 661 PTTFNQSKLN SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TSVDFAVNTE 721 GVYSEPRPIG TRYLTRNL [0540] SEQ ID NO: 2051: AAV4 VP1 capsid reference sequence 1 MTDGYLPDWL EDNLSEGVRE WWALQPGAPK PKANQQHQDN ARGLVLPGYK YLGPGNGLDK 61 GEPVNAADAA ALEHDKAYDQ QLKAGDNPYL KYNHADAEFQ QRLQGDTSFG GNLGRAVFQA 121 KKRVLEPLGL VEQAGETAPG KKRPLIESPQ QPDSSTGIGK KGKQPAKKKL VFEDETGAGD 181 GPPEGSTSGA MSDDSEMRAA AGGAAVEGGQ GADGVGNASG DWHCDSTWSE GHVTTTSTRT 241 WVLPTYNNHL YKRLGESLQS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGMRPK 301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE GSLPPFPNDV 361 FMVPQYGYCG LVTGNTSQQQ TDRNAFYCLE YFPSQMLRTG NNFEITYSFE KVPFHSMYAH 421 SQSLDRLMNP LIDQYLWGLQ STTTGTTLNA GTATTNFTKL RPTNFSNFKK NWLPGPSIKQ 481 QGFSKTANQN YKIPATGSDS LIKYETHSTL DGRWSALTPG PPMATAGPAD SKFSNSQLIF 541 AGPKQNGNTA TVPGTLIFTS EEELAATNAT DTDMWGNLPG GDQSNSNLPT VDRLTALGAV 601 PGMVWQNRDI YYQGPIWAKI PHTDGHFHPS PLIGGFGLKH PPPQIFIKNT PVPANPATTF 661 SSTPVNSFIT QYSTGQVSVQ IDWEIQKERS KRWNPEVQFT SNYGQQNSLL WAPDAAGKYT 721 EPRAIGTRYL THHL [0541] SEQ ID NO: 2052: AAV7 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD NGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVEEGAKTAP AKKRPVEPSP QRSPDSSTGI GKKGQQPARK RLNFGQTGDS 181 ESVPDPQPLG EPPAAPSSVG SGTVAAGGGA PMADNNEGAD GVGNASGNWH CDSTWLGDRV 241 ITTSTRTWAL PTYNNHLYKQ ISSETAGSTN DNTYFGYSTP WGYFDFNRFH CHFSPRDWQR 301 LINNNWGFRP KKLRFKLFNI QVKEVTTNDG VTTIANNLTS TIQVFSDSEY QLPYVLGSAH 361 QGCLPPFPAD VFMIPQYGYL TLNNGSQSVG RSSFYCLEYF PSQMLRTGNN FEFSYSFEDV 421 PFHSSYAHSQ SLDRLMNPLI DQYLYYLART QSNPGGTAGN RELQFYQGGP STMAEQAKNW 481 LPGPCFRQQR VSKTLDQNNN SNFAWTGATK YHLNGRNSLV NPGVAMATHK DDEDRFFPSS 541 GVLIFGKTGA TNKTTLENVL MTNEEEIRPT NPVATEEYGI VSSNLQAANT AAQTQVVNNQ 601 GALPGMVWQN RDVYLQGPIW AKIPHTDGNF HPSPLMGGFG LKHPPPQILI KNTPVPANPP 661 EVFTPAKFAS FITQYSTGQV SVEIEWELQK ENSKRWNPEI QYTSNFEKQT GVDFAVDSQG 721 VYSEPRPIGT RYLTRNL Page 151 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0542] SEQ ID NO: 2053: AAV10 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVEEAAKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPAKK RLNFGQTGES 181 ESVPDPQPIG EPPAGPSGLG SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWAL PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA 361 HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFEFSYTFED 421 VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQSTGGTQGT QQLLFSQAGP ANMSAQAKNW 481 LPGPCYRQQR VSTTLSQNNN SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS 541 GVLMFGKQGA GRDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQAN TGPIVGNVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP 661 PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TNVDFAVNTE 721 GTYSEPRPIG TRYLTRNL [0543] SEQ ID NO: 2054: AAV11 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVEEGAKTAP GKKRPLESPQ EPDSSSGIGK KGKQPARKRL NFEEDTGAGD 181 GPPEGSDTSA MSSDIEMRAA PGGNAVDAGQ GSDGVGNASG DWHCDSTWSE GKVTTTSTRT 241 WVLPTYNNHL YLRLGTTSSS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGLRPK 301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE GSLPPFPNDV 361 FMVPQYGYCG IVTGENQNQT DRNAFYCLEY FPSQMLRTGN NFEMAYNFEK VPFHSMYAHS 421 QSLDRLMNPL LDQYLWHLQS TTSGETLNQG NAATTFGKIR SGDFAFYRKN WLPGPCVKQQ 481 RFSKTASQNY KIPASGGNAL LKYDTHYTLN NRWSNIAPGP PMATAGPSDG DFSNAQLIFP 541 GPSVTGNTTT SANNLLFTSE EEIAATNPRD TDMFGQIADN NQNATTAPIT GNVTAMGVLP 601 GMVWQNRDIY YQGPIWAKIP HADGHFHPSP LIGGFGLKHP PPQIFIKNTP VPANPATTFT 661 AARVDSFITQ YSTGQVAVQI EWEIEKERSK RWNPEVQFTS NYGNQSSMLW APDTTGKYTE 721 PRVIGSRYLT NHL [0544] SEQ ID NO: 2055: AAV12 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NGRGLVLPGY KYLGPFNGLD 61 KGEPVNEADA AALEHDKAYD KQLEQGDNPY LKYNHADAEF QQRLATDTSF GGNLGRAVFQ 121 AKKRILEPLG LVEEGVKTAP GKKRPLEKTP NRPTNPDSGK APAKKKQKDG EPADSARRTL 181 DFEDSGAGDG PPEGSSSGEM SHDAEMRAAP GGNAVEAGQG ADGVGNASGD WHCDSTWSEG 241 RVTTTSTRTW VLPTYNNHLY LRIGTTANSN TYNGFSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGLRPKS MRVKIFNIQV KEVTTSNGET TVANNLTSTV QIFADSTYEL PYVMDAGQEG 361 SFPPFPNDVF MVPQYGYCGV VTGKNQNQTD RNAFYCLEYF PSQMLRTGNN FEVSYQFEKV 421 PFHSMYAHSQ SLDRMMNPLL DQYLWHLQST TTGNSLNQGT ATTTYGKITT GDFAYYRKNW 481 LPGACIKQQK FSKNANQNYK IPASGGDALL KYDTHTTLNG RWSNMAPGPP MATAGAGDSD 541 FSNSQLIFAG PNPSGNTTTS SNNLLFTSEE EIATTNPRDT DMFGQIADNN QNATTAPHIA 601 NLDAMGIVPG MVWQNRDIYY QGPIWAKVPH TDGHFHPSPL MGGFGLKHPP PQIFIKNTPV 661 PANPNTTFSA ARINSFLTQY STGQVAVQID WEIQKEHSKR WNPEVQFTSN YGTQNSMLWA 721 PDNAGNYHEL RAIGSRFLTH HL [0545] SEQ ID NO: 2056: AAV13 VP1 capsid reference sequence 1 MTDGYLPDWL EDNLSEGVRE WWALQPGAPK PKANQQHQDN ARGLVLPGYK YLGPGNGLDK 61 GEPVNAADAA ALEHDKAYDQ QLKAGDNPYL KYNHADAEFQ ERLQEDTSFG GNLGRAVFQA Page 152 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 121 KKRILEPLGL VEEAAKTAPG KKRPVEQSPA EPDSSSGIGK SGQQPARKRL NFGQTGDTES 181 VPDPQPLGQP PAAPSGVGST TMASGGGAPM ADNNEGADGV GNSSGNWHCD SQWLGDRVIT 241 TSTRTWALPT YNNHLYKQIS SQSGATNDNH YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN 301 NNWGFRPKRL NFKLFNIQVK EVTQNDGTTT IANNLTSTVQ VFTDSEYQLP YVLGSAHQGC 361 LPPFPADVFM VPQYGYLTLN NGSQAVGRSS FYCLEYFPSQ MLRTGNNFQF SYTFEDVPFH 421 SSYAHSQSLD RLMNPLIDQY LYYLNRTQTA SGTQQSRLLF SQAGPTSMSL QAKNWLPGPC 481 YRQQRLSKQA NDNNNSNFPW TGATKYHLNG RDSLVNPGPA MASHKDDKEK FFPMHGTLIF 541 GKEGTNANNA DLENVMITDE EEIRTTNPVA TEQYGTVSNN LQNSNAGPTT GTVNHQGALP 601 GMVWQDRDVY LQGPIWAKIP HTDGHFHPSP LMGGFGLKHP PPQIMIKNTP VPANPPTNFS 661 AAKFASFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS NYNKSVNVDF TVDTNGVYSE 721 PRPIGTRYLT RNL [0546] SEQ ID NO: 2057: AAVrh74 VP1 capsid reference sequence 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD NGRGLVLPGY KYLGPFNGLD 61 KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121 AKKRVLEPLG LVESPVKTAP GKKRPVEPSP QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS 181 ESVPDPQPIG EPPAGPSGLG SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV 241 ITTSTRTWAL PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE YQLPYVLGSA 361 HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY FPSQMLRTGN NFEFSYNFED 421 VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR TQSTGGTAGT QQLLFSQAGP NNMSAQAKNW 481 LPGPCYRQQR VSTTLSQNNN SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS 541 GVLMFGKQGA GKDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL IKNTPVPADP 661 PTTFNQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE IQYTSNYYKS TNVDFAVNTE 721 GTYSEPRPIG TRYLTRNL [0547] The disclosure is further illustrated by the following example. An example is provided for illustrative purposes only. It is not to be construed as limiting the scope or content of the disclosure in any way. [0548] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. Page 153 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Numbered embodiments: [0549] Embodiment 1. A recombinant adeno-associated virus (rAAV) particle comprising: [0550] (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: [0551] (i) the peptide insertion comprises a sequence of: [0552] (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0553] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0554] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0555] (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0556] (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0557] (VI) RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0558] (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0559] (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0560] (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or [0561] (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and Page 154 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0562] (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and [0563] (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0564] Embodiment 2. A recombinant adeno-associated virus (rAAV) particle comprising: [0565] (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: [0566] (i) the peptide insertion comprises a sequence provided in Table 1; and [0567] (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and [0568] (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. [0569] Embodiment 3. The rAAV particle of embodiment 1 or 2, wherein the insertion site is located between two adjacent amino acids in the variable region of the parental AAV capsid protein. [0570] Embodiment 4. The rAAV particle of embodiment 1 or 2, wherein the insertion site is located between two non-adjacent amino acids in the variable region of the parental AAV capsid protein [0571] Embodiment 5. The rAAV particle of any one of the preceding embodiments, wherein the insertion of the heterologous peptide replaces a contiguous stretch of amino acids of the parental AAV capsid protein. [0572] Embodiment 6. The rAAV particle of any one of embodiments 1-4, wherein the insertion of the heterologous peptide does not replace a contiguous stretch of amino acids of the parental AAV capsid protein. Page 155 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0573] Embodiment 7. The rAAV particle of any one of the preceding embodiments, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein. [0574] Embodiment 8. The rAAV particle of any one of the preceding embodiments, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein. [0575] Embodiment 9. The rAAV particle of embodiment 8, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII of AAV9 comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. [0576] Embodiment 10. The rAAV particle of embodiment 8 or 9, wherein the insertion site is located between amino acids 588 and 589 of VP1 of an AAV9 capsid protein or the corresponding position in the VP1 of another parental AAV capsid protein. [0577] Embodiment 11. The rAAV particle of any one of embodiments 8- 10, wherein the insertion site is located between amino acids 588 and 589 of VP2 of an AAV9 capsid protein or the corresponding position in the VP2 of another parental AAV capsid protein. [0578] Embodiment 12. The rAAV particle of any one of embodiments 8- 11, wherein the insertion site is located between amino acids 588 and 589 of VP3 of an AAV9 capsid protein or the corresponding position in the VP3 of another parental AAV capsid protein. [0579] Embodiment 13. The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. [0580] Embodiment 14. The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the sequence of SEQ ID NO: 1551, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. [0581] Embodiment 15. The rAAV particle of any one of embodiments 1-13, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the Page 156 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sequence of SEQ ID NO: 1825, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. [0582] Embodiment 16. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 1. [0583] Embodiment 17. The rAAV particle of embodiment 16, wherein the peptide insertion comprises a sequence provided in Table 2. [0584] Embodiment 18. The rAAV particle of embodiment 16 or 17, wherein the peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0585] Embodiment 19. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 2. [0586] Embodiment 20. The rAAV particle of embodiment 19, wherein the peptide insertion comprises a sequence provided in Table 3. [0587] Embodiment 21. The rAAV particle of embodiment 19 or 20, wherein the peptide insertion does not comprise the sequence of RGDYQAV (SEQ ID NO: 1764). [0588] Embodiment 22. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 3. [0589] Embodiment 23. The rAAV particle of embodiment 22, wherein the peptide insertion comprises a sequence provided in Table 4. [0590] Embodiment 24. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 4. [0591] Embodiment 25. The rAAV particle of embodiment 24, wherein the peptide insertion comprises a sequence provided in Table 5. [0592] Embodiment 26. The rAAV particle of embodiment 24 or 25, wherein the peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0593] Embodiment 27. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 5. Page 157 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0594] Embodiment 28. The rAAV particle of embodiment 27, wherein the peptide insertion comprises a sequence provided in Table 6. [0595] Embodiment 29. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 6. [0596] Embodiment 30. The rAAV particle of embodiment 29, wherein the peptide insertion comprises a sequence provided in Table 7. [0597] Embodiment 31. The rAAV particle of embodiment 29 or 30, wherein the peptide insertion comprises the sequence of RGDPQRW (SEQ ID NO: 802). [0598] Embodiment 32. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 7. [0599] Embodiment 33. The rAAV particle of embodiment 30, wherein the peptide insertion comprises a sequence provided in Table 8. [0600] Embodiment 34. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 8. [0601] Embodiment 35. The rAAV particle of embodiment 34, wherein the peptide insertion comprises a sequence provided in Table 9. [0602] Embodiment 36. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 9. [0603] Embodiment 37. The rAAV particle of embodiment 36, wherein the peptide insertion comprises a sequence provided in Table 10. [0604] Embodiment 38. The rAAV particle of any one of embodiments 1-13, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 10. [0605] Embodiment 39. The rAAV particle of embodiment 38, wherein the peptide insertion comprises a sequence provided in Table 11. [0606] Embodiment 40. The rAAV particle of embodiment 38 or 39, wherein the peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551). Page 158 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0607] Embodiment 41. The rAAV particle of embodiment 38 or 39, wherein the peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825). [0608] Embodiment 42. The rAAV particle of embodiment 38 or 39, wherein the peptide insertion comprises the sequence of RGDYREV (SEQ ID NO: 1829). [0609] Embodiment 43. The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein confers increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0610] Embodiment 44. The rAAV particle of embodiment 43, wherein the variant AAV capsid protein confers at least 5-fold increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. [0611] Embodiment 45. The rAAV particle of any one of the preceding embodiments, wherein the muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof. [0612] Embodiment 46. The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site. [0613] Embodiment 47. The rAAV particle of embodiment 46, wherein the peptide insertion site is located at or between amino acids 581 and 593 of VP1 of AAV9 or the corresponding position in the capsid protein of another parental AAV capsid protein. [0614] Embodiment 48. The rAAV particle of embodiment 46, wherein the peptide insertion site is located between amino acids 588 and 589 of VP1 of AAV9 or the corresponding position in the capsid protein of another parental AAV capsid protein. [0615] Embodiment 49. The rAAV particle of any one of embodiments 46-48, wherein the one or more modifications are within about 5 to 10 amino acids upstream or downstream of the location of the peptide insertion site. Page 159 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0616] Embodiment 50. The rAAV particle of embodiment 49, wherein the one or more modifications are within about 5 amino acids upstream or downstream of the location of the peptide insertion site. [0617] Embodiment 51. The rAAV particle of any one of embodiments 46-50, wherein the one or more modifications are located in a variable region IV (VR-IV) of AAV9 a VP (e.g., VP1, VP2, and/or VP3), or a variable region V (VR-V) of AAV9 a VP (e.g., VP1, VP2, and/or VP3), or both. [0618] Embodiment 52. The rAAV particle of embodiment 51, wherein the AAV9 VP1 VR-IV comprises amino acids 451-475 of VP1 of AAV9. [0619] Embodiment 53. The rAAV particle of embodiment 51 or 52, wherein the AAV9 VP1 VR-V comprises amino acids 488-506 of VP1 of AAV9. [0620] Embodiment 54. The rAAV particle of any one of embodiments 46-53, wherein the one or more modifications comprises an insertion, deletion, mutation, or a combination thereof. [0621] Embodiment 55. The rAAV particle of any one of the preceding embodiments wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. [0622] Embodiment 56. The rAAV particle of embodiment 55, wherein the one or more modifications reduces glycan binding. [0623] Embodiment 57. The rAAV particle of embodiment 55 or 56, wherein the glycan is galactose. [0624] Embodiment 58. The rAAV particle of any one of embodiments 46-57, wherein the one or more modifications is at or between amino acids: [0625] (a) 271 and 272 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; [0626] (b) 446 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; Page 160 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0627] (c) 470 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; [0628] (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; [0629] (e) 489 and 545 of VP1 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; [0630] (f) 591 and 621 of VP1 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein; or [0631] (g) any combination or all of (a)-(f). [0632] Embodiment 59. The rAAV particle of any one of the preceding embodiments, wherein the variant AAV capsid protein has at least 90% identity relative to a parental AAV capsid protein. [0633] Embodiment 60. The rAAV particle of embodiment 59, wherein percent identity is determined by comparing the sequence of the variant AAV capsid protein without the peptide insertion, with the parental AAV capsid protein. [0634] Embodiment 61. The rAAV particle of embodiment 60, wherein the variant AAV capsid protein and the parental AAV capsid protein have 100% identity when: [0635] (a) the peptide insertion in the variant AAV capsid protein is not taken into account in the sequence comparison; and [0636] (b) the variant AAV capsid protein does not have one or more modifications other than the peptide insertion. [0637] Embodiment 62. The rAAV particle of embodiment 60, wherein the variant AAV capsid protein and the parental AAV capsid protein have less than 100% identity when: [0638] (a) the peptide insertion in the variant AAV capsid protein is not taken into account in the sequence comparison; and Page 161 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0639] (b) the variant AAV capsid protein comprises one or more modifications other than the peptide insertion. [0640] Embodiment 63. The rAAV particle of any one of the preceding embodiments, wherein the parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001. [0641] Embodiment 64. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002. [0642] Embodiment 65. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003. [0643] Embodiment 66. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2050. [0644] Embodiment 67. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV5 capsid protein of SEQ ID NO: 2004. [0645] Embodiment 68. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV6 capsid protein of SEQ ID NO: 2005. [0646] Embodiment 69. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006. [0647] Embodiment 70. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV4 capsid protein of SEQ ID NO: 2051. [0648] Embodiment 71. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2052. [0649] Embodiment 72. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2053. [0650] Embodiment 73. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2054. [0651] Embodiment 74. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2055. Page 162 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0652] Embodiment 75. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2056. [0653] Embodiment 76. The rAAV particle of any one of embodiments 1-62, wherein the parental AAV capsid protein is an AAVrh74 capsid protein of SEQ ID NO: 2057. [0654] Embodiment 77. The rAAV particle of any one of the preceding embodiments, wherein the payload is a polypeptide. [0655] Embodiment 78. The rAAV particle of embodiment 77, wherein the polypeptide is or comprises: [0656] (i) a CRISPR-Cas protein or a variant or a fragment thereof; [0657] (ii) a Zinc finger protein, or a variant or a fragment thereof; [0658] (iii) a TAL, or a variant or a fragment thereof; [0659] (iv) a base editor, or a variant or a fragment thereof; [0660] (v) a prime editor, or a variant or a fragment thereof; and/or [0661] (vi) a meganuclease, or a variant or a fragment thereof. [0662] Embodiment 79. The rAAV particle of embodiment 78, wherein the CRISPR-Cas protein is or comprises a Type II, Type V or Type VI CRISPR-Cas protein, e.g., a Cas9 protein, a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment of any of the foregoing. [0663] Embodiment 80. The rAAV particle of embodiment 78 or 79, wherein the polypeptide is associated with a muscle disorder, or a glycogen or sugar storage disorder. [0664] Embodiment 81. The rAAV particle of embodiment 80, wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. Page 163 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0665] Embodiment 82. The rAAV particle of embodiment 77, wherein the polypeptide is an enzyme. [0666] Embodiment 83. The rAAV particle of embodiment 82, wherein the enzyme is a lysosomal enzyme or an adenosine deaminase enzyme. [0667] Embodiment 84. The rAAV particle of embodiment 77, wherein the polypeptide is an antibody. [0668] Embodiment 85. The rAAV particle of embodiment 77, wherein the polypeptide is a secreted protein. [0669] Embodiment 86. The rAAV particle of any one embodiments 1-76, wherein the payload is an RNA molecule. [0670] Embodiment 87. The rAAV particle of embodiment 86, wherein the RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, circular RNA, a snRNA, or an aptamer. [0671] Embodiment 88. The rAAV particle of embodiment 86 or 87, wherein the RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder. [0672] Embodiment 89. The rAAV particle of embodiment 88, wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0673] Embodiment 90. The rAAV particle of any one embodiments 1-76, wherein the payload is a DNA molecule. [0674] Embodiment 91. The rAAV particle of any one of the preceding embodiments, wherein the nucleic acid sequence encoding a payload comprises a promoter. [0675] Embodiment 92. The rAAV particle of embodiment 91, wherein the promoter is or comprises a muscle-specific promoter. Page 164 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0676] Embodiment 93. The rAAV particle of embodiment 92, wherein the muscle- specific promoter is chosen from: MHCK7, CK8, desmin, tMCK, dMCK, or CK6. [0677] Embodiment 94. The rAAV particle of embodiment 91, wherein the promoter is or comprises a dual muscle-liver promoter. [0678] Embodiment 95. The rAAV particle of embodiment 94, wherein the dual muscle-liver promoter is SPc5-12. [0679] Embodiment 96. A pharmaceutical composition comprising: [0680] (a) a rAAV particle of any one of the preceding embodiments; and [0681] (b) a pharmaceutically acceptable excipient. [0682] Embodiment 97. A method of delivering a payload to a muscle cell, comprising administering the pharmaceutical composition of embodiment 96 to the muscle cell. [0683] Embodiment 98. The method of embodiment 97, wherein the muscle cell is in vitro. [0684] Embodiment 99. The method of embodiment 97, wherein the muscle cell is in vivo. [0685] Embodiment 100. The method of embodiment 98 or 99, wherein the muscle cell is from a subject that has, or has been determined to have, a muscle disorder. [0686] Embodiment 101. A method of treating a subject having a muscle disorder and/or ameliorating a symptom of a muscle disorder in a subject, the method comprising [0687] administering to the subject the pharmaceutical composition of embodiment 96. [0688] Embodiment 102. The method of embodiment 100 or 101, wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie- Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. [0689] Embodiment 103. The method of any one of embodiments 97-102, wherein the pharmaceutical composition is administered via a route of administration chosen from: Page 165 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) intramuscular, intra-cisterna magna, intravenous, intraarterial, intracoronoary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, or limb perfusion. [0690] Embodiment 104. The method of any one of embodiments 101-103, wherein the subject is a human. [0691] Embodiment 105. An isolated cell transduced with the rAAV particle of any one of embodiments 1-95. [0692] Embodiment 106. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: [0693] (i) the peptide insertion comprises a sequence of: [0694] (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0695] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0696] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0697] (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0698] (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0699] (VI) RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0700] (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0701] (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; Page 166 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0702] (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or [0703] (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and [0704] (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein. [0705] Embodiment 107. The isolated nucleic acid of embodiment 106, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein. [0706] Embodiment 108. The isolated nucleic acid of embodiment 106 or 107, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein. [0707] Embodiment 109. The isolated nucleic acid of embodiment 108, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. [0708] Embodiment 110. An isolated cell comprising the nucleic acid of any one of embodiments 106-109. [0709] Embodiment 111. A variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: [0710] (i) the peptide insertion comprises a sequence of: [0711] (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0712] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0713] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; Page 167 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0714] (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0715] (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0716] (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0717] (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0718] (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0719] (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or [0720] (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and [0721] (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein. [0722] Embodiment 112. The variant AAV capsid protein of embodiment 111, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR- VIII or VR-IX of the parental AAV capsid protein. [0723] Embodiment 113. The variant AAV capsid protein of embodiment 111 or 112, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein. [0724] Embodiment 114. The variant AAV capsid protein of embodiment 113, wherein the parental AAV capsid protein is an AAV9 capsid protein and the VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein. [0725] Embodiment 115. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence of Page 168 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0726] (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0727] (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0728] (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0729] (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0730] (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0731] (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0732] (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0733] (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; [0734] (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or [0735] (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I. [0736] Embodiment 116. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence provided in Table 1. [0737] Embodiment 117. The composition of embodiment 115 or 116, wherein the targeting moiety is conjugated to the payload. [0738] Embodiment 118. The composition of any one of embodiments 115-117, wherein the targeting moiety is inserted into a viral protein. Page 169 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0739] Embodiment 119. The composition of embodiment 118, wherein the viral protein is an AAV capsid protein. [0740] Embodiment 120. The composition of any one of embodiments 115-117, wherein the targeting moiety is part of, e.g., incorporated into, a vector. [0741] Embodiment 121. The composition of any one of embodiments 115-117, wherein the targeting moiety is not part of a vector. [0742] Embodiment 122. The composition of any one of embodiments 115-121, wherein the targeting moiety is a muscle-targeting moiety. EXAMPLES [0743] The following example is put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their discovery nor are they intended to represent that the experiments below are all or the only experiments performed. Example 1: Identification of RGD-containing capsids with enhanced skeletal muscle tropism [0744] This Example describes the identification of RGD-containing capsids obtained by screening an AAV library having RGD-containing variants in non-human primates (NHPs). [0745] Materials and Methods [0746] Round 1 DNA library construction. The plasmid backbone for Round 1 library consists of an AAV2 inverted terminal (ITR2)-flanked sequence containing a CBh promoter, AAV2 P40 promoter, a partial AAV9 VP1 coding sequence, and SV40 polyadenylation signal. Oligonucleotides encoding RGDxxxx and xRGDxxx peptide insertions were synthesized by Integrated DNA Technologies, where x represents standard trinucleotide 19 mix without cysteine. Oligonucleotides were pooled and used as PCR primers to amplify DNA fragments encoding the AAV9 VP1 C-terminus with RGD peptides inserted between amino acid residues 588 and 589 (AAV9 VP1 numbering), which were cloned into AgeI-linearized plasmid Page 170 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) backbone via Gibson assembly. Assembled products were electroporated into NEB 10-beta cells for DNA library production. [0747] AAV Library Production. Virus production was performed by transfecting HEK293T cells with an Ad helper plasmid, a plasmid supplying Rep2, and a limited amount of DNA library at ~2500 copies per cell using PEI Max. Virus was harvested 3 days post- transfection, and purified through iodixanol density gradient. Virus preparations were buffer exchanged into phosphate-buffered saline (pH 7.4) with 0.001% pluronic F-68, and subsequently subjected to digital droplet PCR (ddPCR)-based titration, purity analysis by SYPRO-Ruby staining of PAGE gels, and endotoxin measurement. [0748] In vivo library screening. In vivo library screening was performed on adult/juvenile cynomolgus macaques sero-negative for AAV9 neutralizing antibodies. For each round of library screening, two animals were injected intravenously, each with 1E14 genome copies (GC) of AAV library. Necropsies were performed 21 days post-injection, and major organs collected for analysis. [0749] Trizol-based RNA isolation was performed from skeletal muscle and heart. To enrich capsid mRNA, hybridization-based mRNA capture was performed using biotinylated antisense oligos and streptavidin paramagnetic beads. Captured RNA was reverse transcribed, and cDNA used for PCR amplification for NGS analysis as well as subsequent round library cloning. [0750] Round 2 DNA library construction. A synthetic oligo pool consisting of 24,000 oligos, with two oligos encoding each peptide insertion for the top 12,000 enriched Round 1 variants, was synthesized by Twist Biosciences. Round 1 muscle amplicons as well as this oligo pool were used to clone Round 2 DNA library. Recovered Round 1 muscle amplicons were inserted into the AgeI-digested plasmid library backbone by Gibson assembly whereas the synthetic oligo pool underwent low-cycle high-fidelity PCR amplification for subsequent insertion into a modified plasmid backbone with BsaI sites flanking the insertion site by Golden Gate assembly. [0751] NGS and bioinformatics analysis. A custom AAV amplicon-sequencing pipeline was developed to process the NGS raw data. Briefly, paired-end reads were merged, followed by Page 171 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) sequential trimming on constant regions allowing maximum 10% error rate. Variable and flanking regions were quality filtered with minimum Phred score of 20. Sequences matching RGDxxxx or xRGDxxx trimer nucleotide sequences are retained for further analysis. Variant fraction in a given AAV or tissue amplicon library is normalized to reads per million (rpm) plus a pseudo count of 0.1. [0752] For Variant i, log-transformed enrichment score = Log2(Vi_tissue * Vaav9_inputAAV / (Vi_inputAAV * Vaav9_tissue)) [0753] False discovery rate (FDR) was calculated by performing student’s t test using log-transformed enrichments scores from all biological and technical replicates, and adjusting p values using Benjamini-Hochberg method. [0754] Results: One thousand nine hundred and ninety one (1991) capsids showing over 5-fold or higher skeletal muscle transduction in cynomolgus macaque compared to AAV9 were identified from Round 2 screening (see sequences in Table 12). [0755] Table 12: VR-VIII peptide insertion sequences for top muscle-targeting capsids with >5 fold enhanced skeletal muscle transduction compared to AAV9. Peptide SEQ ID NO Fold change over AAV9 Page 172 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 17 C Page 173 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 55 A Page 174 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 93 A Page 175 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 131 A Page 176 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 169 B Page 177 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 207 A Page 178 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 245 A Page 179 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 283 A Page 180 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 321 A Page 181 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 359 A Page 182 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 397 A Page 183 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 435 A Page 184 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 473 A Page 185 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 511 A Page 186 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 549 A Page 187 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 587 A Page 188 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 625 A Page 189 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 663 A Page 190 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 701 A Page 191 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 739 A Page 192 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 777 A Page 193 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 815 A Page 194 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 853 A Page 195 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 891 A Page 196 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 929 A Page 197 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 967 A Page 198 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1005 A Page 199 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1043 A Page 200 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1081 A Page 201 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1119 A Page 202 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1157 A Page 203 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1195 B Page 204 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1233 A Page 205 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1271 A Page 206 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1309 A Page 207 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1347 A Page 208 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1385 A Page 209 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1423 A Page 210 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1461 A Page 211 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1499 B Page 212 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1537 B Page 213 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1575 A Page 214 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1613 A Page 215 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1651 A Page 216 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1689 A Page 217 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1727 A Page 218 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1765 A Page 219 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1803 A Page 220 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1841 A Page 221 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1879 A Page 222 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1917 A Page 223 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 1955 A Page 224 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0756] Enrichment scores observed from CBh and MHCK7 promoters were highly concordant, suggesting the bulk of transgene expression was derived from myotubes. Notably, it was observed that RGD tripeptide predominantly resides at the very N-terminus of peptide positions (1951 out of 1991). There were 8 significantly enriched peptide motifs among enhanced variants: RGDxxRW (SEQ ID NO: 2070), RGDYQxx (SEQ ID NO: 2071), RGDHxxW (SEQ ID NO: 2072), RGDPxxW (SEQ ID NO: 2073), RGDYxxV (SEQ ID NO: 2074), RGDxQxW (SEQ ID NO: 2075), RGDYxSx (SEQ ID NO: 2076), and LRGDxQ[W/F] (SEQ ID NO: 2077). In particular, 86.4% of RGDxxRW (SEQ ID NO: 2070) variants showed enhanced muscle transduction (FDR < 0.1) compared to WT AAV9 (FIG.1). [0757] Discussion: A pool of RGD-containing capsid candidates with enhanced skeletal muscle tropism was identified through a combinatorial approach based on rational design and directed evolution. Several distinct peptide motifs emerged among top candidates, suggesting that a comprehensive functional interrogation was achieved within the RGD peptide space. In some embodiments, RGD-containing capsids disclosed herein can be used to target muscle cells for delivery of a payload to muscle cells. [0758] The data presented in this Example demonstrates that RGD-containing capsids have enhanced targeting to a muscle cell as compared to a WT AAV9 capsid. Accordingly, in some embodiments, an AAV capsid protein having an RGD-peptide has increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, an AAV capsid protein having an RGD-peptide can be used to deliver a payload to a muscle cell, e.g., to treat a muscle disorder, or a glycogen or sugar storage disorder. Example 2: Identification of RGD-containing capsids with enhanced skeletal muscle tropism [0759] This Example describes the identification of RGD-containing capsids obtained by screening an AAV library having RGD-containing variants in non-human primates (NHPs). Page 225 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0760] Materials and Methods [0761] Round 3 DNA library construction. The Round 3 library includes 73 capsid variants selected from the Round 2 library. Two capsid variants (RGDPQRW (SEQ ID NO: 802) and RGDYGLH (SEQ ID NO: 1574)) were also included on a liver-detargeted W503R scaffold to evaluate potential for peripheral organ detargeting. In addition, wtAAV8, wtAAV9 and five previously identified myotropic capsids (having SNSRGDYNSL (SEQ ID NO: 2017), STVRGDYTSV (SEQ ID NO: 2019), QERRGDYTSM (SEQ ID NO: 2014), ASTRGDHGVL (SEQ ID NO: 2048), or ENRRGDFNNT (SEQ ID NO: 2049) peptide insertions) were added as controls. The sequence encoding the capsid gene was inserted into a RepCap plasmid backbone for AAV production by adherent triple transfection. The plasmid containing the expression cassette included a ubiquitous CAG promoter-driven H2B-EGFP transgene and a bGH polyadenylation signal. To enable capsid identification, a unique 16 bp barcode was inserted into the 3’ UTR (FIG.2). Each capsid variant was produced independently in a 2-layer Cellstack and packaged with three unique barcodes. [0762] AAV library and single capsid production. Capsid variants in the Round 3 library were individually produced by adherent triple transfection in HEK293T cells. Producer cells were transfected with an Ad helper plasmid, a plasmid supplying the ITR-flanked barcoded transgene cassette, and the RepCap plasmid supplying the capsid coding sequence. Three days following transfection, AAV vectors were harvested and pooled for purification through a discontinuous iodixanol density gradient. The AAV library was then buffer exchanged and concentrated into phosphate-buffered saline (pH 7.4) with 0.001% Pluronic F-68. Genome titer was performed using digital droplet PCR (ddPCR), purity was determined by SDS-PAGE, and endotoxin measurements were obtained using LAL method. [0763] Single capsids were packaged with CAG-mCherry-3xFLAG-hGHpA and produced individually using the same purification method as above. [0764] In vivo library screening. For the Round 3 screening study in cynomolgus macaque, one male and one female animal was administered 6e13 genome copies (GC) of the AAV library followed by 21 days in-life. Several different muscle tissues including gastrocnemius, biceps brachii, quadriceps, soleus, diaphragm, pectoralis major, esophageal Page 226 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) muscles, rectus abdominis and heart were collected for individual analysis. In addition, liver, kidney and lumbar dorsal root ganglia were also collected for analysis of peripheral transduction. [0765] For mouse studies, Round 3 AAV library was administered into adult C57BL/6J and bALB/cJ mice at a dose of 4e11 genome copies (GC) per mouse by tail-vein injection. Necropsies were performed 21 days after injection and major organs were collected for analysis. [0766] For all Round 3 in vivo studies, tissues were homogenized in Buffer RLT and total RNA was isolated using QIAGEN RNeasy Kits. RNA was reverse transcribed, and cDNA was used for PCR amplification of the barcode sequences for NGS analysis. NGS raw data was processed and analyzed as described in Example 1. [0767] Results: The Round 3 library, which included top variants selected from RGD Round 2, was administered into both cynomolgus macaque and mice , allowing for cross-species comparison. In the cynomolgus macaque, a set of variants were found to be enriched over AAV9 across all muscle groups analyzed (FIG.3 and FIG.4). [0768] Twenty-eight (28) capsids showing over 2-fold or higher overall skeletal muscle transduction in cynomolgus macaques compared to AAV9 were identified from Round 3 screening (see sequences in Table 13). All of the peptides provided in Table 13 have a 2-fold or more enhanced skeletal muscle transduction compared to AAV9. The fold change data is presented in categories E, F, G, and H. Peptides in category E have a 2-fold or more enhanced skeletal muscle transduction compared to AAV9, peptides in category F have a more than 3-fold enhanced skeletal muscle transduction compared to AAV9, peptides in category G have a more than 4-fold enhanced skeletal muscle transduction compared to AAV9, and peptides in category H have a more than 5-fold enhanced skeletal muscle transduction compared to AAV9. [0769] Table 13: Skeletal muscle transduction of Round 3 RGD capsid variants relative to AAV9. Peptide SEQ ID NO Fold change over Page 227 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) RGDQDRW 862 F RGDRDGL 978 E [0770] ll 2021). ^ On liver-detargeted W503R backbone. [0771] Comparison of Round 3 library overall muscle transduction in cynomolgus macaque and mice (inbred C57BL/6J and bALB/cJ strains) revealed that the top-performing Round 3 capsids in cynomolgus macaque also achieved enhanced transduction in mouse muscle tissue (FIG.5). [0772] To narrow down the candidate pool for a single capsid validation study, an assessment of production fitness and amenability to existing AAV purification workflows was conducted for ten capsids: RGDYLNT (SEQ ID NO: 1696), RGDYESR (SEQ ID NO: 1556), RGDYERI (SEQ ID NO: 1551), RGDPSPW (SEQ ID NO: 833), RGDSERW (SEQ ID NO: 1088), RGDYLSV (SEQ ID NO: 1713), RGDYREI (SEQ ID NO: 1825), RGDPQRW (SEQ ID Page 228 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) NO: 802), RGDYREV (SEQ ID NO: 1829), and RGDPIRW (SEQ ID NO: 772). Most of the selected capsids produced reasonably well relative to AAV9 based on the pre-purification genome titer; however, following purification, some of the candidates exhibited a decrease in genome titer (FIG.6). The best recovery was achieved with capsid variants RGDYREI (SEQ ID NO: 1825), RGDYERI (SEQ ID NO: 1551), and RGDYREV (SEQ ID NO: 1829). Capsid variants RGDYREI (SEQ ID NO: 1825), RGDYERI (SEQ ID NO: 1551), and RGDYREV (SEQ ID NO: 1829) exhibited enhanced transduction of skeletal muscle tissue based on the RGD Round 3 pooled screening data (FIGs.7A-7J) and were deemed the most promising based on muscle transduction and production fitness evaluation. [0773] Discussion: A pool of RGD-containing capsid candidates with enhanced skeletal muscle tropism was identified through a combinatorial approach based on rational design and directed evolution. Several distinct peptide motifs emerged among top candidates, suggesting that a comprehensive functional interrogation was achieved within the RGD peptide space. In some embodiments, RGD-containing capsids disclosed herein can be used to target muscle cells for delivery of a payload to muscle cells. Example 3: Characterization of RGD-containing capsids with enhanced skeletal muscle tropism. [0774] This Example describes the characterization of RGD-containing capsids obtained by screening an AAV library having RGD-containing variants in non-human primates (NHPs), as described in Examples 1 and 2. [0775] Materials and Methods [0776] Vector production. AAV particles having a wildtype AAV9 capsid and AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid packaged with a CAGG-driven FLAG- tagged mCherry transgene were produced through suspension transient transfection of HEK293T cells. Crude lysates were harvested 3 days post-transfection and purified using POROS CaptureSelect AAV9 affinity resin. AAV preparations were then concentrated and buffer Page 229 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) exchanged into phosphate-buffered saline (pH 7.4) with 0.001% Poloxamer 188 by tangential flow filtration (TFF). Genome titer quantification was performed using digital droplet PCR (ddPCR). Purity was assessed by SDS-PAGE followed by SYPRO Ruby staining, and endotoxin was measured using a limulus amebocyte lysate (LAL) assay. [0777] Non-human primates studies. Six adult cynomolgus macaques of mixed sexes were included in the NHP study. Animals were orally dosed daily with immunosuppressants composed of either prednisolone (3 mg/kg) alone or dexamethasone (0.5 mg/kg) plus Tacrolimus (1 mg/kg), from 2 days prior to AAV dosing to 20 days post-AAV injection. AAV vectors (also referred to herein as AAV particles) were administered through a single intravenous bolus injection at 5x1013 vg/kg. Animals were euthanized 21 days post-injection, and tissues were collected for analysis. [0778] Mice studies. Male C57Bl/6 mice were obtained from Charles River Laboratories. B6 albino mice (JAX000058) were purchased from Jackson Laboratory.5-week-old C57Bl/6 were treated via tail vein intravenous (i.v.) injection with AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid, AAV particles having a RGDYERI (SEQ ID NO: 1551) capsid or AAV particles having a wildtype AAV9 capsid, which were all packaged with a CAG-mCherry- 3xFLAG-hGHpA reporter, at the indicated doses (low dose or high dose) or PBS (control group) (n=4 mice per group). Mouse tissues were harvested 4 weeks post AAV injection and were processed for FFPE or snap-frozen in liquid nitrogen for mRNA analysis. Mice were housed on a 12-h light/dark cycle and given food and water ad libitum. [0779] In vivo bioluminescence imaging studies. B6 albino mice were injected intravenously (IV) with 5E12 vg/kg of AAV capsids packaged with a CBA-SV40 intron- mCherry-GT2A-Fluc2-WPRE-hGHpA reporter at 8 weeks old, followed by bioluminescence imaging (BLI) sessions using an IVIS SpectrumCT In Vivo Imaging System (PerkinElmer) at day 14 post dose. Mice were injected intraperitoneally (IP) with 150 mg/kg of d-luciferin at 0 minutes, placed in an induction chamber containing 2.5% isoflurane at 2 minutes, and positioned in nose cones on the IVIS imaging platform at 6 minutes. An image was captured at 10 minutes for analysis. Total radiance was measured from same size of region of interest (ROI) across animals, using Living Image software (version 4.7.2, PerkinElmer). Page 230 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0780] Vector genome biodistribution and transgene mRNA quantification. NHP tissues were homogenized in Qiagen Buffer RLT Plus using Genogrinder. Tissue homogenates were aliquoted for DNA extraction using Qiagen DNeasy kit, and RNA extraction using TRIzol LS reagent following manufacturer’s protocol. Vector genome biodistribution was performed via qPCR using primer-probe sets against the WPRE element in the vector genome and the endogenous RPP30 gene. Transgene mRNA quantification was performed via RT-qPCR, using primer-probe sets against the WPRE element, as well as the endogenous GAPDH as control. Transgene mRNA level was normalized against the endogenous GAPDH mRNA level per sample for cross-sample comparison. [0781] Immunohistochemistry studies. Tissues were drop-fixed in 10% neutral-buffer formalin for 2 days, washed in PBS, and paraffin blocked. For immunohistochemical (IHC) staining of mCherry, staining was conducted on the Leica Bond RXm platform using standard chromogenic methods. For antigen retrieval (HIER), slides were heated in a pH9 EDTA-based buffer for 25 minutes at 94C, followed by a 30-minute antibody incubation (1:6000, Abcam ab167453). Antibody binding was detected using an HRP-conjugated secondary polymer (Biocare, MACH 2 goat anti-rabbit, Ref# RHRP520MM, ready to use at room temperature), followed by chromogenic visualization with diaminobenzidine (DAB). A hematoxylin counterstain was used to visualize nuclei. [0782] In vitro validation in 2D human skeletal muscle cells. For 2D culture of human muscle cells, human primary myoblasts and immortalized human myoblasts derived from vastus lateralis muscles were used. Three independent primary myoblast lines were used: hSkMDC_con1, SK-1111-P01431-18F; hSkMDC_con2, SK-1111-P03005-49F; hSkMDC_con3 SK-1111-P01547-29M. Additionally, one human immortalized myoblast line (AB1190, ImmSkMDC) was used that was obtained from Institut de Myologie. Primary myoblasts were grown in growth medium (Cook Myosite Myotonic Basal Medium and growth supplement cat # MB-2222 and MS-8888, with 20% HI FBS (Hyclone cat# SH30070.01HI), bFGF 5ng/ml (Invitrogen cat# 13256-029) and insulin 10 µg/ml (Sigma Aldrich cat# I9278-5ML)). Immortalized myoblasts were grown in growth medium (PromoCell cat # C-39360 with 15% HI FBS (Hyclone cat# SH30070.01HI)). Differentiation to myotubes was performed by plating primary myoblasts at high confluency 40,000-50,000 cells/cm2 and replacing growth medium Page 231 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) after 24hr to differentiation medium (Cook Myosite Myotonic serum free differentiation medium, cat# MD-9999 with 2% horse serum (Invitrogen) and 1% FBS). On day 6 of differentiation, cells were transduced with AAV packaged with a CAG-mCherry-3xFLAG- hGHpA or a CBA-SV40 intron-mCherry-GT2A-Fluc2-WPRE-hGHpA reporter at the indicated doses or PBS (control). After 24hr, medium with AAVs was replaced with fresh differentiation medium. Cells were placed in an Incucyte SX5 Live Cell Analysis System (Sartorius), that imaged each well at 20X every 4 hours for 5 days. Analysis of mCherry positive cells was performed using the Incucyte Software to identify the mCherry integrated intensity per well. For quantification of luciferase activity, the Pierce Firefly Luc One-Step Glow Assay Kit was used in accordance with manufacturer’s guidelines at day 5 post-transduction (Thermo Scientific cat# 16197) [0783] In vitro validation in 3D human myobundles. For 3D culture of human muscle cells, human immortalized myoblasts (AB1190) obtained from Institut de Myologie were used. Cells were grown in growth medium (PromoCell cat # C-39360 with 15% HI FBS (Hyclone cat# SH30070.01HI)).3D myobundles were established using CuriBio’s Mantarray casting platform. Briefly, 750,000 dissociated myoblasts were resuspended in growth medium (PromoCell cat # C- 39360 with 5% HI FBS (Gibco cat# 10082-147)) supplemented with Matrigel (Corning cat# 356231, 33.3%), Fibrinogen (Sigma cat# F8630-1G, 50 mg/mL) and Thrombin (Sigma cat# T4648-1KU, 100 U/mL) to create a total seeding volume of 150 µl. This cell suspension was applied to Mantarray casting wells with the two-post array and incubated at 37°C, 5% CO2 for 90 min to facilitate hydrogel formation. Following incubation, 1 mL of growth medium, with 5% HI FBS, was added to the casting wells to aid separation of the gel from the substrate. The two- post array supporting the newly formed hydrogels was then removed from the casting plate and transferred to a fresh 24-well plate (Greiner cat# M8812) containing 2 mL of Lonza growth medium (Lonza cat# CC-3245) supplemented with 2.5 g/L 6-aminocaproic acid (Sigma cat# A2504-100G) to inhibit fibrinolysis. One day after plating, myobundles were transferred to 3D Primary Skeletal Muscle Differentiation Media (CuriBio cat# SKM-MED-PRI-D) for 7 days to induce myoblast fusion. On day 7 of differentiation, cells were transduced with AAV packaged with a CAG-mCherry-3xFLAG-hGHpA reporter at the indicated doses (MOI 1E5 vg/cell) or PBS (control) in 3D Skeletal Muscle Maintenance Medium (CuriBio cat# SKM-MED-IPS-M). Page 232 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) After 48hr, medium with AAVs was replaced with fresh maintenance medium. Seven days post- infection, myobundles were embedded in frozen in OCT and snap frozen in isopentane pre- cooled in liquid nitrogen.10 µm cryosections from each condition were arranged onto the same coverslip to reduce batch effects from staining and imaging. Air dried sections were blocked in PBS/3% BSA for 1 hr at room temperature and immunostained using antibodies to α-Actinin (Cell Signaling Technology #3134, 1:50), MyHC4 (Thermo Fisher Scientific #14-6503-82, 1:500), mCherry (Cell Signaling Technologies, cat#43590) diluted in PBS/3% BSA overnight at 4°C. Secondary antibodies (Jackson ImmunoResearch Laboratories, 1:500) were diluted in PBS/3% BSA were applied for 1 hr at room temperature and then nuclei were counterstained with DAPI. Images were acquired using an EVOS M5000 Imaging system (Thermo Fisher Scientific) with 4x and 10x objectives and individual fields were stitched and analyzed using the Evos Image Analysis application. [0784] RNA isolation and qRT-PCR analysis. Trizol-based RNA isolation was performed for skeletal muscles (diaphragm, gastrocnemius, triceps, tibialis anterior, soleus), heart, liver, lung, kidney, and brain. RNA was reversed transcribed, and cDNA was used for qRT-PCR analysis of mCherry levels in tissues. qRT-PCR was performed using TaqMan Assays (Applied Biosystems) in a QuantStudio 12k Flex Real-Time PCR System (Thermo Fisher Scientific). To quantify relative transcript levels, 2−ΔΔCt was used to compare samples to GAPDH as a housekeeping gene and then to the AAV9-CAG-mCherry group. For Taqman based qRT-PCR, the following primer/probe sequences were used: mCherry fw: 5'- TTGGACATCACCTCCCACAAC-3' (SEQ ID NO: 2060); mCherry rev: 5'- CCTCGGCGCGTTCGTA-3’(SEQ ID NO: 2061); mCherry probe: 5'-6FAM- ACTACACCATCGTGGAAC-MGB/NFQ-3' (SEQ ID NO: 2062); mouse GAPDH endogenous control FAM/MGB probe (Mm99999915_g1; Applied Biosystems), NHP GAPDH FAM/MGB probe (Mf04392546_g1; Applied Biosystems). SEQ ID NO: 2062 is an oligonucleotide probe used in TaqMan real-time PCR for mCherry transgene mRNA detection. It has a 6- carboxyfluorescein (6-FAM) conjugated at the 5’-end, and a nonfluorescent quencher-minor groove binder (NFQ-MGB) conjugated at the 3’-end. [0785] Results: Top capsid variants showed enhanced transduction in human myotubes and myobundles: To confirm that capsid variants RGDYREI (SEQ ID NO: 1825) and Page 233 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) RGDYERI (SEQ ID NO: 1551) were capable of transducing human myotubes, individually produced RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variant AAVs packaged with a mCherry fluorescent reporter transgene were added to primary human myotubes in culture. RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variants substantially outperformed AAV9 based on mCherry fluorescence (FIGS.8A-8C and FIGS.9A-D). [0786] Additionally, capsid variant RGDYREV (SEQ ID NO: 1829), packaged with a dual reporter containing mCherry and luciferase, was tested on immortalized human myoblasts differentiated to myotubes. Capsid variant RGDYREV (SEQ ID NO: 1829) also outperformed AAV9 based on mCherry fluorescence and luciferase activity (FIGS.10B-10C). Furthermore, transduction of capsids variants was assessed in contractile human muscle tissues (myobundles). These engineered muscle tissues can respond to electrical and chemical signals to contract, and are more mature than 2D cultures resembling human muscle tissues better. Human myobundles treated with capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) showed enhanced expression of mCherry fluorescent reporter as shown by immunofluorescence analysis of myobundle cross-sections 7 days post-treatment compared to AAV9 (FIGS.11A- 11F). [0787] Top capsid variants showed enhanced transduction in mouse muscles following intravenous administration. Additionally, the ability of AAV particles having capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) to transduce mouse quadriceps muscles was tested. Transduction levels were evaluated by observing reporter gene (mCherry) expression: red coloration of the muscle indicated expression of mCherry with darker red associated with higher mCherry expression. The data shows that AAV particles having capsid variants RGDYREI and RGDYERI transduced mouse quadriceps muscles more efficiently than AAV9 (FIGS.12A-12C). Analysis using mCherry IHC of tissue sections also showed enhanced transduction levels in myofibers and myonuclei of skeletal muscles (gastrocnemius and diaphragm) and heart of AAV particles having capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) compared to AAV9 (FIGS.13A-13C). [0788] Furthermore, mCherry expression was analyzed at 4 weeks post-treatment by qPCR in skeletal muscle tissues, heart, lung, kidney, brain and liver of C57Bl/6 mice treated with Page 234 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) AAVs containing CAG promoter driven mCherry. Two doses of virus were used for AAV9 (low dose and high dose) and one low dose was used for RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551). As shown in FIGS.14A-14J, mCherry expression was comparable or higher in skeletal muscles of mice treated with RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsids at 5X lower dose compared to AAV9 (high dose). Biodistribution analysis showed that AAV particles having capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) delivered vector genomes to skeletal muscles (Gastrocnemius, Tibialis anterior and diaphragm), with no significant enhancement over AAV9 (FIGS.15A-15F). Without wishing to be bound by any particular theory, in some embodiments, vector genome distribution may not be directly correlated with AAV transduction (e.g., as measured by payload expression). For example, see Martino RA et al., “Vector Affinity and Receptor Distribution Define Tissue-Specific Targeting in an Engineered AAV Capsid.” J Virol 97:e00174-23 (2023) which is incorporated herein by reference in its entirety. [0789] To evaluate muscle transduction for AAV particle having capsid variant RGDYREV (SEQ ID NO: 1829) in vivo, B6 albino mice were i.v. dosed at 5E12 vg/kg with AAV particles having a wildtype AAV9 capsid or capsid variant RGDYREV (SEQ ID NO: 1829), packaged with a mCherry-luciferase dual reporter. Bioluminescence imaging (BLI) was performed longitudinally for 14 days to monitor luciferase expression. At days 7 and 14 post- injection, mice injected with AAV particles having capsid variant RGDYREV (SEQ ID NO: 1829) showed higher BLI signal in their limbs compared to mice injected with AAV particles having a wildtype AAV9 capsid (FIGS.16A-16D). [0790] Capsid with RGDYREI insertion mediated enhanced muscle transduction in cynomolgus macaques following intravenous administration. AAV capsid variant RGDYREI (SEQ ID NO: 1825) was selected for further validation in NHP. AAV particles having a wildtype AAV9 capsid and AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid were packaged with a CAGG-driven FLAG-tagged mCherry transgene, and the AAV particles were intravenously dosed at 5x10E13 vg/kg to three adult cynomolgus macaques. Dexamethasone in combination with Tacrolimus was orally administered daily to test animals to dampen immune responses. Page 235 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) [0791] AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid were well tolerated with minimal safety or pathology findings. Vector genome biodistribution and transgene mRNA expression were analyzed in muscle and liver by qPCR and RT-qPCR, respectively (FIG.17 and FIGS.18A-18B). Aside from NHP #2, AAV particles having a RGDYREI (SEQ ID NO: 1825) capsid showed similar vector genome biodistribution to that of AAV particles having a wildtype AAV9 capsid in all tested muscles, which is consistent with observations in mice. Although there was no increase in vector genome biodistribution to muscle, AAV particles displaying a RGDYREI (SEQ ID NO: 1825) peptide mediated about 3- fold to about 36- fold enhanced transgene mRNA expression over AAV9 in various muscle tissues including rectus femoris, biceps, triceps, gastrocnemius, and tibialis anterior in NHPs #1 and #3 (FIGS.18A-18B). IHC revealed robust mCherry expression in multiple muscle tissues in NHPs #1 and #3, including rectus femoris, gastrocnemius, and the upper esophageal muscle (FIGS.19A-19C, FIGS.20A-20C, and FIGS.21A-21C) as compared to tissues from NHPs administered AAV particles having a wildtype AAV9 capsid (FIGS.22A-22C). [0792] Without wishing to be bound to any particular theory, NHP #2 may have had neutralizing antibodies against AAV9 which could have affected transduction with AAV9 particles displaying RGD-containing peptides. [0793] Discussion: The data presented in this Example demonstrates that RGD- containing capsids, e.g., capsid variants RGDYREI (SEQ ID NO: 1825), RGDYERI (SEQ ID NO: 1551), and RGDYREV (SEQ ID NO: 1829) have enhanced targeting to a muscle cell as compared to a WT AAV9 capsid protein. Accordingly, in some embodiments, an AAV capsid protein having an RGD-peptide disclosed herein has increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. In some embodiments, an AAV capsid protein having an RGD-peptide disclosed herein can be used to deliver a payload to a muscle cell, e.g., to treat a muscle disorder, or a glycogen or sugar storage disorder. [0794] Future experiments to further characterize variant AAV capsid proteins disclosed herein may include evaluation of anti-AAV neutralizing antibodies (e.g., titers of anti-AAV neutralizing antibodies such as titers of anti-AAV9 neutralizing antibodies) in subjects administered AAV particles comprising a variant AAV capsid protein disclosed herein (e.g., Page 236 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) prior to or after administration of AAV particles). In some embodiments, such studies may be useful in evaluating vector distribution and/or payload expression, e.g., in samples from subject administered AAV particles comprising a variant AAV capsid protein disclosed herein. Page 237 of 248 11821383v1

Claims

Attorney Docket No.: 2011256-1804 (P1811WO01) CLAIMS We claim: 1. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of: (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. Page 238 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 2. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence provided in Table 1; and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. 3. The rAAV particle of claim 1 or 2, wherein the insertion site is located between two adjacent amino acids in the variable region of the parental AAV capsid protein. 4. The rAAV particle of any one of the preceding claims, wherein the insertion of the heterologous peptide replaces a contiguous stretch of amino acids of the parental AAV capsid protein. 5. The rAAV particle of any one of the preceding claims, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein. 6. The rAAV particle of any one of the preceding claims, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein. 7. The rAAV particle of claim 6, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII of AAV9 comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. 8. The rAAV particle of claim 6 or 7, wherein the insertion site is located between amino acids 588 and 589 of VP1 of an AAV9 capsid protein or the corresponding position in the VP1, VP2 and/or VP3 of another parental AAV capsid protein. Page 239 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 9. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. 10. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the sequence of SEQ ID NO: 1551, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. 11. The rAAV particle of any one of claims 1-9, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the sequence of SEQ ID NO: 1825, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. 12. The rAAV particle of any one of claims 1-9, wherein the variant AAV capsid protein comprises: (1) a peptide insertion comprising the sequence of SEQ ID NO: 1829, and (2) one or more sequences of a VP (e.g., VP1, VP2, and/or VP3) of the AAV9 capsid protein. 13. The rAAV particle of any one of claims 1-9, wherein the peptide insertion comprises the consensus sequence of SEQ ID NO: 10. 14. The rAAV particle of claim 13, wherein the peptide insertion comprises a sequence provided in Table 11. 15. The rAAV particle of claim 13 or 14, wherein the peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551). 16. The rAAV particle of claim 13 or 14, wherein the peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825). Page 240 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 17. The rAAV particle of claim 13 or 14, wherein the peptide insertion comprises the sequence of RGDYREV (SEQ ID NO: 1829). 18. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein confers at least 5-fold increased infectivity and/or transduction of a muscle cell compared to the infectivity and/or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein. 19. The rAAV particle of any one of the preceding claims, wherein the muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof. 20. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site. 21. The rAAV particle of claim 20, wherein the one or more modifications are within about 5 to 10 amino acids upstream or downstream of the location of the peptide insertion site. 22. The rAAV particle of any one of the preceding claims wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. 23. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein has at least 90% identity relative to a parental AAV capsid protein. 24. The rAAV particle of claim 23, wherein percent identity is determined by comparing the sequence of the variant AAV capsid protein without the peptide insertion, with the parental AAV capsid protein. Page 241 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 25. The rAAV particle of claim 24, wherein the variant AAV capsid protein and the parental AAV capsid protein have 100% identity when: (a) the peptide insertion in the variant AAV capsid protein is not taken into account in the sequence comparison; and (b) the variant AAV capsid protein does not have one or more modifications other than the peptide insertion. 26. The rAAV particle of claim 24, wherein the variant AAV capsid protein and the parental AAV capsid protein have less than 100% identity when: (a) the peptide insertion in the variant AAV capsid protein is not taken into account in the sequence comparison; and (b) the variant AAV capsid protein comprises one or more modifications other than the peptide insertion. 27. The rAAV particle of any one of the preceding claims, wherein the parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001. 28. The rAAV particle of any one of the preceding claims, wherein the payload is a polypeptide. 29. The rAAV particle of claim 28, wherein the polypeptide is or comprises: (i) a CRISPR-Cas protein or a variant or a fragment thereof; (ii) a Zinc finger protein, or a variant or a fragment thereof; (iii) a TAL, or a variant or a fragment thereof; (iv) a base editor, or a variant or a fragment thereof; (v) a prime editor, or a variant or a fragment thereof; and/or (vi) a meganuclease, or a variant or a fragment thereof. 30. The rAAV particle of claim 29, wherein the polypeptide is associated with a muscle disorder, or a glycogen or sugar storage disorder, optionally, wherein the muscle disorder is Page 242 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. 31. The rAAV particle of claim 28, wherein the polypeptide is an enzyme or an antibody. 32. The rAAV particle of any one claims 1-27, wherein the payload is an RNA molecule. 33. The rAAV particle of claim 32, wherein the RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a muscle disorder, or a glycogen or sugar storage disorder, optionally, wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. 34. The rAAV particle of any one claims 1-27, wherein the payload is a DNA molecule. 35. The rAAV particle of any one of the preceding claims, wherein the nucleic acid sequence encoding a payload comprises a promoter. 36. The rAAV particle of claim 35, wherein the promoter is or comprises a muscle-specific promoter, or a dual muscle-liver promoter. 37. A pharmaceutical composition comprising: (a) a rAAV particle of any one of the preceding claims; and (b) a pharmaceutically acceptable excipient. Page 243 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) 38. A method of delivering a payload to a muscle cell, comprising administering the pharmaceutical composition of claim 37 to the muscle cell. 39. The method of claim 38, wherein the muscle cell is from a subject that has, or has been determined to have, a muscle disorder. 40. A method of treating a subject having a muscle disorder and/or ameliorating a symptom of a muscle disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 37. 41. The method of claim 39 or 40, wherein the muscle disorder is chosen from: X-linked myotobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, a Limb-Girdle muscular dystrophy, Emery Dreifuss muscular dystrophy, Facioscapulohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, Nemaline Myopathy, Pompe Disease, or Myotonic Dystrophy. 42. The method of any one of claims 38-41, wherein the pharmaceutical composition is administered via a route of administration chosen from: intramuscular, intra-cisterna magna, intravenous, intraarterial, intracoronoary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, or limb perfusion. 43. The method of any one of claims 40-42, wherein the subject is a human. 44. An isolated cell transduced with the rAAV particle of any one of claims 1-36. 45. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) the peptide insertion comprises a sequence of: Page 244 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein. 46. An isolated cell comprising the nucleic acid of claim 45. 47. A variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, and wherein: (i) the peptide insertion comprises a sequence of: (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; Page 245 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein. 48. The variant AAV capsid protein of claim 47, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein. 49. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; Page 246 of 248 11821383v1 Attorney Docket No.: 2011256-1804 (P1811WO01) (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3 is V or I. 50. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence provided in Table 1. Page 247 of 248 11821383v1
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