Rgd-containing peptides for delivering payloads

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 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 X3is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1and X2are independently any amino acid, and X3is Y, W, or F; (VI) RGDX1QX2X3(SEQ ID NO: 6), wherein X1and X2are 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 X1and X2are independently any amino acid, and X3is 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 11821383v1Attorney 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 X1and X2are independently any amino acid and X3is 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 X3is 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, X3is Y. In some embodiments, X3is W. In some embodiments, X3is F. Page 3 of 248 11821383v1Attorney 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 X3is 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 11821383v1Attorney 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 X3is 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, X3is Y. In some embodiments, X3is W. In some embodiments, X3is 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, X3is Y. In some embodiments, X3is W. In some embodiments, X3is 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) comprising a nucleotide sequence encoding a payload. In some embodiments, X3is 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 X3is 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 11821383v1Attorney 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 X3is 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, X3is Y. In some embodiments, X3is W. In some embodiments, X3is 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 X1and X2are independently any amino acid, and X3is 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, X3is 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 11821383v1Attorney 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 X1and X2are 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, X3is 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 11821383v1Attorney 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 X3is 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, X3is Y. In some embodiments, X3is W. In some embodiments, X3is 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, X3is W. In some embodiments, X3is 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, X3is W. In some embodiments, X3is F. Page 9 of 248 11821383v1Attorney 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 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 X3is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1and X2are independently any amino acid, and X3is 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 X3is Y, W, or F; or (VIII) LRGDX1X2X3(SEQ ID NO: 8), wherein X1and X2are 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, X2is E or R, and X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 X3is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein X1and X2are 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 X1and X2are independently any amino acid, and X3is Y, W, or F; or Page 13 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) (VIII) LRGDX1X2X3(SEQ ID NO: 8), wherein X1and X2are 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 X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 log2fold-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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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) / cm2of culture medium or vc / mL. Page 31 of 248 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 AAV9Page 61 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RDHA173 CPage 62 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RDRD L978 BPage 63 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RDYI1667 BPage 64 of 248 11821383v1Attorney 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 X3is 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 AAV9Page 65 of 248 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) NO: 2027), wherein X1and X2are 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 AAV9Page 67 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RDY H1795 APage 68 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RD M1452 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 X3is 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 X1and X2are 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3is 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 X1and X2are 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 X1and X2are 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 X1and X2are 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 AAV9Page 70 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9Page 71 of 248 11821383v1Attorney 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 X1and X2are Page 72 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3is 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 X1and X2are 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 X3is 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 X1and X2are independently any amino acid, and X3is 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3is 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 X1and X2are 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 X1and X2are 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 X1and X2are 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 AAV9Page 74 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9Page 75 of 248 11821383v1Attorney 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 X3is 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 AAV9Page 76 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYERV1554 BPage 77 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYSIV1856 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 X1and X2are 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDAQRW61 APage 79 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDFQSY125 APage 80 of 248 11821383v1Attorney 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 X3is 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 X1and X2are independently any amino acid, and X3is 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) Peptide SEQ ID NO Fold change over AAV9 RGDYASH 1518 APage 82 of 248 11821383v1Attorney 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3is 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 X1and X2are 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 X3is 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 X1and X2are independently any amino acid, and X3is 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3is 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 X1and X2are 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 X3is 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 X1and X2are independently any amino acid, and X3is 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 X1and X2are 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) independently any amino acid, and X3is 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 X1and X2are 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 X3is 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 X1and X2are independently any amino acid, and X3is 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 X1and X2are 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 11821383v1Attorney 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 X1and X2are 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 X1and X2are 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 disclosedherein comprises a sequence of RGDX1GLX2(SEQ ID NO: 9), wherein X1is Y, W, or F, and X2is 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 AAV9Page 87 of 248 11821383v1Attorney 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. PeptideSEQ IDNO Fold change over AAV9

[0352] In rotein disclosedherein 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 11821383v1Attorney 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 X1is E or R, X2is 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 X1is E or R, X2is 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 X1is E or R, X2is 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 X1is E or R, X2is E or R, and X3is 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 11821383v1Attorney 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 X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 X1and X2are independently any amino acid and X3is Y, W, or F; (2) a peptide Page 94 of 248 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 X1and X2are independently any amino acid, and X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 X1is E or R, and X2is 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 104viable cells (vc) / cm2, e.g., at a density of about 1.0 x 104vc / cm2to about 2.0 x 104vc / cm2, e.g., about 1.0 x 104vc / cm2, about 1.1 x 104vc / cm2, about 1.2 x 104vc / cm2, about 1.3 x 104vc / cm2, about 1.4 x 104vc / cm2, about 1.5 x 104vc / cm2, about 1.6 x 104vc / cm2, about 1.7 x 104vc / cm2, about 1.8 x 104vc / cm2, about 1.9 x 104vc / cm2, or about 2.0 x 104vc / 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 106vc / cm2+ / - 15%, e.g., at a density of 1.0 x 106vc / cm2+ / - 15% to 2.0 x 106vc / cm2+ / - 15%, e.g., 1.0 x 106vc / cm2+ / - 15%, 1.1 x 106vc / cm2+ / - 15%, 1.2 x 106vc / cm2+ / - 15%, 1.3 x 106vc / cm2+ / - 15%, 1.4 x 106vc / cm2+ / - 15%, 1.5 x 106vc / cm2+ / - 15%, 1.6 x 106vc / cm2+ / - 15%, 1.7 x 106vc / cm2+ / - 15%, 1.8 x 106vc / cm2+ / - 15%, 1.9 x 106vc / cm2+ / - 15%, or 2.0 x 106vc / 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 ...

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 X1and X2are independently any amino acid, and X3is 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 X1and X2are 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 X3is 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 X1and X2are independently any amino acid, and X3is 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 X1is Y, W, or F, and X2is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein X1 is E or R, X2 is E or R, and X3is 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) (I) RGDX1X2RX3(SEQ ID NO: 1), wherein X1and X2are 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 X3is 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 X1and X2are 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 X3is Y, W, or F; (VI) RGDX1QX2X3(SEQ ID NO: 6), wherein X1and X2are 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 X3is 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 X1is Y, W, or F, and X2is 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 X3is Y, W, or F; Page 245 of 248 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) (II) RGDX3QX1X2(SEQ ID NO: 2), wherein X1and X2are 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 X3is 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 X1and X2are 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 X3is Y, W, or F; (VII) RGDX3X1SX2(SEQ ID NO: 7), wherein X1and X2are 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 X3is 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 X1is E or R, X2is E or R, and X3is 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 11821383v1Attorney Docket No.: 2011256-1804 (P1811WO01) (II) RGDX3QX1X2(SEQ ID NO: 2), wherein X1and X2are 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 X3is 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 X1and X2are 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 X3is Y, W, or F; (VII) RGDX3X1SX2(SEQ ID NO: 7), wherein X1and X2are 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 X3is 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 X1is E or R, X2is E or R, and X3is 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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