RGD-containing peptides for delivering payloads

Abstract

The present disclosure relates to muscle-targeting moieties comprising RGD-containing peptides, where the peptides can be inserted into the capsid of recombinant adeno-associated viral (rAAV) vectors. Also disclosed herein are compositions comprising the muscle-targeting moieties disclosed herein, as well as methods for making and using the same. For example, the present disclosure identifies a significant challenge: the lack of targeting moieties that can specifically deliver a payload to target cells or tissues and reduce non-specific delivery to other cells or tissues. Another challenge identified by the present disclosure is the low efficacy associated with delivery vectors, such as recombinant adeno-associated viral vectors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 446,649, filed February 17, 2023, and U.S. Provisional Patent Application No. 63 / 547,663, filed November 7, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated herein by reference in its entirety. The Sequence Listing, created on February 14, 2024, is named 2011256-1804.xml and is 1,834,072 bytes in size. [Background technology]

[0003] Targeted delivery of payloads to cells or tissues (eg, using recombinant adeno-associated viruses) to treat and / or prevent disease remains a challenge. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure identifies certain challenges associated with existing targeted delivery of payloads. For example, the present disclosure identifies a significant challenge: the lack of a targeting moiety that can specifically deliver a payload to a target cell or tissue and reduce non-specific delivery to other cells or tissues. Another challenge identified by the present disclosure is the low efficacy associated with delivery vectors, such as recombinant adeno-associated virus vectors. In some embodiments, improving the efficacy of delivery vectors can be beneficial in achieving clinically relevant outcomes.

[0005] In particular, the present disclosure provides techniques that can address certain limitations identified in existing targeted delivery of payloads. The techniques provided herein are particularly useful for specifically delivering a payload to a target cell or tissue. In some embodiments, by specifically delivering a payload to a target cell or tissue, the techniques provided herein can also increase the efficacy of the payload. The techniques provided herein are useful for delivering a payload to a target cell or tissue, for example, a muscle cell or tissue.

[0006] The present disclosure encompasses targeting moieties comprising peptides containing 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, the targeting moiety, e.g., a muscle-targeting moiety, in a variant AAV capsid is also referred to as a "peptide insert." In some embodiments, for example, the targeting moiety, e.g., a muscle-targeting moiety, in a variant AAV capsid provides muscle cell tropism. In some embodiments, rAAV particles comprising variant capsids with peptide inserts disclosed herein bind to and / or recognize targets on cells, e.g., muscle cells. Also disclosed herein are compositions comprising rAAV particles and uses thereof.

[0007] Disclosed herein are recombinant adeno-associated virus (rAAV) particles comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion is selected from the group consisting of: (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F; X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (III) RGDHX1X2X3 (SEQ ID NO: 3) (wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (IV) RGDX1X2X3 (SEQ ID NO: 4) (wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (V) RGDX3X1X2V (SEQ ID NO: 5) (wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (VI) RGDX1QX2X3 (SEQ ID NO: 6) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), (VII) RGDX3X1SX2 (SEQ ID NO: 7) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), or (VIII) LRGDX1X2X3 (SEQ ID NO: 8) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), (IX) RGDX1GLX2 (SEQ ID NO: 9) (wherein X1 is Y, W, or F, and X2 is any amino acid), or (X)RGDYX1X2X3 (SEQ ID NO: 10) (wherein X1 is E or R, X2 is E or R, and X3 is V or I), (ii) a variant AAV capsid protein as described above, wherein the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a nucleotide sequence encoding a payload.

[0008] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDX1X2RX3 (SEQ ID NO: 1), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0009] In some embodiments, the peptide insert comprises a sequence presented in Table 2. In some embodiments, the peptide insert comprises the sequence RGDQPRW (SEQ ID NO: 802).

[0010] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDX1X2RX3 (SEQ ID NO: 1), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0011] In some embodiments, the peptide insert consists of a sequence presented in Table 2. In some embodiments, the peptide insert consists of the sequence RGDPQRW (SEQ ID NO: 802).

[0012] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0013] In some embodiments, the peptide insert comprises a sequence presented in Table 3. In some embodiments, the peptide insert does not comprise the sequence of RGDYQAV (SEQ ID NO: 1764). In some embodiments, the peptide insert does not comprise the sequence of RGDYQTL (SEQ ID NO: 1814). In some embodiments, the peptide insert does not comprise RGDYQEL (SEQ ID NO: 2008).

[0014] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent 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, the peptide insert consists of a sequence presented in Table 3.

[0016] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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, the peptide insert comprises a sequence presented in Table 4. In some embodiments, the peptide insert does not comprise the sequence of RGDHASW (SEQ ID NO: 191). In some embodiments, the peptide insert does not comprise the sequence of RGDHSGW (SEQ ID NO: 322). In some embodiments, the peptide insert does not comprise the sequence of RGDHSTW (SEQ ID NO: 333). In some embodiments, the peptide insert does not comprise the sequence of RGDHTQW (SEQ ID NO: 349). In some embodiments, the peptide insert does not comprise the sequence of RGDHQNF (SEQ ID NO: 283).

[0018] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0019] In some embodiments, the peptide insert consists of a sequence presented in Table 4.

[0020] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDPX1X2X3 (SEQ ID NO: 4), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein); and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0021] In some embodiments, the peptide insert comprises a sequence provided in Table 5. In some embodiments, the peptide insert comprises the sequence RGDPQRW (SEQ ID NO: 802).

[0022] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDPX1X2X3 (SEQ ID NO: 4), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0023] In some embodiments, the peptide insert consists of a sequence presented in Table 5. In some embodiments, the peptide insert consists of the sequence RGDPQRW (SEQ ID NO: 802).

[0024] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDX3X1X2V (SEQ ID NO: 5), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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, the peptide insert comprises a sequence presented in Table 6.

[0026] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDX3X1X2 (SEQ ID NO: 5), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent 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, the peptide insert consists of a sequence presented in Table 6.

[0028] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0029] In some embodiments, the peptide insert comprises a sequence presented in Table 7. In some embodiments, the peptide insert comprises the sequence RGDPQRW (SEQ ID NO: 802).

[0030] In some embodiments, the peptide insert does not include the sequence of RGDVQNF (SEQ ID NO: 2011). In some embodiments, the peptide insert does not include the sequence of RGDHQNF (SEQ ID NO: 283).

[0031] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0032] In some embodiments, the peptide insert consists of a sequence presented in Table 7. In some embodiments, the peptide insert consists of the sequence RGDPQRW (SEQ ID NO: 802).

[0033] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein); and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0034] In some embodiments, the peptide insert comprises a sequence presented in Table 8. In some embodiments, the peptide insert does not comprise the sequence of RGDYVSV (SEQ ID NO: 1948). In some embodiments, the peptide insert does not comprise the sequence of RGDYSSV (SEQ ID NO: 1882). In some embodiments, the peptide insert does not comprise the sequence of RGDYHSF (SEQ ID NO: 1623). In some embodiments, the peptide insert does not comprise the sequence of RGDYTSM (SEQ ID NO: 1906). In some embodiments, the peptide insert does not comprise the sequence of RGDYASL (SEQ ID NO: 2015). In some embodiments, the peptide insert does not comprise the sequence of RGDYNSL (SEQ ID NO: 2016). In some embodiments, the peptide insert does not comprise the sequence of RGDYTSV (SEQ ID NO: 2018). In some embodiments, the peptide insert does not comprise the sequence of RGDYTST (SEQ ID NO: 2021). In some embodiments, the peptide insert does not comprise the sequence of RGDYTSL (SEQ ID NO: 2023).

[0035] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0036] In some embodiments, the peptide insert consists of a sequence presented in Table 8. In some embodiments, the peptide insert does not consist of the sequence of RGDYVSV (SEQ ID NO: 1948).

[0037] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence LRGDX1X2X3 (SEQ ID NO: 8), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0038] In some embodiments, the peptide insert comprises a sequence presented in Table 9.

[0039] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence LRGDX1X2X3 (SEQ ID NO: 8), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0040] In some embodiments, the peptide insert consists of a sequence presented in Table 9.

[0041] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, the variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence 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 within a variable region (VR) of the parent AAV capsid protein (e.g., an AAV9 capsid protein); and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F.

[0042] In some embodiments, the peptide insert comprises a sequence presented in Table 10.

[0043] In some embodiments, the rAAV particles comprise (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion consists of the sequence RGDX1GLX2 (SEQ ID NO: 9), where X1 is Y, W, or F, and X2 is any amino acid, and (ii) the peptide insertion site is within a variable region (VR) of the parent 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.

[0044] In some embodiments, the peptide insert consists of a sequence presented in Table 10.

[0045] Provided herein are rAAV particles comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises the sequence 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 within a variable region (VR) of the parent AAV capsid protein (e.g., AAV9 capsid protein); and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

[0046] In some embodiments, the peptide insert comprises a sequence presented in Table 11. In some embodiments, the peptide insert comprises the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, the peptide insert comprises the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, the peptide comprises the sequence of RGDYREV (SEQ ID NO: 1829).

[0047] In some embodiments, the rAAV particles comprise: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, (i) the peptide insertion consists of the sequence RGDYX1X2X3 (SEQ ID NO: 10), where X1 is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is within a variable region (VR) of the parent 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, the peptide insert consists of a sequence presented in Table 11. In some embodiments, the peptide insert consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, the peptide consists of the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, the peptide consists of the sequence of RGDYREV (SEQ ID NO: 1829).

[0049] The present disclosure further provides rAAV particles comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion comprises a sequence set forth in Table 1, and (ii) the peptide insertion site is within a variable region of the parent 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 that comprises a peptide insertion relative to a corresponding parent AAV capsid protein, wherein (i) the peptide insertion is selected from the group consisting of: (I) RGDX1X2RX3 (SEQ ID NO: 1) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F); (II) RGDX3QX1X2 (SEQ ID NO: 2) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F); (III) RGDHX1X2X3 (SEQ ID NO: 3) (wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (IV) RGDPX1X2X3 (SEQ ID NO: 4) (wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (V) RGDX3X1X2V (SEQ ID NO: 5) (wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F), (VI) RGDX1 QX2X3 (SEQ ID NO: 6) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), (VII) RGDX3X1SX2 (SEQ ID NO: 7) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), or (VIII) LRGDX1X2X3 (SEQ ID NO: 8) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), (IX) RGDX1G X1 is Y, W, or F, and X2 is any amino acid; or (X)RGDYX1X2X3 (SEQ ID NO: 10) (wherein X1 is E or R, X2 is E or R, and X3 is V or I); and (ii) the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein (e.g., AAV9 capsid protein).

[0051] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the insertion site is located between two adjacent amino acids in the variable region of the parent AAV capsid protein (e.g., AAV9 capsid protein).

[0052] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the insertion site is located between two non-adjacent amino acids in the variable region of the parent AAV capsid protein (e.g., AAV9 capsid protein).

[0053] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the peptide insertion is within VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, or VR-IX of the parent AAV capsid protein (e.g., an AAV9 capsid protein).

[0054] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the peptide insertion is within VR-VIII of the parent AAV capsid protein (e.g., AAV9 capsid protein). In some embodiments, the parent AAV capsid protein is selected 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 580-601 of VP1, VP2, or VP3 of the AAV9 capsid protein, or the corresponding position in a capsid protein (e.g., VP1, VP2, or VP3) of another parent 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, the parent AAV capsid protein is an AAV9 capsid protein, and VR-VIII comprises amino acids 580-601 of AAV9 capsid protein VP1, VP2, or VP3. In some embodiments, the peptide insertion site is located between amino acids 588 and 589 of AAV9 capsid protein VP1, VP2, or VP3.

[0056] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the heterologous peptide insertion replaces a contiguous stretch of amino acids in the parent AAV capsid protein (e.g., AAV9 capsid protein).

[0057] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the heterologous peptide insertion does not replace a contiguous stretch of amino acids in the parent AAV capsid protein (e.g., AAV9 capsid protein).

[0058] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the insertion site is located at amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein, or at the corresponding positions in the capsid protein (e.g., VP1, VP2, or VP3) of another parent AAV capsid protein.

[0059] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the variant AAV capsid protein comprises (1) a peptide insert comprising the consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) the sequence of one or more of the VPs of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3).

[0060] In some embodiments of the rAAV particles disclosed herein or the variant AAV capsid proteins disclosed herein, the variant AAV capsid protein comprises (1) a peptide insert comprising any one of the sequences presented in any one of Tables 1-11, and (2) the sequence of one or more of the VPs of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3).

[0061] In some embodiments, disclosed herein are targeting moieties, e.g., muscle-targeting moieties, conjugated to a payload. In some embodiments, the targeting moiety, e.g., muscle-targeting moiety, comprises a peptide sequence set forth in any one of Tables 1-11. In some embodiments, the targeting moiety, e.g., 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, the targeting moiety, e.g., muscle-targeting moiety, encapsidates a payload, e.g., described herein. In some embodiments, the targeting moiety, e.g., muscle-targeting moiety, is engaged to a payload, e.g., described herein. In some embodiments, the peptide insert in a targeting moiety disclosed herein comprises the sequence of SEQ ID NO: 1825. In some embodiments, the peptide insert in a targeting moiety disclosed herein comprises the sequence of SEQ ID NO: 1551. In some embodiments, the peptide insert in a targeting moiety disclosed herein comprises the 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 disclosed herein.

[0063] Further disclosed herein are isolated cells transduced with the rAAV particles disclosed herein. Also disclosed herein are cells containing an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein disclosed herein.

[0064] The present disclosure provides compositions comprising a targeting moiety, e.g., a muscle-targeting moiety, and a payload, wherein the targeting moiety, e.g., the muscle-targeting moiety, is selected from the group consisting of: (I) RGDX1X2RX3 (SEQ ID NO: 1) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F); (II) RGDX3QX1X2 (SEQ ID NO: 2) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F); (III) RGDHX1X2X3 (SEQ ID NO: 3) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F); (IV) RGDPX1X2X3 (SEQ ID NO: 4) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F); (V) RGDX3X1X2V (SEQ ID NO: 5) (wherein X1 and X2 are independently any amino acid). X1 and X2 are independently any amino acid and X3 is Y, W, or F), (VI) RGDX1QX2X3 (SEQ ID NO: 6) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), (VII) RGDX3X1SX2 (SEQ ID NO: 7) (wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), or (VIII) LRGDX1X2X3 (SEQ ID NO: 8) (wherein wherein X1 and X2 are independently any amino acid and X3 is Y, W, or F), (IX) RGDX1GLX2 (SEQ ID NO: 9) (wherein X1 is Y, W, or F and X2 is any amino acid), or (X) RGDYX1X2X3 (SEQ ID NO: 10) (wherein X1 is E or R, X2 is E or R, and X3 is V or I).

[0065] Also provided herein are compositions comprising a targeting moiety, e.g., a muscle-targeting moiety, and a payload, wherein the targeting moiety, e.g., the muscle-targeting moiety, comprises a peptide comprising a sequence set forth in any one of Tables 1-11. In some embodiments, the peptide insert in the targeting moiety, e.g., the muscle-targeting moiety, comprises the sequence of SEQ ID NO: 1825. In some embodiments, the peptide insert in the targeting moiety, e.g., the muscle-targeting moiety, comprises the sequence of SEQ ID NO: 1551. In some embodiments, the peptide comprises the sequence of SEQ ID NO: 1829.

[0066] In some embodiments, a targeting moiety, for example, a muscle-targeting moiety, is conjugated to a payload.

[0067] In some embodiments, the targeting moiety, eg, a muscle-targeting moiety, is inserted into a viral protein, eg, an AAV capsid protein.

[0068] In some embodiments, the targeting moiety, e.g., a muscle-targeting moiety, is part of, e.g., incorporated within, a vector.

[0069] In some embodiments, the targeting moiety, e.g., the muscle targeting moiety, is not part of the vector.

[0070] The present disclosure further provides a pharmaceutical composition comprising (a) the rAAV particles disclosed herein and (b) a pharmaceutically acceptable excipient.

[0071] Also provided herein is a method of delivering a payload to a muscle cell, the method comprising administering to the muscle cell a pharmaceutical composition disclosed herein.

[0072] In some embodiments, the muscle cells are in vitro.

[0073] In some embodiments, the cell is in vivo.

[0074] In some embodiments, the muscle cells are derived from a subject who has or has been determined to have a muscle disorder.

[0075] The present disclosure provides a method of treating a subject having a myopathic disorder and / or ameliorating symptoms of a myopathic disorder in a subject, comprising administering to the subject a pharmaceutical composition disclosed herein.

[0076] In some embodiments, the muscle disorder is selected from X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Lim-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

[0077] In some embodiments, the pharmaceutical composition is administered via a route of administration selected from intramuscular, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, intracisternal, or limb perfusion.

[0078] In some embodiments, the subject is a human.

[0079] In some embodiments of the rAAV particles, variant AAV capsids, compositions, or methods disclosed herein, the variant AAV capsid proteins confer increased infectivity and / or transduction of muscle cells compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parent AAV capsid proteins.

[0080] In some embodiments of the rAAV particles, variant AAV capsids, compositions, or methods disclosed herein, the variant AAV capsid proteins confer an increase in infectivity and / or transduction of muscle cells of 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 compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parental AAV capsid proteins.

[0081] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the variant AAV capsid proteins confer an increase in infectivity and / or transduction of muscle cells of 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 compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parent AAV capsid protein.

[0082] In some embodiments of the rAAV particles, variant AAV capsids, compositions, or methods disclosed herein, the variant AAV capsid proteins confer an increase in infectivity and / or transduction of muscle cells of 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 compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parent AAV capsid protein.

[0083] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the muscle cells are selected from cardiomyocytes, smooth muscle cells, skeletal muscle cells, muscle stem cells (e.g., muscle satellite cells), or combinations thereof.

[0084] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the peptide insertion site is located at or between amino acids 581 and 593 of VP1, VP2, or VP3 of AAV9, or at a corresponding position within the capsid protein (e.g., VP1, VP2, or VP3) of another parent AAV capsid protein.

[0085] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the peptide insertion site is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein, or at a corresponding position within the capsid protein of another parent AAV capsid protein. In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the variant AAV capsid protein further comprises one or more modifications in the amino acid sequence adjacent to the peptide insertion site. In some embodiments, the one or more modifications are within about 10 amino acids upstream or downstream of the location of the peptide insertion site. In some embodiments, the one or more modifications are within about 5 amino acids upstream or downstream of the location of the peptide insertion site.

[0086] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, one or more modifications are located within a variable region of the parent AAV capsid protein. In some embodiments, the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, one or more modifications are located within the AAV9 capsid protein variable region, for example, 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, the one or more modifications are located in VR-VIII of a VP (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein, VR-IV of a VP (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein, or VR-V of a VP (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein, or any combination thereof.

[0088] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, VR-IV comprises amino acids 451 to 475 of AAV9 capsid protein VP1, VP2, or VP3.

[0089] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the VR-V comprises amino acids 488 to 506 of AAV9 capsid protein VP1, VP2, or VP3.

[0090] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the one or more modifications comprise an insertion, a deletion, a mutation, or a combination thereof.

[0091] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the variant AAV capsid proteins further comprise one or more modifications to the amino acid sequence at or near the glycan binding region.

[0092] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, one or more modifications reduce glycan binding. In some embodiments, the glycan is galactose.

[0093] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the one or more modifications are at the following amino acids: (a) 271 and 272 of VP1, VP2, or VP3 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (b) 446 of VP1, VP2, or VP3 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (c) 470 of VP1, VP2, or VP3 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (d) 471 of VP1, VP2, or VP3 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (e) 501 and 505 of VP1, VP2, or VP3 of AAV1 or a corresponding position in the capsid protein of another parent AAV capsid protein (e.g., any one or all of residues 501, 502, 503, 504, or 505, or a combination thereof), or a corresponding position in the capsid protein of another parent AAV capsid protein; (f) 591 and 621 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (g) any combination, or all of (a) through (f). In some embodiments, the parent AAV capsid protein is an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV9 capsid protein.

[0094] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the 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 to the parent AAV capsid protein. In some embodiments, the percent identity is determined by comparing the sequence of the variant capsid, excluding the peptide insert, to the parent AAV capsid protein (e.g., AAV9 capsid protein).

[0095] In some embodiments, the variant capsid protein and the parent AAV capsid protein have 100% identity when (a) the peptide insert 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 insert.

[0096] In some embodiments, the variant capsid protein and the parent AAV capsid protein have less than 100% identity when (a) the peptide insert in the variant capsid protein is not taken into account in the sequence comparison, and (b) the variant capsid protein contains one or more modifications other than the peptide insert.

[0097] In some embodiments, the parent AAV capsid protein is the AAV9 capsid protein of SEQ ID NO:2001.

[0098] In some embodiments, the parent AAV capsid protein is the AAV1 capsid protein of SEQ ID NO:2002.

[0099] In some embodiments, the parent AAV capsid protein is the AAV2 capsid protein of SEQ ID NO:2003.

[0100] In some embodiments, the parent AAV capsid protein is the AAV3B capsid protein of SEQ ID NO: 2050.

[0101] In some embodiments, the parent AAV capsid protein is the AAV4 capsid protein of SEQ ID NO: 2051.

[0102] In some embodiments, the parent AAV capsid protein is the AAV5 capsid protein of SEQ ID NO:2004.

[0103] In some embodiments, the parent AAV capsid protein is the AAV6 capsid protein of SEQ ID NO:2005.

[0104] In some embodiments, the parent AAV capsid protein is the AAV7 capsid protein of SEQ ID NO: 2052.

[0105] In some embodiments, the parent AAV capsid protein is the AAV8 capsid protein of SEQ ID NO:2006.

[0106] In some embodiments, the parent AAV capsid protein is the AAV10 capsid protein of SEQ ID NO: 2053.

[0107] In some embodiments, the parent AAV capsid protein is the AAV11 capsid protein of SEQ ID NO: 2054.

[0108] In some embodiments, the parent AAV capsid protein is the AAV12 capsid protein of SEQ ID NO: 2055.

[0109] In some embodiments, the parent AAV capsid protein is the AAV13 capsid protein of SEQ ID NO: 2056.

[0110] In some embodiments, the parent AAV capsid protein is the AAVrh74 capsid protein of SEQ ID NO:2057.

[0111] In some embodiments of the rAAV particles, variant AAV capsids, compositions, or methods disclosed herein, the payload is a polypeptide encoded by a nucleic acid sequence within the rAAV particle.

[0112] In some embodiments, the polypeptide is or comprises a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein is selected 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, a gRNA, an sgRNA, or a crRNA / tracrRNA that interacts with the CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein is fused to one or more domains, such as an activator domain and / or a repressor domain. In some embodiments, the CRISPR-Cas protein is a nuclease. In some embodiments, the CRISPR-Cas protein is a nickase and cleaves only one strand of the target nucleic acid molecule, hi some embodiments, the CRISPR-Cas protein inactivates the target nucleic acid molecule, binding to it but not cleaving it.

[0113] In some embodiments, the polypeptide is or comprises a zinc finger protein, or a variant or fragment thereof. In some embodiments, the zinc finger protein is selected from a zinc finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeting zinc finger DNA-binding entity optionally fused to a nuclease domain (e.g., a Fok1 nuclease domain), or a variant, fragment, or combination of any of the foregoing. In some embodiments, the zinc finger protein is fused to one or more domains, such as an activator domain and / or a repressor domain.

[0114] In some embodiments, the polypeptide is or comprises a transcription activator-like effector (TAL) protein, or a variant or fragment thereof. In some embodiments, the TAL comprises a TAL effector DNA-binding domain (e.g., a TAL effector DNA-binding domain isolated from a Xanthomonas species), a transcription activator-like effector nuclease (TALEN), e.g., a TAL effector DNA-binding domain fused to a nuclease domain (e.g., a Fok1 nuclease domain), or a variant, fragment, or combination of any of the foregoing. In some embodiments, the TAL protein is fused to one or more domains, e.g., an activator domain and / or a repressor domain.

[0115] In some embodiments, the polypeptide is or comprises a base editor, or a variant or fragment thereof. In some embodiments, the base editor is a deaminase, adenosine deaminase enzyme (ABE), cytosine deaminase enzyme (CBE), APOBEC1, APOBEC3A, APOBEC3G, evoAPOBEC, BE4-YE1, CDA1, activation-induced cytidine deaminase (AID), mutant TadA, adenosine deaminase (TadA*), E. coli The payload may comprise a tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA-binding domain monomer, an RNA-guided base editing enzyme, a DNA glycosylase 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 targeted-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax, a circular permutation base editor, an adenosine deaminase enzyme (ADAR), a programmable RNA editing for adenosine to inosine substitution (REPAIR), an exploited endogenous ADAR for programmable editing of RNA (LEAPER), or a variant, 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, the polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising same. In some embodiments, the prime editor and / or system comprising same comprises a reverse transcriptase, a prime editing enzyme, an editing enzyme comprising a reverse transcriptase domain, avian myeloblastosis virus (AMV) reverse transcriptase, murine leukemia virus (MLV) reverse transcriptase, HIV-1 reverse transcriptase, bacterial reverse transcriptase, a DNA-binding domain and / or a reverse transcriptase associated with a 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, 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, the polypeptide is or comprises a meganuclease, or a variant or fragment thereof. In some embodiments, the meganuclease is selected from a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His-cyst box family meganuclease, or a HNH family endonuclease, I-SeeI, I-CeuI, PI-PspI, PI-SceI, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, I-TevIII, or a variant or fragment or combination of any of the foregoing.

[0118] In some embodiments, the polypeptide is associated with a muscle disorder or a glycogen or sugar storage disorder, in some embodiments, the muscle disorder is selected from X-linked myositis, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Lim-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

[0119] In some embodiments, the polypeptide is an enzyme. In some embodiments, the enzyme is a lysosomal enzyme or an adenosine deaminase enzyme.

[0120] In some embodiments, the polypeptide is an antibody.

[0121] In some embodiments, the polypeptide is a secreted protein.

[0122] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the payload is an RNA molecule. In some embodiments, the RNA molecule is an siRNA, miRNA, gRNA, antisense RNA, circular RNA, snRNA, or an aptamer. In some embodiments, the 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, the muscle disorder is selected from X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Lim-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

[0123] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the payload is a DNA molecule, e.g., a donor DNA molecule that is integrated into the host genome via homologous recombination. In some embodiments, the DNA molecule comprises a nucleic acid sequence up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900 nt, up to about 4,800 nt, up to about 4,700 nt, up to about 4,600 nt, up to about 4,500 nt, up to about 4,400 nt, etc. In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the nucleotide sequence encoding the payload comprises a promoter.

[0124] In some embodiments, the promoter is or comprises a muscle-specific promoter, hi some embodiments, the muscle-specific promoter is selected from MHCK7, CK8, desmin, tMCK, dMCK, or CK6, or a variant or fragment of any of the foregoing.

[0125] In some embodiments, the promoter is or comprises a dual muscle-liver promoter. In some embodiments, the dual muscle-liver promoter is Spc5-12 or a variant or fragment thereof.

[0126] As will be appreciated by those skilled in the art, the peptide sequences disclosed herein, for example, peptides comprising an RGD motif disclosed in any one of Tables 1-11, can be used as targeting moieties, for example, muscle-targeting moieties, to deliver payloads. In some embodiments, the targeting moiety, for example, the muscle-targeting moiety, can be conjugated to a payload. In some embodiments, the targeting moiety, for example, the muscle-targeting moiety, is inserted into a viral protein, for example, an AAV capsid protein.

[0127] Other features, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of example only, and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0128] This patent or patent application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0129] The figures described below, which together constitute the drawings, are for purposes of illustration only and not limitation. [Brief explanation of the drawings]

[0130] [Figure 1] 1 is a graph showing enhanced transduction of skeletal muscle in cynomolgus monkeys by RGDxxRW variants.

[0131] [Figure 2] Schematic diagram of the transgene cassette used for the round 3 library. Gene expression is initiated by a ubiquitous CAG promoter driving transcription of the histone-2B (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] [Figure 3] Graph showing mean capsid enrichment in skeletal muscle tissues in cynomolgus monkeys (n=2) after round 3 library screening. Enrichment values ​​were calculated from the average log2 fold change (log2FC) relative to AAV9 in all analyzed skeletal muscle tissues (gastrocnemius, biceps brachii, quadriceps, soleus, diaphragm, pectoralis major, and rectus abdominis) from each of two animals.

[0133] [Figure 4] Graph showing mean capsid enrichment in heart tissue after round 3 of library screening in cynomolgus monkeys (n=2). Enrichment values ​​were calculated from the mean log2 fold change (log2FC) relative to AAV9 in heart tissue from both animals.

[0134] [Figure 5] 1 is a graph showing that the best-performing round 3 capsid in cynomolgus monkeys also achieved enhanced skeletal muscle transduction in C57BL / 6J mice. Log2 fold change (log2FC) enrichment values ​​represent the average of all analyzed muscle tissues in the two species. Error bars represent the standard error of the mean.

[0135] [Figure 6] Figure 1 shows the productivity of selected capsid variants before and after purification (n=1 per capsid). Before purification, the productivity is represented by the genome copies (GC) measured in the lysate collected after harvest tangential flow filtration (hTFF). After purification, the productivity is represented by the genome titer measured in the final formulation after iodixanol discontinuous gradient ultracentrifugation and formulation TFF (fTFF).

[0136] [Figure 7-1] 1 is a graph showing enhanced transduction observed in multiple muscle tissue regions of cynomolgus monkeys. The dotted line represents AAV9 transduction levels. [Figure 7-2] 1 is a graph showing enhanced transduction observed in multiple muscle tissue regions of cynomolgus monkeys. The dotted line represents AAV9 transduction levels.

[0137] [Figure 8A]This shows that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. AAV9 and two RGD capsid variants (with specific peptides inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated primary myotubes derived from healthy human donors. Images were taken 1 and 5 days after transduction. A is color. [Figure 8B] AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. AAV9 and two RGD capsid variants (with specific peptides inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated primary myotubes derived from healthy human donors. Images were acquired 1 and 5 days after transduction. B, Grayscale. [Figure 8C] This shows that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. AAV9 and two RGD capsid variants (with specific peptides inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated primary myotubes derived from healthy human donors. Images were acquired 1 and 5 days post-transduction. C, Black and white.

[0138] [Figure 9A] We 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), which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits, were packaged with a CAG-mCherry reporter. [Figure 9B] We show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. Three independent primary myoblast cell lines (hSKMDC_con1, hSKMDC_con2, and hSKMDC_con3) were differentiated into 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 AAV transduction (Figures 9B-9D). [Figure 9C] We show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. Three independent primary myoblast cell lines (hSKMDC_con1, hSKMDC_con2, and hSKMDC_con3) were differentiated into 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 AAV transduction (Figures 9B-9D). [Figure 9D] We show that AAV particles displaying RGD-containing peptides efficiently transduced primary human myotubes in culture. Three independent primary myoblast cell lines (hSKMDC_con1, hSKMDC_con2, and hSKMDC_con3) were differentiated into 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 AAV transduction (Figures 9B-9D).

[0139] [Figure 10A] We show that AAV particles displaying an RGD-containing peptide efficiently transduced immortalized human myotubes (ImmSkMDC_AB1190) in culture. AAV9 and the capsid variant RGDYREV (SEQ ID NO: 1829; all viral protein subunits have a specific peptide inserted between positions 588 and 589 of AAV9 VP1) were packaged with the CBA-mCherry-Fluc2 dual reporter. [Figure 10B] We show that AAV particles displaying RGD-containing peptides efficiently transduced immortalized human myotubes (ImmSkMDC_AB1190) in culture. Quantification of mCherry fluorescence (Figure 10B) was performed 5 days after transduction with AAV particles at the indicated doses (1E4 vg / cell, 3E4 vg / cell, or 1E5 vg / cell). [Figure 10C] We show that AAV particles displaying RGD-containing peptides efficiently transduced immortalized human myotubes (ImmSkMDC_AB1190) in culture. Quantification of luciferase activity (Figure 10C) was performed 5 days after transduction with AAV particles at the indicated doses (1E4 vg / cell, 3E4 vg / cell, or 1E5 vg / cell).

[0140] [Figure 11A] These results show that AAV particles displaying RGD-containing peptides transduced 3D muscle bundles more efficiently than AAV particles with wild-type AAV9 capsids. AAV9 and the capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) (which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated muscle bundles generated from immortalized myotubes derived from healthy human donors. Muscle bundles were transduced with AAV particles at a multiplicity of infection (MOI) of 1E5 vg / cell 7 days after differentiation, and images were acquired 7 days after viral transduction. Each channel was imaged with the same exposure time across samples. Figures 11A, 11C, and 11E show images acquired at 4x magnification (scale bar = 250 μm). Figures 11B, 11D, and 11F show images acquired at 10x magnification (scale bar = 250 μm). Figures 11A-11B are in color, Figures 11C-11D are in grayscale, and Figures 11E-11F are in black and white. [Figure 11B]These results show that AAV particles displaying RGD-containing peptides transduced 3D muscle bundles more efficiently than AAV particles with wild-type AAV9 capsids. AAV9 and the capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) (which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated muscle bundles generated from immortalized myotubes derived from healthy human donors. Muscle bundles were transduced with AAV particles at a multiplicity of infection (MOI) of 1E5 vg / cell 7 days after differentiation, and images were acquired 7 days after viral transduction. Each channel was imaged with the same exposure time across samples. Figures 11A, 11C, and 11E show images acquired at 4x magnification (scale bar = 250 μm). Figures 11B, 11D, and 11F show images acquired at 10x magnification (scale bar = 250 μm). Figures 11A-11B are in color, Figures 11C-11D are in grayscale, and Figures 11E-11F are in black and white. [Figure 11C]These results show that AAV particles displaying RGD-containing peptides transduced 3D muscle bundles more efficiently than AAV particles with wild-type AAV9 capsids. AAV9 and the capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) (which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated muscle bundles generated from immortalized myotubes derived from healthy human donors. Muscle bundles were transduced with AAV particles at a multiplicity of infection (MOI) of 1E5 vg / cell 7 days after differentiation, and images were acquired 7 days after viral transduction. Each channel was imaged with the same exposure time across samples. Figures 11A, 11C, and 11E show images acquired at 4x magnification (scale bar = 250 μm). Figures 11B, 11D, and 11F show images acquired at 10x magnification (scale bar = 250 μm). Figures 11A-11B are in color, Figures 11C-11D are in grayscale, and Figures 11E-11F are in black and white. [Figure 11D]These results show that AAV particles displaying RGD-containing peptides transduced 3D muscle bundles more efficiently than AAV particles with wild-type AAV9 capsids. AAV9 and the capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) (which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated muscle bundles generated from immortalized myotubes derived from healthy human donors. Muscle bundles were transduced with AAV particles at a multiplicity of infection (MOI) of 1E5 vg / cell 7 days after differentiation, and images were acquired 7 days after viral transduction. Each channel was imaged with the same exposure time across samples. Figures 11A, 11C, and 11E show images acquired at 4x magnification (scale bar = 250 μm). Figures 11B, 11D, and 11F show images acquired at 10x magnification (scale bar = 250 μm). Figures 11A-11B are in color, Figures 11C-11D are in grayscale, and Figures 11E-11F are in black and white. [Figure 11E]These results show that AAV particles displaying RGD-containing peptides transduced 3D muscle bundles more efficiently than AAV particles with wild-type AAV9 capsids. AAV9 and the capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) (which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated muscle bundles generated from immortalized myotubes derived from healthy human donors. Muscle bundles were transduced with AAV particles at a multiplicity of infection (MOI) of 1E5 vg / cell 7 days after differentiation, and images were acquired 7 days after viral transduction. Each channel was imaged with the same exposure time across samples. Figures 11A, 11C, and 11E show images acquired at 4x magnification (scale bar = 250 μm). Figures 11B, 11D, and 11F show images acquired at 10x magnification (scale bar = 250 μm). Figures 11A-11B are in color, Figures 11C-11D are in grayscale, and Figures 11E-11F are in black and white. [Figure 11F]These results show that AAV particles displaying RGD-containing peptides transduced 3D muscle bundles more efficiently than AAV particles with wild-type AAV9 capsids. AAV9 and the capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) (which have a specific peptide inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits) were packaged with a CAG-mCherry reporter. Individual purified capsids were added to differentiated muscle bundles generated from immortalized myotubes derived from healthy human donors. Muscle bundles were transduced with AAV particles at a multiplicity of infection (MOI) of 1E5 vg / cell 7 days after differentiation, and images were acquired 7 days after viral transduction. Each channel was imaged with the same exposure time across samples. Figures 11A, 11C, and 11E show images acquired at 4x magnification (scale bar = 250 μm). Figures 11B, 11D, and 11F show images acquired at 10x magnification (scale bar = 250 μm). Figures 11A-11B are in color, Figures 11C-11D are in grayscale, and Figures 11E-11F are in black and white.

[0141] [Figure 12A] This figure shows visualization of mCherry expression in the quadriceps muscle of transduced mice. AAV9 and two RGD capsid variants (with a specific peptide inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits) were packaged with a CAG-mCherry reporter, and the corresponding AAV particles were administered by intravenous injection. Images were acquired 4 weeks after transduction. Grid = 10 mm. A is color. [Figure 12B] Visualization of mCherry expression in the quadriceps muscle of transduced mice. AAV9 and two RGD capsid variants (with a specific peptide inserted between positions 588 and 589 of the VP1 of AAV9 in all viral protein subunits) were packaged with a CAG-mCherry reporter, and the corresponding AAV particles were administered intravenously. Images were acquired 4 weeks after transduction. Grid = 10 mm. B, Grayscale. [Figure 12C] Visualization of mCherry expression in the quadriceps muscle of transduced mice. AAV9 and two RGD capsid variants (with a specific peptide inserted between positions 588 and 589 of the VP1 of AAV9 in all viral protein subunits) were packaged with a CAG-mCherry reporter, and the corresponding AAV particles were administered intravenously. Images were acquired 4 weeks after transduction. Grid = 10 mm. C, black and white.

[0142] [Figure 13A] This is a series of images showing mCherry expression in transduced striated muscle via IHC of tissues from mice treated with AAV particles displaying an RGD-containing peptide compared with mice treated with AAV particles carrying wild-type AAV9 capsids 4 weeks after injection. AAV9 and the RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variants (with a specific peptide inserted between positions 588 and 589 of AAV9 VP1 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 carrying RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) showed mCherry expression comparable to tissues from animals administered a high dose (1E14 vg / kg) of AAV particles carrying wild-type AAV9 capsids. Tissues were stained for mCherry using a hematoxylin counterstain. A is color. [Figure 13B]This is a series of images showing mCherry expression in transduced striated muscle via IHC of tissues from mice treated with AAV particles displaying an RGD-containing peptide compared with mice treated with AAV particles carrying wild-type AAV9 capsids 4 weeks after injection. AAV9 and the RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variants (with a specific peptide inserted between positions 588 and 589 of AAV9 VP1 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 carrying RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) showed mCherry expression comparable to tissues from animals administered a high dose (1E14 vg / kg) of AAV particles carrying wild-type AAV9 capsids. Tissues were stained for mCherry using a hematoxylin counterstain. B is grayscale. [Figure 13C] This is a series of images showing mCherry expression in transduced striated muscle via IHC of tissues from mice treated with AAV particles displaying an RGD-containing peptide compared with mice treated with AAV particles carrying wild-type AAV9 capsids 4 weeks after injection. AAV9 and the RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) capsid variants (with a specific peptide inserted between positions 588 and 589 of AAV9 VP1 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 carrying RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551) showed mCherry expression comparable to tissues from animals administered a high dose (1E14 vg / kg) of AAV particles carrying wild-type AAV9 capsids. Tissues were stained for mCherry using a hematoxylin counterstain. C is black and white.

[0143] [Figure 14]Figures A-J show that in samples from mice administered AAV particles, administration of a low dose of AAV particles displaying the RGDYREI (SEQ ID NO: 1825) peptide or the RGDYERI (SEQ ID NO: 1551) peptide resulted in similar or enhanced mCherry mRNA expression compared to a high dose of AAV particles with a wild-type AAV9 capsid. The specific peptide was inserted between positions 588 and 589 of the VP1 of AAV9 in all viral protein subunits. Non-muscle targets (lung, kidney, brain, and liver) showed reduced 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.

[0144] [Figure 15A] Figure 1 shows quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles containing wild-type AAV9 capsids or AAV particles displaying the RGDYREI (SEQ ID NO: 1825) or RGDYERI (SEQ ID NO: 1551) peptides (with a CAG-mCherry reporter). The specific peptide was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Vector genome values ​​were normalized to the RPP30 gene using a standard curve determined from a pUC57 plasmid control. A: Anterior tibia. [Figure 15B] Figure 1 shows quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles containing wild-type AAV9 capsids or AAV particles displaying the RGDYREI (SEQ ID NO: 1825) or RGDYERI (SEQ ID NO: 1551) peptides (with a CAG-mCherry reporter). The specific peptides were inserted between positions 588 and 589 of the VP1 region of AAV9 in all viral protein subunits. Vector genome values ​​were normalized to the RPP30 gene using a standard curve determined from a pUC57 plasmid control. B: Gastrocnemius muscle. [Figure 15C]Quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles containing wild-type AAV9 capsids or AAV particles displaying the RGDYREI (SEQ ID NO: 1825) or RGDYERI (SEQ ID NO: 1551) peptides (with a CAG-mCherry reporter) is shown. The specific peptide was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Vector genome values ​​were normalized to the RPP30 gene using a standard curve determined from a pUC57 plasmid control. C: Diaphragm. [Figure 15D] Figure 1 shows quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles containing wild-type AAV9 capsids or AAV particles displaying the RGDYREI (SEQ ID NO: 1825) or RGDYERI (SEQ ID NO: 1551) peptides (with a CAG-mCherry reporter). The specific peptides were inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Vector genome values ​​were normalized to the RPP30 gene using a standard curve determined from a pUC57 plasmid control. D: Brain. [Figure 15E] Figure 1 shows quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles containing wild-type AAV9 capsids or AAV particles displaying the RGDYREI (SEQ ID NO: 1825) or RGDYERI (SEQ ID NO: 1551) peptides (with a CAG-mCherry reporter). The specific peptides were inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Vector genome values ​​were normalized to the RPP30 gene using a standard curve determined from a pUC57 plasmid control. E: Kidney. [Figure 15F]Figure 1 shows quantification of vector genomes per diploid genome in tissues from mice treated with AAV particles containing wild-type AAV9 capsids or AAV particles displaying the RGDYREI (SEQ ID NO: 1825) or RGDYERI (SEQ ID NO: 1551) peptides (with a CAG-mCherry reporter). The specific peptide was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Vector genome values ​​were normalized to the RPP30 gene using a standard curve determined from a pUC57 plasmid control. F: Liver.

[0145] [Figure 16A] This shows that AAV particles displaying the RGDYREV (SEQ ID NO: 1829) peptide have higher hindlimb transduction in mice compared to AAV particles with wild-type AAV9 capsids. RGDYREV was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Representative images of bioluminescence imaging 14 days after IV injection of AAV particles with capsids containing 5E12 vg / kg of the dual reporter (CBA-mCherry-Fluc2) are shown. The red circle represents the region of interest (ROI) for measurement. Two-way ANOVA and Sidak's multiple comparison test. ****p<0.001. [Figure 16B] Figure 1 shows that AAV particles displaying the RGDYREV (SEQ ID NO: 1829) peptide have higher hindlimb transduction in mice compared to AAV particles with wild-type AAV9 capsids. RGDYREV was inserted between positions 588 and 589 of VP1 of AAV9, a subunit of all viral proteins. B-D show the average intensity of each ROI over time. Data are presented as mean ± SEM (n = 5 per group). Two-way ANOVA and Sidak's multiple comparison test. ****p<0.001. [Figure 16C]Figure 1 shows that AAV particles displaying the RGDYREV (SEQ ID NO: 1829) peptide have higher hindlimb transduction in mice compared to AAV particles with wild-type AAV9 capsids. RGDYREV was inserted between positions 588 and 589 of VP1 of AAV9, a subunit of all viral proteins. B-D show the average intensity of each ROI over time. Data are presented as mean ± SEM (n = 5 per group). Two-way ANOVA and Sidak's multiple comparison test. ****p<0.001. [Figure 16D] Figure 1 shows that AAV particles displaying the RGDYREV (SEQ ID NO: 1829) peptide have higher hindlimb transduction in mice compared to AAV particles with wild-type AAV9 capsids. RGDYREV was inserted between positions 588 and 589 of VP1 of AAV9, a subunit of all viral proteins. B-D show the average intensity of each ROI over time. Data are presented as mean ± SEM (n = 5 per group). Two-way ANOVA and Sidak's multiple comparison test. ****p<0.001.

[0146] [Figure 17] Figure 1 shows qPCR quantification of vector genome biodistribution in NHPs administered AAV particles with wild-type AAV9 capsids or AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. The Y-axis indicates vector genome copy number per diploid genome (vg / dg).

[0147] [Figure 18A]Figure 1 shows RT-qPCR quantification of mCherry mRNA levels in NHPs administered AAV particles with wild-type AAV9 capsids or AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of AAV9 VP1 in all viral protein subunits. Quantification from tissues harvested in both the AAV9 and RGDYREI capsid groups is shown, with the y-axis indicating relative mCherry mRNA levels compared to those from AAV9 transduction in the same tissues. [Figure 18B] Figure 1 shows RT-qPCR quantification of mCherry mRNA levels in NHPs administered AAV particles with wild-type AAV9 capsids or AAV particles displaying the RGDYREI peptide packaged with the CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Quantification from tissues harvested only from the RGDYREI capsid group is shown, and the y-axis indicates relative mCherry levels normalized to those from AAV9 transduction in the gastrocnemius muscle.

[0148] [Figure 19A]This is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered with AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide that expressed mCherry into the tongue, upper esophagus, and heart of NHP#1 and NHP#3 (upper and lower panels, respectively). Immunohistochemistry images show that mCherry expression was moderate in the liver relative to muscle tissue in all NHPs. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. A is color. [Figure 19B] A series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered with AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide that expressed mCherry into the tongue, upper esophagus, and heart of NHP#1 and NHP#3 (upper and lower panels, respectively). Immunohistochemistry images show that mCherry expression was moderate in the liver relative to muscle tissue in all NHPs. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. B, grayscale. [Figure 19C]This is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered with AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide that expressed mCherry into the tongue, upper esophagus, and heart of NHP#1 and NHP#3 (top and bottom panels, respectively). Immunohistochemistry images show that mCherry expression was moderate in the liver relative to muscle tissue in all NHPs. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. C, black and white.

[0149] [Figure 20A] This is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide, which expressed mCherry in the triceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, soleus, and vastus lateralis muscles of NHP#1 and NHP#3. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. A is color. [Figure 20B]A series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide, which expressed mCherry in the triceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, soleus, and vastus lateralis muscles of NHP#1 and NHP#3. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. B, Grayscale. [Figure 20C] This is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide, which expressed mCherry in the triceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, soleus, and vastus lateralis muscles of NHP#1 and NHP#3. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. C, black and white.

[0150] [Figure 21A]This is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide, which expressed mCherry in the rectus femoris, tibialis anterior, biceps brachii, pectoralis, and diaphragm of NHP#1 and NHP#3. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. A is color. [Figure 21B] A series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles displaying the RGDYREI peptide packaged with the CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide, which expressed mCherry in the rectus femoris, tibialis anterior, biceps brachii, pectoralis, and diaphragm of NHP#1 and NHP#3. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. B, Grayscale. [Figure 21C]This is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles displaying the RGDYREI peptide packaged with a CAG-mCherry reporter. RGDYREI was inserted between positions 588 and 589 of VP1 of AAV9 in all viral protein subunits. Animals were administered AAV particles displaying the RGDYREI peptide, which expressed mCherry in the rectus femoris, tibialis anterior, biceps brachii, pectoralis, and diaphragm of NHP#1 and NHP#3. NHP#1 and #2 were male, and NHP#3 was female. Tissues were stained for mCherry using a hematoxylin counterstain. Scale bar = 500 μm. C, black and white.

[0151] [Figure 22A]

[0023] Figure 1 is a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles with wild-type AAV9 capsids packaged with a CAG-mCherry reporter. NHPs #4 and #5 were male, and NHP #6 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500 μm. A is color. [Figure 22B] (A) and (B) are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles with wild-type AAV9 capsids packaged with a CAG-mCherry reporter. NHPs #4 and #5 were male, and NHP #6 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500 μm. (B) Grayscale. [Figure 22C](a) and (b) are a series of images showing mCherry expression in transduced tissues via IHC of tissues from NHPs administered AAV particles with wild-type AAV9 capsids packaged with a CAG-mCherry reporter. NHPs #4 and #5 were male, and NHP #6 was female. Tissues were stained for mCherry with a hematoxylin counterstain. Scale bar = 500 μm. (c) is black and white. DETAILED DESCRIPTION OF THE INVENTION

[0152] definition In this application, unless otherwise clear from the 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 the listed components or steps, whether presented by themselves or together with one or more additional components or steps; (iv) the terms "about" and "approximately" may be understood to allow for standard variation as understood by one of ordinary skill in the art; and (v) when ranges are stated, both endpoints are included.

[0153] 5' and 3': The terms "5'" and "3'" are relative terms used to define the spatial relationship or orientation between two or more segments of a nucleic acid sequence. Thus, 3' of a nucleic acid refers to a segment of the nucleic acid that is downstream of another segment, and 5' refers to a segment of the nucleic acid that is upstream of another segment. For example, 3' can indicate that a segment is in the 3' half of a nucleic acid sequence, or even at the 3' end of the nucleic acid sequence. Similarly, 5' can indicate that a segment is in the 5' half of a nucleic acid sequence, or even at the 5' end of the nucleic acid sequence. Unless otherwise indicated, the directionality of a nucleic acid is in the 5' to 3' translational direction.

[0154] About or Approximately: As used herein, the term "about" or "approximately" when referring to a number is generally interpreted as including numbers within 5%, 10%, 15%, or 20% in either direction (greater or lesser) of that number, unless otherwise stated or clear from the context (except where such number is less than 0% or greater than 100% of a possible value).

[0155] Adeno-associated virus (AAV): As used herein, the terms "adeno-associated virus" and "AAV" refer to all or part of a viral particle of the family Parvoviridae and genus Dependoparvovirus. AAV is a small, replication-deficient, non-enveloped virus. AAV may include, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAVrh74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, non-human primate AAV, e.g., rhesus monkey, and any variant of any of the foregoing. Wild-type AAV is replication-deficient and requires co-infection of cells with a helper virus, such as an adenovirus, herpes, or vaccinia virus, e.g., Ad2 or Ad5 virus, or complementation with helper virus genes, in order to replicate.

[0156] Ad2 helper: As used herein, the term "Ad2 helper" refers to adenovirus serotype 2 (Ad2) helper viruses (e.g., wild-type or recombinantly engineered Ad2 helper viruses) and various Ad2 helper genes and / or 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., a 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 for the generation of functional rAAV particles. In certain embodiments, the Ad2 helper vector is transfected into an E1-complementing cell line (e.g., HEK293). The nucleotide sequences of the Ad2 helper vector and Ad2 helper virus genes can be derived from the adenovirus 2 genome (Genbank accession number J01917.1).

[0157] Ad5 helper: As used herein, the term "Ad5 helper" refers to adenovirus serotype 5 (Ad5) helper virus (e.g., wild-type 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., a plasmid) contains the Ad5 helper genes (e.g., one, two, three, or four of E1 (e.g., E1a and / or E1b), E2A, E4, or VA RNA) necessary for the generation of functional rAAV particles. In certain embodiments, the Ad5 helper vector is transfected into an E1-complementing cell line (e.g., HEK293). The nucleotide sequences of the Ad5 helper vector and Ad5 helper genes may be derived from the adenovirus 5 genome (Genbank accession number AY601635).

[0158] Administration: As used herein, the term "administration" refers to the administration of a composition comprising the rAAV particles described herein to a subject. Administration can be by any suitable route. For example, in some embodiments, administration can be local or systemic (e.g., to a mammal, e.g., a human, e.g., a patient). Compositions of the present disclosure can be administered by injection or infusion by any route. For example, compositions may be administered by retinal, subretinal, intravitreal, suprachoroidal, intrathecal, intracisternal, or intrathecal injection or infusion. Additional exemplary routes of administration can include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, intradermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreous.

[0159] Bioreactor: As used herein, the term "bioreactor" refers to any vessel used for growing cell cultures (e.g., mammalian cell cultures). Bioreactors can be of any size and / or shape, so long as they are useful for culturing cell cultures (e.g., mammalian cell cultures).

[0160] Cap polypeptide: As used herein, the term "Cap polypeptide" refers to a structural protein that forms a functional AAV capsid, which can then package DNA and infect or transduce a target cell. In some embodiments, the Cap polypeptide comprises a variant AAV capsid disclosed herein. In some embodiments, the Cap polypeptide comprises all of the AAV capsid subunits, but can consist of fewer than all of the capsid subunits, so long as a functional capsid is produced. In some embodiments, the nucleic acid sequence encoding the Cap polypeptide is present on a single vector (e.g., a plasmid). In some embodiments, the Cap polypeptide comprises the Cap polypeptide of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh74, or a variant of any of the foregoing. AAV capsid genes and proteins are described, for example, in Knipe et al., Fields Virology, Volume 1, (6th ed., Lippincott-Raven Publishers), which is incorporated herein by reference in its entirety.

[0161] Cell density: As used herein, the term "cell density" refers to the 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 is expressed as viable cells (vc) / cm of culture medium. 2 or may be expressed as vc / mL.

[0162] Culture: As used herein, the terms "culture" and "cell culture" refer to a population of cells (e.g., a eukaryotic cell population) suspended in or covered by a medium under conditions suitable for the survival and / or growth of the cell population. As will be apparent to one of skill in the art, these terms can also refer to a combination comprising a cell population and a medium.

[0163] Fragment: As used herein, the term "fragment" or "portion" refers to a structure that includes a discrete portion of a whole, but lacks one or more portions found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or includes a characteristic structural element or portion 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 acid) found throughout the nucleotide sequence. 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 throughout the nucleotide. The entire material or entity may, in some embodiments, be referred to as the "parent" of the entire.

[0164] Gene: As used herein, the term "gene" refers to a DNA sequence that encodes 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 can include both coding sequence (e.g., exon sequence) and non-coding sequence (e.g., intron sequence). In some embodiments, a gene can include one or more regulatory elements that can, for example, 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 the insertion or deletion of specific genomic DNA sequences to treat or prevent a disorder or condition for which such treatment is sought. In some embodiments, the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells). The target cells may be of mammalian origin and / or may be cells within a mammalian subject. Mammals include, but are not limited to, humans, dogs, cats, cows, sheep, pigs, llamas, etc. In some embodiments, heterologous DNA is transferred to the target cells. The heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and the therapeutic product encoded thereby is produced. Additionally or alternatively, the heterologous DNA may in some way mediate expression of the DNA encoding the therapeutic product or may encode a product, such as a peptide or RNA, that in some way mediates or regulates, directly or indirectly, expression of the therapeutic product. Gene therapy may also be used to deliver nucleic acids encoding gene products that replace defective genes or complement gene products produced by the mammal or cell into which it has been introduced. Heterologous DNA encoding a therapeutic product may be modified before introduction into the cells of an affected host to enhance or otherwise alter the product or its expression. Gene therapy may also involve the delivery of inhibitors, repressors, or other regulators of gene expression. Such inhibitors, repressors, or other regulators may be polypeptides, peptides, or nucleic acids (e.g., DNA or RNA). Gene therapy may involve in vivo or ex vivo techniques. In some embodiments, viral and non-viral-based gene transfer methods can be used to introduce nucleic acids encoding polypeptides of interest or to introduce therapeutic nucleic acids into mammalian cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as poloxamers or liposomes. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes after delivery to cells.For reviews 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 incorporated herein 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. Upon reading this disclosure, those skilled in the art 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 subsequent 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 herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences).

[0167] Identity: As used herein, the term "identity" refers to the overall relatedness between polymers, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymers are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the aligned sequences 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 the reference sequence. Nucleotides at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between 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 that needs to be introduced for optimal alignment of the two sequences. Sequence comparison 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 Meyers and Miller algorithm (CABIOS, 1989, 4:11-17), which is incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weighted 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 the NWSgapdna.CMP matrix.

[0168] Improve, increase, inhibit, or reduce: As used herein, the terms "improve," "increase," "inhibit," "reduce," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., in a single sample of culture medium) under other comparative conditions in the absence, presence, or presence of a particular agent or treatment (e.g., before and / or after), or in the presence of a suitable comparator agent. In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment.

[0169] Culture medium: As used herein, the terms "culture medium," "culture medium," and "growth medium" refer to a solution containing nutrients to nourish cells (e.g., proliferating cells, e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, an energy source, lipids, and trace elements necessary for cell survival and / or minimal growth. The solution may also contain components that enhance survival and / or growth beyond a minimal rate, including hormones and growth factors. The solution may be formulated to an optimal pH and concentration of one or more salts for cell survival and / or growth. For example, the medium may also be a "defined medium" or "chemically defined medium," e.g., a serum-free medium that does not contain proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components, and all components have known chemical structures. Those skilled in the art will appreciate that defined media may also contain recombinant polypeptides, such as, but not limited to, hormones, cytokines, interleukins, and / or other signaling molecules.

[0170] Muscle-targeting moiety: As used herein, the phrase "muscle-targeting moiety" refers to a peptide containing an RGD motif that is effective in targeting muscle cells or muscle tissue. In some embodiments, the muscle-targeting moiety can target muscle cells or tissue by (i) contacting the muscle cell or muscle tissue (e.g., binding to one or more receptors expressed on the muscle cell or tissue), (ii) contacting a cell in contact with the muscle cell or tissue (e.g., binding to one or more receptors expressed on the cell in contact with the muscle cell or tissue), (iii) delivering a payload to the muscle cell or tissue, or (iv) any combination of (i)-(iii). In some embodiments, delivering a payload to the muscle cell or muscle tissue comprises transducing the muscle cell or muscle tissue. In some embodiments, delivering a payload to the muscle cell or muscle tissue results in expression (e.g., detectable expression) of the payload in the muscle cell or muscle tissue. In some embodiments, the 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, the muscle-targeting moiety can be conjugated to a payload. In some embodiments, the muscle-targeting moiety can be incorporated into a vector, such as a viral vector or a non-viral vector. In some embodiments, the muscle-targeting moiety can be inserted into an AAV capsid to form the variant AAV capsid disclosed herein.

[0171] Nucleic Acid: The term "nucleic acid" includes any nucleotide, its analogs, and polymers thereof. The term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. These terms include, by equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides, such as, but 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 combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids containing ribose moieties, nucleic acids containing deoxyribose moieties, nucleic acids containing both ribose and deoxyribose moieties, and nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix "poly" refers to nucleic acids 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 nucleic acids containing 2 to about 200 nucleotide monomer units. According to the methods and compositions described herein, in some embodiments, RNA includes short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR / Cas guide RNA, microRNA (miRNA), and / or precursors thereof.

[0172] Payload: As used herein, the term "payload" refers to a nucleic acid sequence of interest (e.g., including a sequence encoding 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 containing the cell, or a polypeptide. A target payload can be a heterologous protein having a therapeutic purpose, such as an enzyme or an antibody. A target payload can be a heterologous nucleic acid having a therapeutic purpose, such as an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR / Cas guide RNA, or a precursor thereof. Those skilled in the art will recognize that a target payload can be selected from any heterologous protein or nucleic acid of interest. As used herein, "encode" or "encodes" means to direct expression or to be processed. 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 (when further processed into) a further processed version of the nucleic acid (e.g., a mature miRNA).

[0173] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition comprising rAAV particles suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen. In some embodiments, the therapeutic regimen comprises one or more doses administered according to a schedule determined (e.g., with a statistically significant probability) to achieve a desired therapeutic effect when administered to a subject or population in need thereof. Pharmaceutical compositions may be specifically formulated for administration in solid or liquid form. In some embodiments, pharmaceutical compositions are formulated for administration by parenteral administration, such as subcutaneous, intramuscular, intravenous, or epidural injection. In some embodiments, pharmaceutical compositions are formulated as a sterile solution or suspension, for example, in a sustained-release formulation. Pharmaceutical compositions of the present disclosure may be formulated for administration by injection or infusion (e.g., subcutaneous, intramuscular, intravenous, or epidural injection or infusion). For example, the composition may be formulated for administration by retinal, subretinal, intravitreal, suprachoroidal, intrathecal, intracisternal, or intrathecal injection or infusion. In some embodiments, the pharmaceutical composition is intended for and suitable for administration to a human subject. In some embodiments, the pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free). Pharmaceutical composition formulations include, but are not limited to, oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., buccal, sublingual, and those targeted for systemic absorption), boluses, powders, granules, pastes for application to the tongue; topical application, e.g., creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., pessaries, creams, or foams; sublingual; intraocular; transdermal; or intranasal, pulmonary, and other mucosal surface formulations.

[0174] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art will understand that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences described herein, but also polypeptides representing functional fragments of such complete polypeptides (e.g., fragments that retain at least one activity). Furthermore, those of skill in the art will understand that protein sequences generally tolerate some degree of substitution without destroying activity. Thus, any polypeptide that retains activity, shares at least about 30-40%, often greater than about 50%, 60%, 70%, or 80% overall sequence identity, and usually contains at least one region of much higher identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%, often in one or more highly conserved regions, and typically encompasses at least 3-4, and often up to 20 or more amino acids with another polypeptide of the same class, is encompassed within the related 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, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides 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 a polypeptide that is designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant combinatorial human polypeptide library, a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise engineered to express a gene(s) or genetic component(s) that encodes and / or directs the expression of a polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof, and / or a polypeptide that is prepared, expressed, created, or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise producing a nucleic acid or one or more component(s), portion(s), element(s), or domain(s) thereof that encodes and / or directs the expression of a polypeptide. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements derived from natural or synthetic sources, such as the germline of a source organism of interest.

[0176] Recombinant AAV (rAAV) particle: As used herein, "recombinant AAV particle" or "rAAV particle" refers to an infectious, replication-deficient viral particle comprising an AAV protein shell enclosing a payload flanked on both sides by ITRs. AAV particles are produced in suitable host cells (e.g., HEK293 cells). For example, the host cells are 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., a polypeptide expression or therapeutic nucleic acid), thereby enabling the host cells to produce the Rep and Cap polypeptides necessary for packaging the rAAV particles. The rAAV particles can then be used for gene delivery.

[0177] Rep polypeptide: As used herein, the term "Rep polypeptide" refers to an AAV nonstructural protein that mediates AAV replication to produce AAV particles. AAV replication genes and proteins are described, for example, in Knipe et al., FIELDS VIROLOGY, Volume 1, (6th ed., Lippincott-Raven Publishers), which is incorporated herein by reference in its entirety.

[0178] RGD motif: As used herein, the phrase "RGD motif" refers to a peptide containing the amino acids R, G, and D, in consecutive order. Peptides containing the RGD motif are presented in Tables 1-11.

[0179] Seeding: As used herein, the term "seeding" 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 growing the cells in a separate bioreactor or vessel prior to providing to the bioreactor or other vessel. The cells are frozen and thawed immediately prior to providing 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, e.g., 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, the human subject is an adult, an adolescent, or a pediatric subject. In some embodiments, the subject suffers 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 neurological disorder or a cancer or tumor described herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or exhibits an increased risk (compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms of the disease, disorder, or condition. In some embodiments, the subject does not exhibit a particular symptom (e.g., a clinical sign of a disease) or characteristic of a disease, disorder, or condition. In some embodiments, the subject does not exhibit symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom and / or who has been diagnosed and / or treated.

[0181] Titer: As used herein, the term "titer" refers to the amount of virus in a given volume. Titer can be expressed, for example, as viral genome copies (vg) per given volume or plaque-forming units (pfu) per given volume. In some embodiments, titer can be expressed as the number of capsids per given volume.

[0182] Transfection: As used herein, the term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, such as a eukaryotic cell (e.g., a mammalian cell). For example, transfection can include vector-based transfection, virus-based transfection, electroporation, lipofection (e.g., using cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and / or cationic polymer (e.g., DEAE-dextran or polyethyleneimine)-based transfection. In some embodiments, virus-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. Treatment can be provided to ameliorate, alleviate, inhibit, prevent or reduce the likelihood of progression of a disease, disorder, or condition, or to ameliorate, alleviate, inhibit, or prevent progression of a disease, disorder, or condition, or to prevent or reduce the likelihood of one or more symptoms or signs. "Preventing" refers to preventing a disease, disorder, condition, or symptom or sign from occurring in at least some individuals, at least for a period of time. Treating can include, for example, administering an agent to a subject after the onset of one or more symptoms or signs indicative of a condition, disease, or disorder to ameliorate, alleviate, or reduce the severity of the condition and / or inhibit or prevent the progression of the condition and / or ameliorate, alleviate, or reduce the severity of the condition and / or suppress one or more symptoms or signs of the condition. The compositions comprising the rAAV particles of the present disclosure can be administered to subjects who have developed a disorder or who are at a higher risk of developing such a disorder than members of the general population. The compositions of the present disclosure can be administered prophylactically or before the onset of any symptoms or signs of a pathological condition. Typically, in this case, the subject is at risk of developing a pathological 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 exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., at the level of the presence or absence of one or more chemical moieties compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule possesses certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. To give some examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function. A nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to each other 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 covalent components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity to a reference polypeptide or nucleic acid. 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 biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to that of the reference but has a small number of sequence changes at specific positions. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues of the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference. Often, a variant polypeptide or nucleic acid will contain a very small (e.g., less than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted functional residues (i.e., residues responsible for a particular biological activity) relative to the reference. In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, the variant polypeptide or nucleic acid contains less 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 usually less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is one found in nature.

[0185] Vector: As used herein, the term "vector" refers to a molecule that contains a nucleic acid molecule, and the vector is capable of transporting the nucleic acid molecule into a cell. As a non-limiting example, one type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments may be packaged into a viral capsid and transferred to 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. Furthermore, certain vectors are capable of directing the expression of genes to which they are operably 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 specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference in its entirety.

[0187] VP: As used herein, the term "VP" refers to the AAV VP1 capsid protein, the AAV VP2 capsid protein, the AAV VP3 capsid protein, or a variant, fragment, or combination of any of the foregoing. The term "capsid protein" is used interchangeably herein with VP. The numbering used herein when describing exemplary locations of peptide insertion sites in VP1, VP2, or VP3 is relative to the numbering of AAV VP1. For example, the AAV9 capsid proteins VP1, VP2, and VP3 correspond to amino acids 1-736 of VP1, amino acids 138-736 of VP1, and amino acids 203-736 of VP1, respectively. Thus, a 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 the numbering of VP1. Those skilled in the art will be able to readily ascertain corresponding positions in VP2 and VP3 by comparing the sequences of the parent AAV capsid proteins VP1, VP2, and VP3 using methods known in the art, such as, for example, sequence alignment. In some embodiments, the VP capsid protein is a VP1 capsid protein. In some embodiments, the VP capsid protein is a VP2 capsid protein. In some embodiments, the VP capsid protein is a VP3 capsid protein. In some embodiments, the VP protein comprises a peptide insert disclosed herein.

[0188] Variant AAV capsid protein: As used herein, the term "variant AAV capsid protein" refers to a VP capsid protein (e.g., VP1, VP2, or VP3) that contains a peptide insertion relative to the corresponding parent AAV capsid protein (e.g., parent VP1, VP2, or VP3). [Mode for Carrying Out the Invention]

[0189] The present disclosure provides, inter alia, improved recombinant adeno-associated virus (rAAV) particles that can be used to target cells or tissues, such as muscle cells or tissues. Safe and efficient delivery of therapeutic payloads to muscles, such as skeletal and / or cardiac muscles, remains a major challenge in gene therapy. Recombinant adeno-associated viruses (rAAV) have emerged as some of the most promising vectors for in vivo gene therapy and are currently undergoing clinical evaluation for many disorders, including muscle disorders. However, naturally occurring AAV capsids suboptimally target skeletal muscle and require very high doses, at least to achieve efficient transgene expression. This poses daunting manufacturing challenges and safety concerns.

[0190] The present disclosure is based, in part, on the discovery that muscle tropism of AAV can be achieved by inserting a short peptide into an AAV capsid to target the AAV capsid to muscle cells. In some embodiments, rAAV particles comprising variant capsids with peptide inserts disclosed herein bind to and / or recognize targets on muscle cells. Without wishing to be bound by any particular theory, in some embodiments, rAAV particles comprising variant capsids with peptide inserts disclosed herein can enhance vector attachment, internalization, and / or payload expression in muscle cells.

[0191] AAV9 Adeno-associated virus (AAV) is a small, non-enveloped, single-stranded DNA (ssDNA) virus belonging to the Parvoviridae family. At least 12 distinct AAV serotypes have been identified from human and non-human primate sources (see DiMattia MA et al., (2012) J. Virology 86(12):6947 (the entire contents of which are incorporated herein by reference)). AAV9 is one of the human AAV serotypes that has improved transduction efficiency in cardiac and skeletal muscle, liver tissue, pancreatic tissue, and eyes 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 the viral proteins VP1, VP2, and VP3, as well as the assembly activating protein (AAP). All three VP proteins are capsid monomers. Transcription of the Cap gene generates two messenger RNAs: one encoding VP1 and the other encoding VP2 and VP3 (as described in Warrington KH et al., (2004) Journal of Virology volume 78 (12) pages 6595-6609). VP1, VP2, and VP3 exist in a ratio of 1:1:10, respectively. The VP3 region has been observed in the capsid structures of all AAV serotypes that have been studied (DiMattia 2012).

[0193] VPs contain beta strands, alpha helical regions, and structurally variable regions (VRs) within the surface loops connecting the beta strands. Without wishing to be bound by any particular theory, differences in the sequence and / or conformation of the VRs are believed to contribute to variations in cell tropism, tissue transduction efficiency, and / or antigen reactivity among different AAV serotypes. In some embodiments, differences in VR sequence and / or structure among different AAV serotypes enable differential recognition of cell surface glycans and / or tissue-specific protein or lipid receptor interactions for internalization.

[0194] Wild-type AAV9 (WT AAV9) contains 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 acids 262-269. AAV VR-II encompasses amino acids 327-332 and plays a role, for example, in genome packaging. AAV9 VR-III encompasses amino acids 382-386. AAV9 VR-IV encompasses amino acids 452-460 and plays a role, for example, in liver transduction and / or delayed blood clearance phenotypes. AAV9 VR-V encompasses amino acids 488-505 and plays a role, for example, in LamR receptor binding, liver- and / or muscle-specific transduction, and / or delayed blood clearance phenotypes. AAV9 VR-VI includes amino acids 527-539 and plays a role in, for example, LamR receptor binding and / or delayed blood clearance phenotypes. AAV9 VR-VII includes amino acids 545-558 and plays a role in, for example, liver transduction and / or delayed blood clearance phenotypes. AAV9 VR-VIII includes amino acids 581-593 and plays a role in, for example, LamR receptor binding and / or transduction. AAV9 VR-IX includes amino acids 704-714 and plays a role in, for example, cardiac tropism, melanoma tropism, and / or altered tropism.

[0195] In some embodiments, the rAAV particles disclosed herein are recombinant AAV (rAAV) particles. In some embodiments, the rAAV particles comprise a variant AAV9 capsid protein comprising a peptide insert disclosed herein. In some embodiments, the peptide insert is any one or all of, or a combination of, the VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and VR-IX of the parent AAV capsid protein.

[0196] In some embodiments, the parent AAV capsid protein comprises a sequence of a wild-type AAV capsid protein, or a sequence having at least 95% identity to the sequence of a wild-type AAV capsid protein, or a sequence having 20 or fewer mutations (e.g., substitutions) compared to the sequence of a wild-type AAV capsid protein. In some embodiments, the parent 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 wild-type AAV capsid protein. In some embodiments, the parent AAV capsid protein comprises a sequence having 1 or fewer, 2 or fewer, 5 or fewer, 10 or fewer, or 20 or fewer mutations (e.g., substitutions) compared to the sequence of a wild-type AAV capsid protein.

[0197] In some embodiments, the parent 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 wild-type AAV capsid protein and one or more mutations, e.g., as disclosed herein.

[0198] In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in VP2 or VP3, or any combination thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0199] In some embodiments, the parent AAV capsid protein is other than an AAV9 capsid protein and comprises one or more mutations (e.g., liver targeting inhibitory mutations) in VP1, VP2, or VP3 corresponding to positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein that alter the binding profile of the parent AAV capsid protein.

[0200] In some embodiments, the one or more mutations comprise a mutation in an amino acid sequence at or near the glycan binding region. In some embodiments, the one or more mutations reduce glycan binding. In some embodiments, the glycan is galactose.

[0201] In some embodiments, the one or more mutations comprise mutations at the following positions: (a) positions 271 and 272 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (b) positions 446 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (c) positions 470 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; or (d) positions 470 of VP1, VP2, or VP3 of AAV9 capsid protein. (e) 501 and 505 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein (e.g., any one or all of residues 501, 502, 503, 504, or 505, or a combination thereof), or a corresponding position in the capsid protein of another parent AAV capsid protein; (f) 591 and 621 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; or (g) any combination, or all, of (a) through (f).

[0202] In some embodiments, the parent AAV capsid protein is selected 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, the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the 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 20 or fewer mutations (e.g., substitutions) compared to SEQ ID NO:2001. In some embodiments, the 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, the AAV9 capsid protein comprises a sequence having 1 or fewer, 2 or fewer, 5 or fewer, 10 or fewer, or 20 or fewer mutations (e.g., substitutions) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001.

[0204] In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.

[0205] In some embodiments, the one or more mutations include mutations that alter the binding profile (e.g., binding to one or more tissues) of the parent AAV capsid protein. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein include mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. Exemplary mutations, including liver-targeting-inhibiting mutations, are disclosed in Pulicherla N. et al., (2011) Molecular Therapy volume 19, pages 1070-1078, the entire contents of which are incorporated herein by reference.

[0206] In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibitory mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in VP2 or VP3, or any combination thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibitory mutations) comprise a mutation at position 503, e.g., a W503R mutation. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibitory mutations) comprise a mutation at position 595, e.g., a W595C mutation. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibitory mutations) comprise a mutation at position 457, e.g., a N457H mutation. In some embodiments, a mutation that alters the binding profile of a parent AAV capsid protein (e.g., a liver-targeting-inhibitory mutation) comprises a mutation at position 574, e.g., a T574S mutation. In some embodiments, a mutation that alters the binding profile of a parent AAV capsid protein (e.g., a liver-targeting-inhibitory mutation) comprises a mutation at position 592, e.g., a Q592L mutation. In some embodiments, a mutation that alters the binding profile of a parent AAV capsid protein (e.g., a liver-targeting-inhibitory mutation) comprises a mutation at position 498, e.g., an N498Y or N498I mutation. In some embodiments, a mutation that alters the binding profile of a parent AAV capsid protein (e.g., a liver-targeting-inhibitory mutation) comprises a mutation at position 602, e.g., an L602F mutation. In some embodiments, a mutation that alters the binding profile of a parent AAV capsid protein (e.g., a liver-targeting-inhibitory mutation) comprises a mutation at position 468, e.g., a P468T mutation. In some embodiments, the mutation that alters the binding profile of the parent AAV capsid protein (e.g., a liver-targeting inhibitory mutation) comprises a mutation at position 500, e.g., an E500D mutation.

[0207] In some embodiments, the one or more mutations comprise a mutation in an amino acid sequence at or near the glycan binding region. In some embodiments, the one or more mutations reduce glycan binding. In some embodiments, the glycan is galactose.

[0208] In some embodiments, the one or more mutations include mutations at the following positions: (a) residues 271 and 272 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (b) residue 446 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (c) residue 470 of VP1, VP2, or VP3 of AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein; (d) residues 501 and 505 of VP1, VP2, or VP3 of AAV9 capsid protein (e.g., residues 501 and 505 of VP1, VP2, or VP3 of AAV9 capsid protein). (e) positions 489 and 545 of VP1, VP2, or VP3 of AAV9 capsid protein, or corresponding positions in the capsid protein of another parent AAV capsid protein; (f) positions 591 and 621 of VP1, VP2, or VP3 of AAV9 capsid protein, or corresponding positions in the capsid protein of another parent AAV capsid protein; or (g) any combination or all of (a)-(f).

[0209] In some embodiments, the parent AAV capsid protein comprises an AAV1 capsid protein. In some embodiments, the AAV1 capsid protein sequence is provided in SEQ ID NO:2002.

[0210] In some embodiments, the parent AAV capsid protein comprises an AAV2 capsid protein. In some embodiments, the AAV2 capsid protein sequence is provided in SEQ ID NO:2003.

[0211] In some embodiments, the parent AAV capsid protein comprises an AAV3B capsid protein. In some embodiments, the AAV3B capsid protein sequence is provided in SEQ ID NO: 2050.

[0212] In some embodiments, the parent AAV capsid protein comprises an AAV4 capsid protein. In some embodiments, the AAV4 capsid protein sequence is provided in SEQ ID NO: 2051.

[0213] In some embodiments, the parent AAV capsid protein comprises an AAV5 capsid protein. In some embodiments, the AAV5 capsid protein sequence is provided in SEQ ID NO:2004.

[0214] In some embodiments, the parent AAV capsid protein comprises an AAV6 capsid protein. In some embodiments, the AAV6 capsid protein sequence is provided in SEQ ID NO:2005.

[0215] In some embodiments, the parent AAV capsid protein comprises an AAV7 capsid protein. In some embodiments, the AAV7 capsid protein sequence is provided in SEQ ID NO: 2052.

[0216] In some embodiments, the parent AAV capsid protein comprises an AAV8 capsid protein. In some embodiments, the AAV8 capsid protein sequence is provided in SEQ ID NO:2006.

[0217] In some embodiments, the parent AAV capsid protein comprises an AAV10 capsid protein. In some embodiments, the AAV10 capsid protein sequence is provided in SEQ ID NO: 2053.

[0218] In some embodiments, the parent AAV capsid protein comprises an AAV11 capsid protein. In some embodiments, the AAV11 capsid protein sequence is provided in SEQ ID NO: 2054.

[0219] In some embodiments, the parent AAV capsid protein comprises an AAV12 capsid protein. In some embodiments, the AAV12 capsid protein sequence is provided in SEQ ID NO: 2055.

[0220] In some embodiments, the parent AAV capsid protein comprises an AAV13 capsid protein. In some embodiments, the AAV13 capsid protein sequence is provided in SEQ ID NO: 2056.

[0221] In some embodiments, the parent AAV capsid protein comprises an AAVrh74 capsid protein. In some embodiments, the AAVrh74 capsid protein sequence is provided in SEQ ID NO:2057.

[0222] In some embodiments, the peptide insertion is in VR-I of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0223] In some embodiments, the peptide insertion is in VR-II of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0224] In some embodiments, the peptide insertion is in VR-III of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0225] In some embodiments, the peptide insertion is in VR-IV of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0226] In some embodiments, the peptide insertion is in a VR-V of a parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0227] In some embodiments, the peptide insertion is in VR-VI of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0228] In some embodiments, the peptide insertion is in VR-VII of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0229] In some embodiments, the peptide insertion is in VR-VIII of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0230] In some embodiments, the peptide insertion is in VR-IX of the parent AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74.

[0231] In some embodiments, the parent AAV capsid protein is selected from the group consisting of 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 VP1, VP2, or VP3 of the AAV9 capsid protein, or the corresponding position within the capsid protein of another parent AAV capsid protein, e.g., the AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74 capsid protein.

[0232] In some embodiments, the parent AAV capsid protein is an AAV9 capsid protein, and VR-VIII comprises amino acids 580-601 of AAV9 capsid protein VP1, VP2, or VP3.

[0233] In some embodiments, the peptide insert is within a VP (e.g., VP1, VP2, and / or VP3) of the parent AAV capsid protein.

[0234] In some embodiments, the peptide insertion site is located between amino acids 588 and 589 of the parent AAV capsid protein VP1, VP2, or VP3 (e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, or AAVrh74).

[0235] In some embodiments, the peptide insert is located between amino acids 588 and 589 of the AAV9 capsid protein VP1, VP2, or VP3.

[0236] In some embodiments, the peptide insert is located between amino acids 588 and 589 of the AAV9 capsid proteins VP1 and VP2.

[0237] In some embodiments, the peptide insert is located between amino acids 588 and 589 of the AAV9 capsid proteins VP1 and VP3.

[0238] In some embodiments, the peptide insert is located between amino acids 588 and 589 of the AAV9 capsid proteins VP2 and VP3.

[0239] In some embodiments, the peptide insert is located between amino acids 588 and 589 of AAV9 capsid proteins VP1, VP2, and VP3.

[0240] In some embodiments, the peptide insertion site is located between two adjacent amino acids of VR-VIII of the parent AAV capsid protein.

[0241] In some embodiments, the peptide insertion site is located between two non-adjacent amino acids of VR-VIII of the parent AAV capsid protein.

[0242] In some embodiments, the heterologous peptide insertion replaces a contiguous stretch of amino acids of the parent AAV capsid protein.

[0243] In some embodiments, the insertion of the heterologous peptide does not replace a contiguous stretch of amino acids of the parent AAV capsid protein.

[0244] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV9 capsid protein, e.g., compared to the WT AAV9 capsid protein. In some embodiments, the AAV9 WT capsid protein sequence is provided in SEQ ID NO: 2001. In some embodiments, the 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%, or at least 99% identity to the WT AAV9 capsid protein.

[0245] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV1 capsid protein, e.g., compared to the WT AAV1 capsid protein. In some embodiments, the AAV1 WT capsid protein is provided in SEQ ID NO: 2002. In some embodiments, the 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%, or at least 99% identity to the WT AAV1 capsid.

[0246] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV2 capsid protein, e.g., compared to the WT AAV2 capsid protein. In some embodiments, the AAV2 WT capsid protein is provided in SEQ ID NO: 2003. In some embodiments, the 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%, or at least 99% identity to the WT AAV2 capsid protein.

[0247] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV3B capsid protein, e.g., compared to the WT AAV3B capsid protein. In some embodiments, the AAV3B WT capsid protein is provided in SEQ ID NO: 2050. In some embodiments, the 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%, or at least 99% identity to the WT AAV3B capsid protein.

[0248] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV5 capsid protein, e.g., compared to the WT AAV5 capsid protein. In some embodiments, the AAV5 WT capsid protein is provided in SEQ ID NO: 2004. In some embodiments, the 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%, or at least 99% identity to the WT AAV5 capsid protein.

[0249] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV6 capsid protein, e.g., compared to the WT AAV6 capsid protein. In some embodiments, the AAV6 WT capsid protein is provided in SEQ ID NO: 2005. In some embodiments, the 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%, or at least 99% identity to the WT AAV6 capsid protein.

[0250] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV8 capsid protein, e.g., compared to the WT AAV8 capsid protein. In some embodiments, the AAV8 WT capsid protein is provided in SEQ ID NO: 2006. In some embodiments, the 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%, or at least 99% identity to the WT AAV8 capsid protein.

[0251] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV4 capsid protein, e.g., compared to the WT AAV4 capsid protein. In some embodiments, the AAV4 WT capsid protein is provided in SEQ ID NO: 2051. In some embodiments, the 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%, or at least 99% identity to the WT AAV4 capsid protein.

[0252] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV7 capsid protein, e.g., compared to the WT AAV7 capsid protein. In some embodiments, the AAV7 WT capsid protein is provided in SEQ ID NO: 2052. In some embodiments, the 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%, or at least 99% identity to the WT AAV7 capsid protein.

[0253] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV10 capsid protein, e.g., compared to the WT AAV10 capsid protein. In some embodiments, the AAV10 WT capsid protein is provided in SEQ ID NO: 2053. In some embodiments, the 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%, or at least 99% identity to the WT AAV10 capsid protein.

[0254] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV11 capsid protein, e.g., compared to the WT AAV11 capsid protein. In some embodiments, the AAV11 WT capsid protein is provided in SEQ ID NO: 2054. In some embodiments, the 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%, or at least 99% identity to the WT AAV11 capsid protein.

[0255] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV12 capsid protein, e.g., compared to the WT AAV12 capsid protein. In some embodiments, the AAV12 WT capsid protein is provided in SEQ ID NO: 2055. In some embodiments, the 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%, or at least 99% identity to the WT AAV12 capsid protein.

[0256] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV13 capsid protein, e.g., compared to the WT AAV13 capsid protein. In some embodiments, the AAV13 WT capsid protein is provided in SEQ ID NO: 2056. In some embodiments, the variant AAV13 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%, or at least 99% identity to the WT AAV13 capsid protein.

[0257] In some embodiments, the rAAV particles disclosed herein comprise a variant AAVrh74 capsid protein, e.g., compared to the WT AAVrh74 capsid protein. In some embodiments, the AAVrh74 WT capsid protein is provided in SEQ ID NO: 2057. In some embodiments, the 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%, or at least 99% identity to the WT AAVrh74 capsid protein.

[0258] Additional modifications to the AAV9 capsid protein (not including the peptide insertions disclosed herein) are possible, including, for example, variants disclosed in International Patent Application No. WO2003 / 052052, filed November 12, 2002, the entire contents of which are incorporated herein by reference. In some embodiments, the rAAV particles disclosed herein comprise a variant AAV9 capsid that includes a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in WO2003 / 052052.

[0259] Several other reports, 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, disclose modifications to the AAV9 capsid protein, the entire contents of each of which are incorporated herein by reference. In some embodiments, the rAAV particles disclosed herein comprise a variant AAV9 capsid containing a peptide insert disclosed herein and one or more AAV9 capsid modifications disclosed in any of the reports referenced herein.

[0260] AAV9 capsid variants containing peptide inserts Among other things, disclosed herein are AAV9 capsid protein variants having one or more peptide insertions, eg, as disclosed herein.

[0261] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV9 capsid comprising a peptide insert disclosed herein, in some embodiments, the peptide insert is within 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, the peptide insert is within VR-I of the AAV9 capsid protein.

[0263] In some embodiments, the peptide insert is within VR-II of the AAV9 capsid protein.

[0264] In some embodiments, the peptide insert is within VR-III of the AAV9 capsid protein.

[0265] In some embodiments, the peptide insert is within VR-IV of the AAV9 capsid protein.

[0266] In some embodiments, the peptide insert is within a VR-V of the AAV9 capsid protein.

[0267] In some embodiments, the peptide insert is within VR-VI of the AAV9 capsid protein.

[0268] In some embodiments, the peptide insert is within VR-VII of the AAV9 capsid protein.

[0269] In some embodiments, the peptide insert is within VR-VIII of the AAV9 capsid protein.

[0270] In some embodiments, the peptide insert is within the VR-IX of the AAV9 capsid protein.

[0271] In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of AAV9 capsid protein VP1, VP2, or VP3. In some embodiments, the peptide insertion site is located between amino acids 580-601 of AAV9 capsid protein VP1, VP2, or VP3. In some embodiments, the peptide insertion site is located between amino acids 580-585, 585-590, 590-595, or 595-601 of AAV9 VP1, VP2, or VP3. In some embodiments, the 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, 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 AAV9 capsid protein VP1, VP2, or VP3.

[0272] In some embodiments, the peptide insertion site is located between amino acids 588 and 589 of the AAV9 capsid protein VP1, VP2, or VP3.

[0273] In some embodiments, the peptide insertion site is located between amino acids 588 and 589 of VP1 of the AAV9 capsid protein. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP2 of the AAV9 capsid protein. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP3 of the AAV9 capsid protein.

[0274] In some embodiments, the peptide insert is located between amino acids 588 and 589 of AAV9 capsid proteins VP1 and VP2. In some embodiments, the peptide insert is located between amino acids 588 and 589 of AAV9 capsid proteins VP2 and VP3. In some embodiments, the peptide insert is located between amino acids 588 and 589 of AAV9 capsid proteins VP1 and VP3. In some embodiments, the peptide insert is located between amino acids 588 and 589 of AAV9 capsid proteins VP1, VP2, and VP3.

[0275] In some embodiments, the peptide insertion site is located between two adjacent amino acids of VR-VIII of the AAV9 capsid protein.

[0276] In some embodiments, the peptide insertion site is located between two non-adjacent amino acids of VR-VIII of the AAV9 capsid protein.

[0277] In some embodiments, the insertion of the heterologous peptide replaces a contiguous stretch of amino acids of the parent AAV capsid protein, for example, the AAV9 parent capsid protein.

[0278] In some embodiments, the insertion of the heterologous peptide does not replace a contiguous stretch of amino acids of the parent AAV capsid protein, for example, the AAV9 parent capsid protein.

[0279] In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising a peptide insert disclosed herein and (2) the sequence of one or more VPs of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the peptide insert comprises a sequence listed in any one of Tables 1-11. In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides listed in Table 1 and (2) the sequence of one or more VPs of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides listed in Table 2 and (2) the sequence of one or more VPs of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 3 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 4 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 5 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 6 and (2) the sequence of one or more of the VPs (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein.In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 7 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 8 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 9 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 10 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3). In some embodiments, the rAAV particles comprise (1) a variant AAV9 capsid protein comprising any one of the peptides presented in Table 11 and (2) one or more sequences of a VP of the AAV9 capsid protein (e.g., VP1, VP2, and / or VP3).

[0280] In some embodiments, the rAAV particles disclosed herein comprise (1) a variant AAV capsid protein 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, or VP3) of an AAV9 capsid protein.

[0281] In some embodiments, the peptide insert does not include additional sequence at the N-terminus of the peptide sequence presented in any one of Tables 1-11.

[0282] In some embodiments, the peptide insert does not include additional sequence at the C-terminus of the peptide sequence presented in any one of Tables 1-11.

[0283] In some embodiments, the peptide insert does not include additional sequences at the N- and C-termini of the peptide sequences presented in any one of Tables 1-11.

[0284] All of the peptides presented in Tables 1-11 have greater than 5-fold enhanced skeletal muscle transduction compared to AAV9. Fold change data are presented in categories A, B, C, and D. Peptides in category A have greater than 5-fold enhanced skeletal muscle transduction compared to AAV9, peptides in category B have greater than 10-fold enhanced skeletal muscle transduction compared to AAV9, peptides in category C have greater than 15-fold enhanced skeletal muscle transduction compared to AAV9, and peptides in category D have greater than 20-fold enhanced skeletal muscle transduction compared to AAV9.

[0285] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0286] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein does not include RGDRDAL (SEQ ID NO: 975).

[0287] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein does not include YGVRGDRDAL (SEQ ID NO: 2025).

[0288] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1X2RX3 (SEQ ID NO: 1), where X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1X2R[W / F] (SEQ ID NO: 2026), where X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 2.

[0289] [Table 2-1] [Table 2-2]

[0290] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGD[W / F]QX1X2 (SEQ ID NO: 2027), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 3.

[0291] [Table 3-1] [Table 3-2] [Table 3-3]

[0292] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYQAV (SEQ ID NO: 1764).

[0293] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYQAV (SEQ ID NO: 1764).

[0294] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYQTL (SEQ ID NO: 1814).

[0295] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises 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 insert is not SKDRGDYQTL (SEQ ID NO: 2007).

[0296] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYQEL (SEQ ID NO: 2008).

[0297] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not LSRRGDYQEL (SEQ ID NO: 2009).

[0298] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDYQAV (SEQ ID NO: 1764), SKDRGDYQTL (SEQ ID NO: 2007), or LSRRGDYQEL (SEQ ID NO: 2009).

[0299] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2[W / F] (SEQ ID NO: 2028), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 4.

[0300] [Table 4-1] [Table 4-2] [Table 4-3]

[0301] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDHASW (SEQ ID NO: 191).

[0302] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDHASW (SEQ ID NO: 191).

[0303] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDHSGW (SEQ ID NO: 322).

[0304] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDHSGW (SEQ ID NO: 322).

[0305] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDHSTW (SEQ ID NO: 333).

[0306] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDHSTW (SEQ ID NO: 333).

[0307] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDHTQW (SEQ ID NO: 349).

[0308] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDHTQW (SEQ ID NO: 349).

[0309] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDHQNF (SEQ ID NO: 283).

[0310] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not ANTRGDHQNF (SEQ ID NO: 2010).

[0311] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert 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, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDPX1X2X3 (SEQ ID NO: 4), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDPX1X2[W / F] (SEQ ID NO: 2029), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 5.

[0313] [Table 5-1] [Table 5-2] [Table 5-3]

[0314] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1X2V (SEQ ID NO: 5), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGD[W / F]X1X2V (SEQ ID NO: 2030), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 6.

[0315] [Table 6-1] [Table 6-2] [Table 6-3]

[0316] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1QX2[W / F] (SEQ ID NO: 2031), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 7.

[0317] [Table 7-1] [Table 7-2] [Table 7-3]

[0318] In some embodiments, the peptide insert in the AAV capsid protein disclosed herein comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDVQNF (SEQ ID NO: 2011).

[0319] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not NGVRGDVQNF (SEQ ID NO: 2012).

[0320] In some embodiments, the peptide insert in the AAV capsid protein disclosed herein comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDHQNF (SEQ ID NO: 283).

[0321] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not ANTRGDHQNF (SEQ ID NO: 2010).

[0322] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence 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 insert is not NGVRGDVQNF (SEQ ID NO: 2012) or ANTRGDHQNF (SEQ ID NO: 2010).

[0323] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGD[W / F]X1SX2 (SEQ ID NO: 2032), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 8.

[0324] [Table 8-1] [Table 8-2]

[0325] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises 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 insert does not include RGDYVSV (SEQ ID NO: 1948).

[0326] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDYVSV (SEQ ID NO: 1948).

[0327] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYSSV (SEQ ID NO: 1882).

[0328] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDYSSV (SEQ ID NO: 1882).

[0329] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYHSF (SEQ ID NO: 1623).

[0330] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDYHSF (SEQ ID NO: 1623).

[0331] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYTSM (SEQ ID NO: 1906).

[0332] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises 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 insert is not STVRGDYTSM (SEQ ID NO: 2013).

[0333] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not QERRGDYTSM (SEQ ID NO: 2014).

[0334] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYASL (SEQ ID NO: 2015).

[0335] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not RGDYASL (SEQ ID NO: 2015).

[0336] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYNSL (SEQ ID NO: 2016).

[0337] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not SNSRGDYNSL (SEQ ID NO: 2017).

[0338] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYTSV (SEQ ID NO: 2018).

[0339] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises 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 insert is not STVRGDYTSV (SEQ ID NO: 2019).

[0340] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not SNSRGDYTSV (SEQ ID NO: 2020).

[0341] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYTST (SEQ ID NO: 2021).

[0342] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not SAMRGDYTST (SEQ ID NO: 2022).

[0343] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert does not include RGDYTSL (SEQ ID NO: 2023).

[0344] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F, with the proviso that the peptide insert is not TSQRGDYVSL (SEQ ID NO: 2024).

[0345] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence 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 insert 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 (SEQ ID NO:2019), SNSRGDYTSV (SEQ ID NO:2020), SAMRGDYTST (SEQ ID NO:2022), or TSQRGDYVSL (SEQ ID NO:2024).

[0346] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence LRGDX1X2X3 (SEQ ID NO: 8), wherein X1 and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence LRGDX1X2[W / F] (SEQ ID NO: 2033), wherein X1 and X2 are independently any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 9.

[0347] [Table 9]

[0348] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGDX1GLX2 (SEQ ID NO: 9), wherein X1 is Y, W, or F, and X2 is any amino acid. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence RGD[W / F]GLX (SEQ ID NO: 2034), wherein X is any amino acid. In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises RGDYGLX (SEQ ID NO: 2035), wherein X is any amino acid. In some embodiments, the peptide insert comprises a sequence presented in Table 10.

[0349] [Table 10]

[0350] In some embodiments, the peptide insert in the AAV capsid protein comprises any one of the peptides disclosed in Table 11.

[0351] [Table 11]

[0352] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDPIRW (SEQ ID NO: 772).

[0353] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDPQRW (SEQ ID NO: 802).

[0354] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDPSPW (SEQ ID NO: 833).

[0355] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDSERW (SEQ ID NO: 1088).

[0356] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYESR (SEQ ID NO: 1556).

[0357] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYLNT (SEQ ID NO: 1696).

[0358] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYLSV (SEQ ID NO: 1713).

[0359] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence 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, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYX1X2X3 (sequence number 2063), wherein X1 is E, X2 is E or R, and X3 is V or I.

[0361] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYX1X2X3 (sequence number 2068), wherein X1 is R, X2 is E or R, and X3 is V or I.

[0362] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYX1X2X3 (sequence number 2064), wherein X1 is E or R, X2 is E, and X3 is V or I.

[0363] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYX1X2X3 (sequence number 2067), wherein X1 is E or R, X2 is R, and X3 is V or I.

[0364] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYX1X2X3 (sequence number 2066), wherein X1 is E or R, X2 is E or R, and X3 is V.

[0365] In some embodiments, the peptide insertion in the AAV capsid protein disclosed herein comprises the sequence RGDYX1X2X3 (sequence number 2065), wherein X1 is E or R, X2 is E or R, and X3 is I.

[0366] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, the peptide insert in an AAV capsid protein disclosed herein consists of the sequence of RGDYERI (SEQ ID NO: 1551).

[0367] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2047, as shown below:

[0368] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYERIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPH TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL.

[0369] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence 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 insert is not NATRGDYREI (SEQ ID NO: 2069).

[0370] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence 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 insert is not RRGDYREIPL (SEQ ID NO: 2059).

[0371] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence of RGDYREII (SEQ ID NO: 1825). In some embodiments, the peptide insert in an AAV capsid protein disclosed herein consists of the sequence of RGDYREI (SEQ ID NO: 1825).

[0372] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2046, as shown below:

[0373] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYREIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPH TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0374] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein that comprises or has the sequence of RGDYREI (SEQ ID NO: 1825) does not include a T residue immediately upstream (or 5') of the peptide insert.

[0375] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein that comprises or has the sequence of RGDYREI (SEQ ID NO: 1825) does not include an N residue at the third position immediately upstream (or 5') of the peptide insert.

[0376] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein that comprises or has the sequence of RGDYREI (SEQ ID NO: 1825) does not include an R residue immediately upstream (or 5') of the peptide insert.

[0377] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein that comprises or has the sequence of RGDYREI (SEQ ID NO: 1825) does not include a P residue immediately downstream (or 3') of the peptide insert.

[0378] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein that comprises or has the sequence of RGDYREI (SEQ ID NO: 1825) does not include an L residue at the second position immediately downstream (or 3') of the peptide insert.

[0379] In some embodiments, the peptide insert in an AAV capsid protein disclosed herein comprises the sequence of RGDYREV (SEQ ID NO: 1829). In some embodiments, the peptide insert in an AAV capsid protein disclosed herein consists of the sequence of RGDYREV (SEQ ID NO: 1829).

[0380] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2058, as shown below:

[0381] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYREVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0382] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV9 capsid protein comprising a peptide insertion disclosed in any one of Tables 1-11 and one or more modifications in the amino acid sequence adjacent to the peptide insertion site. In some embodiments, the 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, the one or more modifications are located in variable region IV (VR-IV) of VP1, VP2, or VP3 of the AAV9 capsid protein, or variable region V (VR-V) of VP1, VP2, or VP3 of the AAV9 capsid protein, or both.

[0384] In some embodiments, the VR-IV of AAV9 capsid protein VP1, VP2, or VP3 comprises amino acids 451 to 475 of AAV9 capsid protein VP1, VP2, or VP3.

[0385] In some embodiments, the VR-V of AAV9 capsid protein VP1, VP2, or VP3 comprises amino acids 488 to 506 of AAV9 capsid protein VP1, VP2, or VP3.

[0386] In some embodiments, the one or more modifications comprise an insertion, a deletion, a mutation, or a combination thereof.

[0387] In some embodiments, the variant AAV capsid protein further comprises one or more modifications in the amino acid sequence at or near the glycan binding region. In some embodiments, the one or more modifications reduce glycan binding. In some embodiments, the glycan is galactose.

[0388] In some embodiments, the one or more modifications are at or between the following amino acids: (a) 271 and 272 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein, (b) 446 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein, (c) 470 of the AAV9 capsid protein, or a corresponding position in the capsid protein of another parent AAV capsid protein, (d) 501 and 505 of the AAV9 capsid protein (e.g., any one or more of residues 501, 502, 503, 504, or 505). (e) positions 489 and 545 of VP1, VP2, or VP3 of AAV9 capsid protein, or corresponding positions in the capsid protein of another parent AAV capsid protein; (f) positions 591 and 621 of VP1, VP2, or VP3 of AAV9 capsid protein, or corresponding positions in the capsid protein of another parent AAV capsid protein; or (g) any combination or all of (a)-(f).

[0389] Exemplary Capsid Variants Containing a Peptide Insertion in VR-VIII In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein, in which (1) the peptide insertion comprises any one of the peptide insertions disclosed herein, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein.

[0390] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 1, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0391] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDX1X2RX3 (SEQ ID NO: 1), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0392] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion listed in Table 2, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0393] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDX3QX1X2 (SEQ ID NO: 2), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0394] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 3, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or any combination thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0395] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDHX1X2X3 (SEQ ID NO: 3), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0396] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 4, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0397] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDPX1X2X3 (SEQ ID NO: 4), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0398] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 5, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile (e.g., binding to one or more tissues) of the parent AAV capsid protein. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0399] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDX3X1X2V (SEQ ID NO: 5), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0400] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 6, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0401] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDX1QX2X3 (SEQ ID NO: 6), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0402] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 7, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0403] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDX3X1SX2 (SEQ ID NO: 7), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0404] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 8, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0405] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence LRGDX1X2X3 (SEQ ID NO: 8), where X1 and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0406] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion listed in Table 9, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0407] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDX1GLX2 (SEQ ID NO: 9), where X1 is Y, W, or F and X2 is any amino acid; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0408] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 10, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0409] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDYX1X2I (SEQ ID NO: 10), where X1 is E or R and X2 is E or R; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0410] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to a parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion as set forth in Table 11, (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0411] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence RGDYX1X2X3 (SEQ ID NO: 2065), where X1 is E or R, X2 is E or R, and X3 is I; (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein; and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0412] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551), (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, the peptide insertion is within VR-VIII of the AAV9 capsid protein. In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0413] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825), (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion consists of the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0414] In some embodiments, the rAAV particles disclosed herein comprise a variant AAV capsid protein comprising a peptide insertion relative to the parent AAV capsid protein. In some embodiments, (1) the peptide insertion comprises the sequence of RGDYREV (SEQ ID NO: 1829). (2) the peptide insertion site is within VR-VIII of the parent AAV capsid protein, and (3) the parent AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the peptide insertion consists of the sequence of RGDYREV (SEQ ID NO: 1829). In some embodiments, VR-VIII of the AAV9 capsid protein comprises amino acids 580-601 of VP1, VP2, or VP3. In some embodiments, the peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, the 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) compared to SEQ ID NO:2001. In some embodiments, the 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, the 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) compared to SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises the sequence provided in SEQ ID NO:2001. In some embodiments, the AAV9 capsid protein comprises a sequence that is at least 95% identical (e.g., at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or at least 99.5% identical) to SEQ ID NO: 2001 and has one or more mutations, e.g., as disclosed herein.In some embodiments, the one or more mutations comprise mutations that alter the binding profile of the parent AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein comprise mutations that reduce binding to one or more tissues, such as liver tissue, e.g., liver-targeting-inhibiting mutations. In some embodiments, the one or more mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise mutations at positions 503, 595, 457, 574, 592, 498, 602, 468, or 500 of VP1 of the AAV9 capsid protein, or corresponding positions in the VP2 or VP3 protein, or combinations thereof. In some embodiments, the mutations that alter the binding profile of the parent AAV capsid protein (e.g., liver-targeting-inhibiting mutations) comprise a W503R mutation.

[0415] Characterization of AAV9 capsid variants The AAV capsid variants disclosed herein have enhanced muscle tropism. In some embodiments, the AAV capsid variants disclosed herein are characterized in that, when administered to a cell or tissue or a subject, the AAV capsid variant confers increased infectivity and / or transduction of muscle cells compared to the infectivity and / or transduction of muscle cells by a control AAV particle comprising the corresponding parent AAV capsid protein.

[0416] In some embodiments, the variant AAV capsid protein confers at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 30-fold, at least a 40-fold, or at least a 50-fold increase in infectivity and / or transduction of muscle cells compared to infectivity and / or transduction of muscle cells by a control AAV particle comprising the corresponding parental AAV capsid protein.

[0417] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the variant AAV capsid proteins confer an increase in infectivity and / or transduction of muscle cells of 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 compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parent AAV capsid protein.

[0418] In some embodiments of the rAAV particles, variant AAV capsid proteins, compositions, or methods disclosed herein, the variant AAV capsid proteins confer an increase in infectivity and / or transduction of muscle cells of 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 compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parent AAV capsid protein.

[0419] In some embodiments, the variant AAV capsid is an AAV9 variant capsid.

[0420] In some embodiments, rAAV particles comprising an AAV capsid variant disclosed herein and a heterologous nucleic acid comprising a nucleotide sequence encoding a payload are characterized in that, when administered to a cell or tissue or a subject, delivery of the payload is enhanced to a muscle cell compared to delivery of the same payload by an otherwise similar AAV particle that does not comprise an AAV capsid variant disclosed herein.

[0421] In some embodiments, the muscle cells are selected from skeletal muscle cells, cardiac muscle cells, smooth muscle cells, muscle stem cells (eg, muscle satellite cells), or combinations thereof.

[0422] Payloads for use with AAV particles containing AAV9 capsid variants rAAV particles containing the AAV capsid variants disclosed herein can also contain a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

[0423] In some embodiments, the payload is a polypeptide, hi some embodiments, the payload polypeptide is selected 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, the polypeptide is or comprises a CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein is selected 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, a gRNA, an sgRNA, or a crRNA / tracrRNA that interacts with the CRISPR-Cas protein. In some embodiments, the CRISPR-Cas protein is fused to one or more domains, such as an activator domain and / or a repressor domain. In some embodiments, the CRISPR-Cas protein is a nuclease. In some embodiments, the CRISPR-Cas protein is a nickase and cleaves only one strand of the target nucleic acid molecule, hi some embodiments, the CRISPR-Cas protein inactivates the target nucleic acid molecule, binding to it but not cleaving it.

[0425] In some embodiments, the polypeptide is or comprises a zinc finger protein, or a variant or fragment thereof. In some embodiments, the zinc finger protein is selected from the following: a zinc finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeting zinc finger DNA-binding entity comprising a nuclease domain (e.g., a Fok1 nuclease domain) fusion protein, or a variant, fragment, or combination of any of the foregoing. In some embodiments, the zinc finger protein is fused to one or more domains, such as an activator domain and / or a repressor domain.

[0426] In some embodiments, the polypeptide is or comprises a transcription activator-like effector (TAL) protein, or a variant or fragment thereof. In some embodiments, the TAL comprises a TAL effector DNA-binding domain (e.g., a TAL effector DNA-binding domain isolated from a Xanthomonas species), a transcription activator-like effector nuclease (TALEN), e.g., a TAL effector DNA-binding domain fused to a nuclease domain (e.g., a Fok1 nuclease domain), or a variant, fragment, or combination of any of the foregoing. In some embodiments, the TAL protein is fused to one or more domains, e.g., an activator domain and / or a repressor domain.

[0427] In some embodiments, the polypeptide is or comprises a base editor, or a variant or fragment thereof. In some embodiments, the base editor is a deaminase, adenosine deaminase enzyme (ABE), cytosine deaminase enzyme (CBE), APOBEC1, APOBEC3A, APOBEC3G, evoAPOBEC, BE4-YE1, CDA1, activation-induced cytidine deaminase (AID), mutant TadA, adenosine deaminase (TadA*), E. coli The payload may comprise a tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA-binding domain monomer, an RNA-guided base editing enzyme, a DNA glycosylase 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 targeted-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax, a circular permutation base editor, an adenosine deaminase enzyme (ADAR), a programmable RNA editing for adenosine to inosine substitution (REPAIR), an exploited endogenous ADAR for programmable editing of RNA (LEAPER), or a variant, 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, the polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising same. In some embodiments, the prime editor and / or system comprising same comprises: a reverse transcriptase, a prime editing enzyme, an editing enzyme comprising a reverse transcriptase domain, avian myeloblastosis virus (AMV) reverse transcriptase, murine leukemia virus (MLV) reverse transcriptase, HIV-1 reverse transcriptase, bacterial reverse transcriptase, a DNA-binding domain and / or a reverse transcriptase associated with a 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, 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, the polypeptide is or comprises a meganuclease, or a variant or fragment thereof. In some embodiments, the meganuclease is selected from a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His-Cyst box family meganuclease, or a HNH family endonuclease, I-SeeI, I-CeuI, PI-PspI, PI-SceI, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, I-TevIII, or a variant or fragment or combination of any of the foregoing.

[0430] In some embodiments, the polypeptide is associated with a muscle disorder or a glycogen or sugar storage disorder.

[0431] In some embodiments, the muscle disorder is selected from X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Lim-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

[0432] In some embodiments, the polypeptide is an enzyme. In some embodiments, the enzyme is a lysosomal enzyme or an adenosine deaminase enzyme.

[0433] In some embodiments, the polypeptide is an antibody.

[0434] In some embodiments, the polypeptide is a secreted protein.

[0435] In some embodiments, the payload is an RNA molecule. In some embodiments, the RNA molecule is an siRNA, miRNA, gRNA, antisense RNA, snRNA, circular RNA, or an aptamer, or a combination thereof.

[0436] In some embodiments, the 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, the muscle disorder is selected from X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Lim-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

[0438] In some embodiments, the payload is a DNA molecule. In some embodiments, the DNA molecule comprises a nucleic acid sequence 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, the nucleotide sequence encoding the payload comprises a promoter. In some embodiments, the promoter is a muscle-specific promoter. In some embodiments, the muscle-specific promoter is selected from MHCK7, CK8, desmin, tMCK, dMCK, or CK6.

[0440] In some embodiments, the promoter is a dual muscle-liver promoter. In some embodiments, the dual muscle-liver promoter is SPc5-12.

[0441] Use of AAV particles containing AAV9 capsid variants The present disclosure provides, inter alia, methods for delivering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein to a cell or tissue, e.g., a muscle cell or tissue. The present disclosure also provides methods for 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.

[0442] In some embodiments, disclosed herein are methods for treating a muscle disorder in a subject, the method comprising administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein.

[0443] In some embodiments, also disclosed herein are methods for ameliorating symptoms of a muscle disorder in a subject, the method comprising administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein.

[0444] In some embodiments, the muscle disorder is selected from (a) a muscular dystrophy, e.g., X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, myotonic dystrophy, or facioscapulohumeral muscular dystrophy (FSHD); (b) a neuromuscular disease, e.g., Charcot-Marie-Tooth disease, myotonic dystrophy, nemaline myopathy, or facioscapulohumeral muscular dystrophy (FSHD); (c) a sugar or glycogen storage disease, e.g., MPS III disease or Pompe disease; (d) an expanded repeat disease, e.g., myotonic dystrophy or facioscapulohumeral muscular dystrophy (FSHD), or (e) a combination of any one or all of (a)-(d).

[0445] In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles 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 described herein is administered to a subject in combination with one or more additional therapeutic agents. In some embodiments, a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein is contacted with an organ, tissue, or cell ex vivo. The organ, tissue, or cell can be introduced into the subject and protected from damage otherwise caused by the recipient's immune system.

[0446] In some embodiments, the methods and kits of the present invention can be used for evaluating and / or monitoring gene therapy. In some embodiments, the gene therapy comprises administering a composition (e.g., a pharmaceutical composition) comprising a plurality of rAAV particles described herein. In some embodiments, a sample for evaluating and / or monitoring gene therapy can be obtained before the initiation of gene therapy. In some embodiments, a sample is obtained after the first gene therapy treatment or administration. In some embodiments, a sample is obtained after the completion of gene therapy. In some embodiments, a sample is taken at specific time points, intervals, or any other metric before, during, or after gene therapy is administered.

[0447] Methods for transfecting host cells using AAV particles containing AAV9 capsid variants The present disclosure provides, inter alia, methods for transfecting a host cell, the methods comprising combining a nucleic acid with a transfection reagent and introducing the mixture into a host cell under conditions that result in transfection of the host cell with the nucleic acid.

[0448] In some embodiments, the nucleic acids used in the methods disclosed herein comprise one or more vectors. In some embodiments, the nucleic acids disclosed herein comprise one or more vectors encoding (i) at least one payload flanked on either side by AAV inverted terminal repeats (ITRs), (ii) at least one AAV Rep polypeptide, (iii) at least one AAV Cap polypeptide, and / or (iv) at least one adenovirus helper polypeptide.

[0449] A host cell (e.g., a mammalian host cell, e.g., HEK293) can be transfected with at least one helper polypeptide (e.g., at least one Ad2 helper polypeptide), at least one Rep polypeptide or fragment thereof, at least one Cap polypeptide or fragment thereof, and at least one payload (e.g., for polypeptide expression or an inhibitor or guide nucleic acid).

[0450] In some embodiments, the transfection method disclosed herein is or comprises transient transfection. In some embodiments, the transient transfection method is suspension transient transfection (sTT). In some embodiments, the transient transfection method is adhesion transient transfection.

[0451] In some embodiments, the present disclosure provides transfected host cells comprising two, three, or four vectors described herein.

[0452] In some embodiments, the method comprises transfecting a host cell with three vectors, in some embodiments, the three vectors comprising: (i) at least one payload flanked on either side by AAV ITRs, (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 adenovirus helper polypeptide.

[0453] In some embodiments, the method comprises transfecting a host cell with two vectors, in some embodiments, the two vectors comprising (i) a first vector encoding at least one AAV Cap polypeptide and at least one payload flanked on either side by AAV ITRs, and (ii) a second vector encoding at least one adenoviral helper polypeptide and at least one AAV Rep polypeptide.

[0454] The transfection methods disclosed herein include transfection of nucleic acids (e.g., containing one or more vectors) with any transfection reagent known to those of skill in the art for introducing nucleic acid molecules into host cells (e.g., mammalian host cells, e.g., HEK293). In some embodiments, the transfection reagent comprises a lipid, a polymer, or a combination thereof. In some embodiments, the transfection reagent is a reagent capable of forming a complex with a nucleic acid.

[0455] In some embodiments, the transfection reagent comprises a polymer, a lipid, or both a polymer and a lipid. In some embodiments, the transfection reagent is or comprises a polymer. In some embodiments, the transfection reagent is or comprises a lipid. In some embodiments, the transfection reagent comprises a polymer and a lipid.

[0456] In some embodiments, the transfection reagent is or includes a polymer, e.g., a cationic polymer. In some embodiments, the transfection reagent includes polyethyleneimine (PEI), FectoVIR, TransIT-VirusGEN, or a combination thereof. In some embodiments, the transfection reagent is or includes polyethyleneimine (PEI).

[0457] In some embodiments, host cells are transfected with PEI. In some embodiments, host cells are transfected at 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, the wt ratio of DNA to transfection reagent depends on the cell culture density (e.g., the density of adherent or suspension host cells).

[0458] In some embodiments, the vector mass ratio of (i) a first vector encoding at least one payload, (ii) a second vector encoding at least one Rep polypeptide and / or at least one Cap polypeptide, and (iii) a third vector encoding at least one helper polypeptide used in the transfection methods disclosed herein is about 1:1:1. In some embodiments, the vector mass ratio of (i) a first vector encoding at least one payload, (ii) a second vector encoding at least one Rep polypeptide and / or at least one Cap polypeptide, and (iii) a third vector encoding at least one helper polypeptide is not about 1:1:1.

[0459] In some embodiments, the vector mass ratio of (i) a first vector encoding at least one payload, (ii) a second vector encoding at least one Rep polypeptide and / or at least one Cap polypeptide, and (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 The ratio is 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, the vector mass ratio of (i) a first vector encoding at least one payload, (ii) a second vector encoding at least one Rep polypeptide and / or at least one Cap polypeptide, and (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.

[0461] host cell The present disclosure provides, inter alia, host cells for transfection with at least one vector described herein for the production of rAAV particles. Host cells include progeny of an original cell transfected with at least one vector described herein. Progeny of a parent cell may not be substantially identical in morphology or genomic content to the parent cell due to natural, accidental, or deliberate mutations.

[0462] The components for the host cell to produce rAAV particles can be provided in trans in at least one vector. A stable host cell can contain at least one polypeptide for producing rAAV particles using methods known to those skilled in the art. In some embodiments, a stable host cell contains at least one polypeptide under the control of an inducible promoter. In other embodiments, a stable host cell contains at least one polypeptide under the control of a constitutive promoter. For example, a stable host cell (e.g., HEK293 cell) can contain a nucleic acid encoding an E1 helper polypeptide under the control of a constitutive promoter. Other stable host cells can be generated by those skilled in the art using routine methods.

[0463] Exemplary host cells include prokaryotes or eukaryotes (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species), mycobacterial 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 kidney cells (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, or BHK), CHO cells (e.g., CHO K1, DXB-1 1CHO, or Veggie-CHO), COS cells (e.g., COS-7), retinal cells, Vero cells, CV1 cells, HepG2 cells, WI38 cells, MRC5 cells, Colo205 cells, HB8065 cells, HL-60 cells (e.g., BHK21), Jurkat cells, Daudi cells, A431 cells (epidermal), CV-1 cells, U937 cells, 3T3 cells, L cells, C127 cells, SP2 / 0 cells, NS-0 cells, MMT060562 cells, Sertoli cells, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, or cell lines derived from the foregoing.

[0465] In some embodiments, the host cell comprises a kidney cell (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, or BHK). In some embodiments, the host cell comprises a HEK293 cell. In some embodiments, the host cell (e.g., a HEK293 cell) comprises or expresses an E1 polypeptide. In some embodiments, the host cell comprises or expresses an E1 polypeptide. In some embodiments, the host cell comprises a CHO cell (e.g., a CHO-K, DXB-1 1CHO, or Veggie-CHO). In some embodiments, the host cell comprises a CHO-K cell.

[0466] In some embodiments, the host cells are or comprise suspension cells, hi 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 of the host cells in culture are suspension.

[0467] In some embodiments, prior to transfection, host cells (e.g., adherent or suspension host cells) are seeded at a particular 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 10 4 Viable cells (vc) / cm 2 For example, at a density of about 1.0 x 10 4 vc / cm 2 ~Approx. 2.0×10 4 vc / cm 2 , for example, about 1.0 x 10 4 vc / cm 2 , about 1.1×10 4 vc / cm 2 , about 1.2×10 4 vc / cm 2 , about 1.3×10 4 vc / cm 2 , about 1.4×10 4 vc / cm 2 , about 1.5×10 4 vc / cm 2 , about 1.6×10 4 vc / cm 2 , about 1.7×10 4 vc / cm 2 , about 1.8×10 4 vc / cm 2 , about 1.9×10 4vc / cm 2 , or approximately 2.0 × 10 4 vc / cm 2 In some embodiments, prior to transfection, host cells (e.g., suspension host cells) are seeded at a density of at least 1.0 x 10 6 vc / cm 2 + / -15% density, e.g., 1.0 x 10 6 vc / cm 2 + / -15%~2.0×10 6 vc / cm 2 + / -15%, e.g., 1.0 x 10 6 vc / cm 2 + / -15%, 1.1 x 10 6 vc / cm 2 + / -15%, 1.2 x 10 6 vc / cm 2 + / -15%, 1.3 x 10 6 vc / cm 2 + / -15%, 1.4×10 6 vc / cm 2 + / -15%, 1.5 x 10 6 vc / cm 2 + / -15%, 1.6×10 6 vc / cm 2 + / -15%, 1.7×10 6 vc / cm 2 + / -15%, 1.8×10 6 vc / cm 2 + / -15%, 1.9 x 10 6 vc / cm 2 + / - 15%, or 2.0 x 10 6 vc / cm 2 They are sown at a density of + / - 15%.

[0468] vector Many forms of vectors can be used in the 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). Vector genetic elements can be delivered by any suitable method known in the art, for example, to those skilled in the art of nucleic acid manipulation, and can 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, NY).

[0469] In some embodiments, the vector encodes at least one helper polypeptide. In some embodiments, the vector encodes at least one Rep polypeptide and / or at least one Cap polypeptide. In some embodiments, the vector encodes at least one payload (e.g., for expression of a polypeptide or as an inhibitor or guide nucleic acid). In some embodiments, the vector encodes at least one helper polypeptide and at least one Rep polypeptide. In some embodiments, the vector encodes at least one Cap polypeptide and at least one payload.

[0470] Vectors can include conventional control elements operably linked to a nucleic acid encoding any of the polypeptides or payloads described herein in a manner that allows transcription, translation, and / or expression in cells transfected with the vectors described herein. 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 increase translation efficiency (e.g., Kozak consensus sequences), sequences that increase protein stability, and, if desired, sequences that increase secretion of the encoded product. Many expression control sequences, including promoters that are native, constitutive, inducible, and / or tissue-specific, are known in the art and can be included in the vectors described herein.

[0471] Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally containing the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally containing the CMV enhancer), the SV40 promoter, and the dihydrofolate reductase promoter.

[0472] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions (e.g., acute phase, a specific differentiation state of cells, or only in replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, tetracycline-repressible systems, tetracycline-inducible systems, RU486-inducible systems, and rapamycin-inducible systems. Still other types of inducible promoters that may be useful are regulated by specific physiological conditions such as temperature, acute phase, a specific differentiation state of cells, or only in replicating cells.

[0473] In another embodiment, the native promoter or a fragment thereof for any of the polypeptides or payload-encoding nucleic acids described herein can be used. In some embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

[0474] Vectors encoding helper polypeptides The present disclosure provides, inter alia, vectors (e.g., plasmids) encoding at least one helper polypeptide. AAV is a helper-dependent DNA parvovirus belonging to the Dependovirus genus. Production of recombinant 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 contain nucleotide sequences for non-AAV-derived viral and / or cellular functions on which AAV replication depends, including, but 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 virus, such as adenovirus, herpesvirus, vaccinia virus, or a combination thereof. Thus, a helper vector (e.g., a plasmid) for host cell culture can contain sufficient helper polypeptides to enable packaging of a recombinant AAV vector into an AAV capsid polypeptide.

[0476] In some embodiments, the helper vector comprises an Ad2 helper vector. In certain embodiments, the nucleic acid sequence of the Ad2 helper vector is derived from the adenovirus 2 genome (Genbank accession number J01917.1). In some embodiments, the helper vector comprises an Ad5 helper vector. In certain embodiments, the nucleic acid sequence of the Ad5 helper vector is derived from the adenovirus 5 genome (Genbank accession number AY601635).

[0477] Helper polypeptides can include at least one, two, three, or four of E1, E2A, E4, or VA RNA. In some embodiments, E1 includes E1a and / or E1b. In some embodiments, one or both of E2A and VA RNAs increase the stability and / or efficiency of AAV mRNA translation, such as cap gene transcripts. In some embodiments, E4 promotes DNA replication. In some embodiments, E1a includes a transactivator (e.g., that controls the activity of at least one Ad gene, the AAV rep gene, and / or the AAV cap gene). In some embodiments, E1b includes a viral mRNA transporter. 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 incorporated herein by reference in its entirety.

[0478] In some embodiments, the helper vector comprises a selection marker. Exemplary selection markers include, but are not limited to, an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene is not a gene encoding penicillin. In some embodiments, the antibiotic resistance gene is not a gene encoding a penicillin derivative. In some embodiments, the antibiotic resistance gene comprises an antibiotic resistance gene selected from kanamycin, puromycin, neomycin, hygromycin, blasticidin, gentamicin, Gr18, or zeocin. In certain embodiments, the antibiotic resistance gene comprises a kanamycin antibiotic resistance gene.

[0479] In some embodiments, the nucleic acids encoding the helper polypeptides are oriented in the same direction (e.g., 5' to 3') on the helper vector. In some embodiments, the nucleic acids encoding the helper polypeptides are transcribed in the same direction on the helper vector. In certain embodiments, the helper polypeptides comprise VA RNA and E4 oriented in the same direction on the helper vector. In certain embodiments, the helper polypeptides comprise E4 and E2A oriented in the same direction on the helper vector. In certain embodiments, the helper polypeptides comprise VA RNA, E4, and E2A oriented in the 5' to 3' direction on the helper vector. In some embodiments, the helper vector does not comprise a nucleic acid sequence encoding a fiber protein or a fragment thereof (e.g., does not comprise the nucleic acid sequence of Genbank Accession No. AP_000226.1 or a fragment thereof).

[0480] Vectors encoding Rep and / or Cap polypeptides The present disclosure provides, inter alia, ve...

Claims

1. (a) a variant AAV capsid protein, said variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein; (i) the peptide insert has the following sequence: (I) RGDX １ X ２ RX ３ (SEQ ID NO: 1) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (II) RGDX ３ QX １ X ２ (SEQ ID NO: 2) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (III)RGDHX １ X ２ X ３ (SEQ ID NO: 3) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IV) RGDPX １ X ２ X ３ (SEQ ID NO: 4) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (V) RGDX ３ X １ X ２ V (SEQ ID NO: 5) (wherein X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VI) RGDX １ QX ２ X ３ (SEQ ID NO: 6) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VII) RGDX ３ X １ SX ２ (SEQ ID NO: 7) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VIII) LRGDX １ X ２ X ３ (SEQ ID NO: 8) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IX) RGDX １ GLX ２ (SEQ ID NO: 9) (wherein, X １ is Y, W, or F, and X ２ is any amino acid), or (X)RGDYX １ X ２ X ３ (SEQ ID NO: 10) (wherein, X １ is E or R, and X ２ is E or R, and X ３ is V or I, (ii) the variant AAV capsid protein, wherein the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

2. (a) a variant AAV capsid protein, said variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein; (i) the peptide insert comprises a sequence set out in Table 1; (ii) the variant AAV capsid protein, wherein the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein; and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

3. 3. The rAAV particle of claim 1, wherein the insertion site is located between two adjacent amino acids within the variable region of the parent AAV capsid protein.

4. rAAV particle according to any one of the preceding claims, wherein the insertion of the heterologous peptide replaces a contiguous stretch of amino acids of the parent AAV capsid protein.

5. 10. The rAAV particle of any one of the preceding claims, wherein the peptide insert is within VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, or VR-IX of the parent AAV capsid protein.

6. 10. The rAAV particle of any one of the preceding claims, wherein the peptide insert is within VR-VIII of the parent AAV capsid protein.

7. The rAAV particle of claim 6, wherein the parent AAV capsid protein is an AAV9 capsid protein, and VR-VIII of AAV9 comprises amino acids 580 to 601 of AAV9 capsid protein VP1, VP2, or VP3.

8. 8. The rAAV particle of claim 6 or 7, wherein the insertion site is located between amino acids 588 and 589 of VP1 of the AAV9 capsid protein, or at a corresponding position within VP1, VP2, and / or VP3 of another parent AAV capsid protein.

9. 10. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises (1) a peptide insert comprising the consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2035, and (2) the sequence of one or more of the VPs (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein.

10. 10. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises (1) a peptide insert comprising the sequence of SEQ ID NO: 1551 and (2) the sequence of one or more of the VPs (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein.

11. 10. The rAAV particle of any one of claims 1 to 9, wherein the variant AAV capsid protein comprises (1) a peptide insert comprising the sequence of SEQ ID NO: 1825 and (2) the sequence of one or more of the VPs (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein.

12. 10. The rAAV particle of any one of claims 1 to 9, wherein the variant AAV capsid protein comprises (1) a peptide insert comprising the sequence of SEQ ID NO: 1829 and (2) the sequence of one or more of the VPs (e.g., VP1, VP2, and / or VP3) of the AAV9 capsid protein.

13. The rAAV particle of any one of claims 1 to 9, wherein the peptide insert comprises the consensus sequence of SEQ ID NO:

10.

14. 14. The rAAV particle of claim 13, wherein the peptide insert comprises a sequence set forth in Table 11.

15. 15. The rAAV particle of claim 13 or 14, wherein the peptide insert comprises the sequence RGDYERI (sequence number 1551).

16. 15. The rAAV particle of claim 13 or 14, wherein the peptide insert comprises the sequence RGDYREI (sequence number 1825).

17. 15. The rAAV particle of claim 13 or 14, wherein the peptide insert comprises the sequence RGDYREV (sequence number 1829).

18. rAAV particles according to any one of the preceding claims, wherein the variant AAV capsid protein confers at least a 5-fold increase in infectivity and / or transduction of muscle cells compared to infectivity and / or transduction of muscle cells by control AAV particles comprising the corresponding parental AAV capsid protein.

19. rAAV particles according to any one of the preceding claims, wherein the muscle cells are selected from cardiac muscle cells, smooth muscle cells, skeletal muscle cells, muscle stem cells (e.g., muscle satellite cells), or combinations thereof.

20. 3. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein further comprises one or more modifications in the amino acid sequence adjacent to 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. rAAV particle according to any one of the preceding claims, wherein the variant AAV capsid protein further comprises one or more modifications in an amino acid sequence at or near the glycan binding region.

23. rAAV particles according to any one of the preceding claims, wherein the variant AAV capsid protein has at least 90% identity to the parent AAV capsid protein.

24. 24. The rAAV particle of claim 23, wherein the percent identity is determined by comparing the sequence of the variant AAV capsid protein without the peptide insert to the parent AAV capsid protein.

25. The variant AAV capsid protein and the parent AAV capsid protein are (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 has 100% identity when it does not have one or more modifications other than the peptide insertion.

26. The variant AAV capsid protein and the parent AAV capsid protein are (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 has less than 100% identity when it contains one or more modifications other than the peptide insertion.

27. rAAV particles according to any one of the preceding claims, wherein the parent AAV capsid protein is the AAV9 capsid protein of SEQ ID NO: 2001.

28. rAAV particles according to any one of the preceding claims, wherein the payload is a polypeptide.

29. the polypeptide (i) a CRISPR-Cas protein or a variant or fragment thereof; (ii) a zinc finger protein or a variant or fragment thereof; (iii) TAL or a variant or fragment thereof; (iv) a base editor or a variant or fragment thereof; (v) a prime editor or a variant or fragment thereof, and / or (vi) The rAAV particle of claim 28, which is or comprises a meganuclease or a variant or fragment thereof.

30. 30. The rAAV particle of claim 29, wherein the polypeptide is associated with a muscle disorder or a glycogen or sugar storage disorder, and optionally the muscle disorder is selected from X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Limm-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral 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 of claims 1 to 27, wherein the payload is an RNA molecule.

33. 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, and optionally the muscle disorder is selected from X-linked myositis, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Limm-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

34. The rAAV particle of any one of claims 1 to 27, wherein the payload is a DNA molecule.

35. rAAV particle according to 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) an rAAV particle according to any one of the preceding claims; and (b) a pharmaceutically acceptable excipient.

38. 38. A method of delivering a payload to a muscle cell, comprising administering to said muscle cell the pharmaceutical composition of claim 37.

39. 39. The method of claim 38, wherein the muscle cells are derived from a subject having or determined to have a muscle disorder.

40. 38. A method of treating a subject having a myopathic disorder and / or ameliorating symptoms of a myopathic disorder in a subject, comprising administering to the subject a pharmaceutical composition of claim 37.

41. 41. The method of claim 39 or 40, wherein the muscle disorder is selected from X-linked myobular myopathy, spinal muscular atrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, Limm-Girdle muscular dystrophy, Emery-Dreifuss muscular dystrophy, faciohumeral muscular dystrophy (FSHD), Charcot-Marie-Tooth disease, nemaline myopathy, Pompe disease, or myotonic dystrophy.

42. 42. The method of any one of claims 38 to 41, wherein the pharmaceutical composition is administered via a route of administration selected from intramuscular, intracisternal, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, or limb perfusion.

43. The method of any one of claims 40 to 42, wherein the subject is a human.

44. An isolated cell transduced with an rAAV particle according to any one of claims 1 to 36.

45. 1. 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 parent AAV capsid protein; (i) the peptide insert has the following sequence: (I) RGDX １ X ２ RX ３ (SEQ ID NO: 1) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (II) RGDX ３ QX １ X ２ (SEQ ID NO: 2) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (III)RGDHX １ X ２ X ３ (SEQ ID NO: 3) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IV) RGDPX １ X ２ X ３ (SEQ ID NO: 4) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (V) RGDX ３ X １ X ２ V (SEQ ID NO: 5) (wherein X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VI) RGDX １ QX ２ X ３ (SEQ ID NO: 6) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VII) RGDX ３ X １ SX ２ (SEQ ID NO: 7) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VIII) LRGDX １ X ２ X ３ (SEQ ID NO: 8) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IX) RGDX １ GLX ２ (SEQ ID NO: 9) (wherein, X １ is Y, W, or F, and X ２ is any amino acid), or (X)RGDYX １ X ２ X ３ (SEQ ID NO: 10) (wherein, X １ is E or R, and X ２ is E or R, and X ３ is V or I, (ii) the isolated nucleic acid, wherein the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein.

46. 46. ​​An isolated cell comprising the nucleic acid of claim 45.

47. a variant AAV capsid protein, said variant AAV capsid protein comprising a peptide insertion relative to a corresponding parent AAV capsid protein; (i) the peptide insert has the following sequence: (I) RGDX １ X ２ RX ３ (SEQ ID NO: 1) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (II) RGDX ３ QX １ X ２ (SEQ ID NO: 2) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (III)RGDHX １ X ２ X ３ (SEQ ID NO: 3) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IV) RGDPX １ X ２ X ３ (SEQ ID NO: 4) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (V) RGDX ３ X １ X ２ V (SEQ ID NO: 5) (wherein X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VI) RGDX １ QX ２ X ３ (SEQ ID NO: 6) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VII) RGDX ３ X １ SX ２ (SEQ ID NO: 7) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VIII) LRGDX １ X ２ X ３ (SEQ ID NO: 8) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IX) RGDX １ GLX ２ (SEQ ID NO: 9) (wherein, X １ is Y, W, or F, and X ２ is any amino acid), or (X)RGDYX １ X ２ X ３ (SEQ ID NO: 10) (wherein, X １ is E or R, and X ２ is E or R, and X ３ is V or I, (ii) the variant AAV capsid protein, wherein the peptide insertion site is within a variable region (VR) of the parent AAV capsid protein.

48. 48. The variant AAV capsid protein of claim 47, wherein the peptide insertion is within VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, or VR-IX of the parent AAV capsid protein.

49. 1. A composition comprising a targeting moiety and a payload, wherein the targeting moiety has the sequence: (I) RGDX １ X ２ RX ３ (SEQ ID NO: 1) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (II) RGDX ３ QX １ X ２ (SEQ ID NO: 2) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (III)RGDHX １ X ２ X ３ (SEQ ID NO: 3) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IV) RGDPX １ X ２ X ３ (SEQ ID NO: 4) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (V) RGDX ３ X １ X ２ V (SEQ ID NO: 5) (wherein X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VI) RGDX １ QX ２ X ３ (SEQ ID NO: 6) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VII) RGDX ３ X １ SX ２ (SEQ ID NO: 7) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (VIII) LRGDX １ X ２ X ３ (SEQ ID NO: 8) (wherein, X １ and X ２ are independently any amino acid, and X ３ is Y, W, or F), (IX) RGDX １ GLX ２ (SEQ ID NO: 9) (wherein, X １ is Y, W, or F, and X ２ is any amino acid), or (X)RGDYX １ X ２ X ３ (SEQ ID NO: 10) (wherein, X １ is E or R, and X ２ is E or R, and X ３ is V or I).

50. 1. A composition comprising a targeting moiety and a payload, wherein the targeting moiety comprises a peptide comprising a sequence set out in Table 1.

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