WO2024184781A1 - Engineered aav capsids - Google Patents

Abstract

The present disclosure provides muscle trophic engineered AAV capsid polypeptides derived from capsid polypeptides from AAV1, AAV6, AAV8 and AAV9 serotypes; nucleic acids encoding same, engineered capsid helper plasmids, engineered AAV vectors comprising the engineered AAV capsid polypeptides, transfected cells, transduced cells, and methods of making and using same.

Description

ENGINEERED AAV CAPSIDS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Nos. 63/488,420, filed March 3, 2023, which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “008073-5244-WO-SEQLIST.xml”, which was created on February 28, 2024, and is 177,219 bytes in size.

FIELD OF THE DISCLOSURE

[0003] The disclosure relates to novel adeno-associated (AAV) capsid polypeptides with enhanced tropism for muscle.

BACKGROUND OF THE DISCLOSURE

[0004] AAV has a 4.7 kb single-stranded DNA genome containing two genes (Rep and Cap) flanked by two inverted terminal repeats (ITRs). The Rep gene produces four nonstructural Rep proteins (Rep78, Rep68, Rep52, and Rep40) from a single open reading frame (ORF) that are essential for viral genome replication and packaging. The Cap gene produces the three structural viral proteins (VPs: VP1, VP2, and VP3) translated from different start codons in a single ORF. Alternative mRNA splicing and the combined use of an ATG codon and an alternative start codon are used for the initiation of VP protein translation (Becerra et al. (1988) J. Virol. 62(8):2745-54; Trempe and Carter (1988) J. Virol. 62(9):3356-63, the disclosure of which is hereby incorporated herein by reference in its entirety). A second +1 frameshifted ORF that encodes a 204 amino acid nonstructural protein named assembly-activating protein (AAP) is important for the assembly of many AAV capsids (Sonntag et al. (201 1 ) ./. Virol.

85(23): 12686-97; Sonntag et al. (2010) Proc. Natl. Acad. Sci. USA 107(22): 10220-5; Earley et al. (2017) J. Virol. 91(3): 12686-97, the disclosures of which are hereby incorporated herein by reference in their entireties). AAV capsids are composed of 60 VP subunits at an approximate ratio of 1 : 1 : 10 of VP1 : VP2: VP3, and each subunit has nine variable regions that are located on the capsid surface and that thereby impact tropism and intracellular trafficking, and are typically the regions recognized by neutralizing antibodies (NAbs) (Xie et al. (2002) Proc. Natl. Acad. Sci. USA 99(16): 10405-10; Govindasamy etal. (2006) J. Virol. 80(23): 11556-70, the disclosure of which is hereby incorporated herein by reference in its entirety).

[0005] AAV capsids have been used extensively as vectors for gene therapy (Li and Samulski (2020) Nat. Rev. Genet. 21(4):255-72; Buning and Srivastava (2019) Mol. Ther. Meth. Clin. Dev. 12:248-65; Weinmann and Grimm (2017) Vir. Genes 53(5):707-13, the disclosures of which are hereby incorporated herein by reference in their entireties). When mice are dosed with AAV intravenously (i.v.), higher vector genomes and transgene expression in muscle have been reported with an AAV9 capsid when compared to an AAV6 capsid (Zincarelli et al. (2008) Mol. Ther. 16(6): 1073-80; Yang et al. (2009) Proc. Natl. Acad. Sci. USA 106(10):3946-51; Weber- Adrian et al. (2021) Sci. Rep. 11(1): 1934, the disclosures of which are hereby incorporated herein by reference in their entireties). Insertion of peptides containing an Arg- Gly-Asp (RGD) sequence into the VR-VIII of AAV9 has been shown to increase tropism to muscle when delivered i.v. to mice and non-human primates (NHPs) (Weinmann et al. (2020) Nat. Commun. 11(1):5432; Tabebordbar et al. (2021) Cell 184(19):4919-38 e22, the disclosures of which are hereby incorporated herein by reference in their entireties). Insertion of an RGD containing peptide into a chimeric AAV capsid (produced from shuffling the sequences of AAV1, AAV6, AAV8 & AAV9) has also been shown to have strong muscle tropism and liver de-targeting when delivered i.v. to mice (El Andari et al. (2022) Science Advances (38):eabn4704, the disclosure of which is hereby incorporated herein by reference in its entirety). However, AAV tropism can vary between animals and delivery routes. Notably, substantial differences between mice and NHPs have been reported in the muscle tropism profiles of AAV9 capsids containing RGD peptides (Tabebordbar et al. (2021) Cell 184(19): 4919-38 e22, the disclosure of which is hereby incorporated herein by reference in its entirety). Similar discrepancies in tropism between mouse and NHPs have been described in the low liver tropism of AAV3B in mice relative to NHPs (Li et al. (2015) Mol. Ther.

23(12): 1867-76; Wang etal. (2015) Mol. Ther. 23(12): 1877-87, the disclosures of which are hereby incorporated herein by reference in their entireties), and the blood brain barrier (BBB)- crossing and central nervous system (CNS) tropism of AAV.PHPeB in only certain strains of mice (Hordeaux et al. (2019) Afo/. Ther. 27(5):912-21; Huang etal. (2019) PLos One

14(1 l):e0225206, the disclosures of which are hereby incorporated herein by reference in their entireties).

[0006] A need therefore exists for AAV vectors that have optimal tropism for muscle cells and muscle tissues in humans, and primate animal models such as old-world NHPs like cynomolgus monkey, for the alleviation human disorders.

SUMMARY OF THE DISCLOSURE

[0007] The present disclosure provides engineered adeno-associated virus (AAV) capsid polypeptides, engineered AAV capsid nucleic acids and expression constructs, engineered capsid helper plasmids, engineered AAV vectors, transfected cells, transduced cells, and compositions thereof. The present engineered AAV vectors comprising the engineered AAV capsid polypeptides demonstrate multiple advantages over wild-type (wt) AAV serotypes, e.g., AAV1, AAV6, AAV8 and AAV9, including (i) enhanced tropism for one or more muscle tissues, (ii) reduced neutralization by host antibodies, and (iii) enhanced transduction in muscle cells and tissues, as measured by both engineered AAV vector DNA delivery and transgene RNA levels.

[0008] As described herein, the present disclosure is, in part, based on identification of one or more segments of amino acid sequence or amino acid residues that render an AAV capsid polypeptide highly specific for muscle tissues. In particular, an engineered AAV capsid polypeptide of the present disclosure has an enhanced tropism for muscle tissues, such as diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue muscle, as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes. As a result, an engineered AAV capsid polypeptide of the present disclosure can be used at a lower dose to achieve a therapeutic effect or a more efficient transfer of therapeutic or prophylactic compounds into muscle cells for use as production units, relative to AAV vectors comprising other, e.g., wild type, AAV capsid polypeptides. This therapeutic effect was demonstrated by the surprisingly high levels of engineered AAV vector DNA or transgene RNA in different cynomolgus NHP muscle cells and muscle tissues when delivered by the engineered AAV vector comprising the present engineered AAV capsid polypeptide. [0009] In one aspect, the present disclosure provides an engineered chimeric AAV capsid polypeptide comprising two or more segments of amino acids derived from different AAV serotypes selected from AAV1, AAV6, AAV8, and AAV9. The engineered chimeric AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide selected from any one of AAV1, AAV6, AAV8, and AAV9. In some embodiments, the two or more segments of amino acids include segment 1 of residues 24-42, segment 2 of residues 125-135, segment 3 of residues 158-169, segment 4 of residues 195-206, or segment 5 of residues 600-605, numbered relative to SEQ ID NO: 103.

[0010] In one embodiment, the engineered chimeric AAV capsid polypeptide comprises five segments of amino acids of which at least two segments are derived from two different AAV serotypes selected from AAV1, AAV6, AAV8 and AAV9. The engineered chimeric AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes.

[0011] According to the present disclosure, engineered AAV vectors with enhanced tropism for a muscle tissue are provided, as well as associated processes for their targeting, preparation, formulation, and use. The five segments of amino acids are segment 1 of residues 24-42, segment 2 of residues 125-135, segment 3 of residues 158-169, segment 4 of residues 195-206, and segment 5 of residues 600-605 numbered relative to SEQ ID NO: 103. As non-limiting examples, segment 5 of residues 600-605 is derived from AAV6. In some examples, segment 3 or segment 4 is derived from AAV1, AAV6, or AAV8. In some embodiments, segment 3 is derived from AAV8. In some embodiments, segment 4 is derived from AAV8. In some embodiments, segment 1 or segment 2 is derived from AAV1, AAV6, AAV8, or AAV9. As non-limiting examples, segment 1 is derived from AAV9. In other examples, segment 2 is derived from AAV9.

[0012] In some embodiments, the present disclosure provides an engineered chimeric AAV capsid polypeptide comprising five segments of amino acids according to:

Sl_a-S2_b-S3_c-S4d-S5e wherein SI is segment 1 of residues 24-42 and a is the AAV serotype from which SI is derived; wherein S2 is segment 2 of residues 125-135 and b is the AAV serotype from which S2 is derived; wherein S3 is segment 3 of residues 158-169 and c is the AAV serotype from which S3 is derived; wherein S4 is segment 4 of residues 195-206 and d is the AAV serotype from which S4 is derived; and wherein S5 is segment 5 of residues 600-605 and e is the AAV serotype from which S5 is derived.

[0013] In some embodiments, the present engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 -S2AAVI-S3AAVI-S4AAVI-S5AAV6 wherein SI comprises the amino acid sequence ELKPGAPKPKANQQKQDDG (SEQ ID NO: 202).

[0014] In some examples, the present engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAVl - S 2AAV1 -S3 AAVl - S4 AAV1 - S 5 AAV6.

[0015] In some examples, the present engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8- S 2AAV6- S 3 AAVl - S4 AAVl - S 5 AAV6.

[0016] In some examples, the present engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV9- S 2AAV9- S 3 AAV8- S4 AAV8- S 5 AAV6.

[0017] In some examples, the present engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV9- S 2AAV9- S 3 AAV8- S4 AAV8- S 5 AAVl .

[0018] In some examples, the present engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8- S2AAV1 -S3 AAV8- S4- S 5 AAV6 wherein S4 comprises amino acid sequence of APSGVGPTTMAS (SEQ ID NO: 203). [0019] In some embodiments, the present disclosure provides an engineered AAV capsid polypeptide with an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes, comprising an amino acid substitution relative to the parent AAV serotype at one or more positions corresponding to residues 24, 163, 169, 195, 197, 198, 587, and 601 of SEQ ID NO: 103. In some embodiments, the substitution at one or more positions are selected from an alanine (A) at position 24; a threonine (T) at position 163; an arginine (R) at position 169; an alanine (A) at position 195; a serine (S) at position 197; a glycine (G) at position 198; a leucine (L) at position 587; or a valine (V) at position 601.

[0020] In some embodiments, the present disclosure provides an engineered AAV capsid polypeptide with an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes, comprising a consensus sequence of SEQ ID NO: 103. In some embodiments, the amino acid residue at position 24 is A, D, or E; the amino acid residue at position 31 is Q or K; the amino acid residue at position 38 is H or K; the amino acid residue at position 41 is N or D; the amino acid residue at position 42 is A or G; the amino acid residue at position 56 is F or G; the amino acid residue at position 84 is K or Q; the amino acid residue at position 125 is L or V; the amino acid residue at position 129 is L or F; the amino acid residue at position 135 is A or G; the amino acid residue at position 152 is absent; the amino acid residue at position 158 is T or S; the amino acid residue at position 163 is K or T; the amino acid residue at position 169 is R or K; the amino acid residue at position 180 is S or T; the amino acid residue at position 195 is A or T; the amino acid residue at position 197 is S or A; the amino acid residue at position 198 is G or A; the amino acid residue at position 202 is N or T; the amino acid residue at position 206 is A or S; the amino acid residue at position 263 is S or N; at the amino acid residue at position 264 is A or S; the amino acid residue at position 265 is S or T; the amino acid residue at position 266 is S or T; the amino acid residue at position 267 is G or absent; the amino acid residue at position 269 is A or S; the amino acid residue at position 274 is H or A; the amino acid residue at position 420 is D or E; the amino acid residue at position 465 is absent; the amino acid residue at position 587 is L or F; the amino acid residue at position 601 is V or A; and the amino acid residue at position 645 is N or H.

[0021] In some embodiments, the present engineered AAV capsid polypeptide with an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes, comprises a consensus sequence of SEQ ID NO: 103, wherein the amino acid residue at position 24 is A; the amino acid residue at position 84 is K; the amino acid residue at position 129 is L; the amino acid residue at position 163 is K; the amino acid residue at position 169 is R; the amino acid residue at position 180 is S; the amino acid residue at position 195 is A; the amino acid residue at position 197 is S; and the amino acid residue at position 198 is G; and the amino acid residue at position 267 is G or is absent.

[0022] In some embodiments, the present engineered AAV capsid polypeptide with an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes, comprises a consensus sequence of SEQ ID NO: 103, wherein the amino acid residue at position 24 is A; at position 56 is F; at position 84 is K; at position 129 is L; at position 163 is K; at position 169 is R; position 180 is S; at position 195 is A; at position 197 is S; at position 198 is G; at position 267 is G or absent; at position 420 is D; at position 587 is L; at position 601 is V; and at position 645 is N. In some embodiments, the amino acid at position 267 is G.

[0023] In some embodiments, the present engineered AAV capsid polypeptide with an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes, comprises a consensus sequence of SEQ ID NO: 103, wherein the amino acid residue at position 56 is F; the amino acid residue at position 180 is S; the amino acid residue at position 263 is S; the amino acid residue at position 264 is A; the amino acid residue at position 265 is S; the amino acid residue at position 266 is T; the amino acid residue at position 267 is absent; the amino acid residue at position 269 is A; the amino acid residue at position 274 is H; the amino acid residue at position 587 is L; and the amino acid residue at position 601 is V.

[0024] In some embodiments, the engineered AAV capsid polypeptide with greater muscle tropism comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-152, or a variant that comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 104-152 wherein the variant maintains muscle tropism. In some embodiments, the engineered AAV capsid polypeptide with greater muscle tropism comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-152, or a variant that comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 104-152 wherein the variant maintains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70, at least 80%, at least 90%, or at least 95% of the level of muscle tropism. In some embodiments, the engineered AAV capsid polypeptide with greater muscle tropism comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-152, or a variant that comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 104-152 wherein the variant maintains the same level of muscle tropism. In one example, the engineered AAV capsid polypeptide with greater muscle tropism comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 105 but maintains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70, at least 80%, at least 90%, or at least 95% of the level of muscle tropism. In one example, the engineered AAV capsid polypeptide with greater muscle tropism comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 105 but maintains the same level of muscle tropism. In one example, the engineered AAV capsid polypeptide with greater muscle tropism comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 112 but maintains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70, at least 80%, at least 90%, or at least 95% of the level of muscle tropism. In one example, the engineered AAV capsid polypeptide with greater muscle tropism comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 112 but maintains the same level of muscle tropism.

[0025] In some embodiments, the engineered AAV capsid polypeptide of the present disclosure results in higher expression levels of transgene mRNA or other RNA transcript of interest, e.g., an siRNA, an shRNA, a guide RNA, a long non-coding RNA, a snoRNA, a pi i- interacting RNA (piRNA), snRNA, circRNA, and a microRNA, in muscle cells and/or muscle tissues as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8 and AAV9. In some embodiments, the engineered AAV capsid polypeptide of the present disclosure increases delivery of engineered AAV vector DNA to muscle cells and/or muscle tissues as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8 and AAV9. In some embodiments, the engineered AAV capsid polypeptide of the present disclosure can result in increased engineered AAV vector DNA delivery and increased expression of transgene mRNA or another RNA transcript of interest in muscle cells and/or muscle tissues.

[0026] In some embodiments, the muscle tissue is muscle of the diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and/or tongue. In some embodiments, the muscle cell can be a cardiac myocyte, a skeletal myocyte, or a smooth muscle cell.

[0027] As non-limiting examples, the engineered AAV capsid polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 105 and 109-115 results in higher expression levels transgene mRNA or another RNA transcript of interest in muscle. In other examples, the engineered AAV capsid polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 111, 112, 115, 117, 118, 120 and 124 can increase delivery of engineered AAV vector DNA to muscle.

[0028] In some embodiments, the engineered AAV capsid polypeptide of the present disclosure has reduced reactivity to neutralizing antibodies, e.g., human antibodies in plasma. [0029] In some embodiments, a nucleic acid sequence that encodes an engineered AAV capsid polypeptide described herein is provided. The nucleic acid encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 104-152, or a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to any one of SEQ ID NOs: 104-152. In some embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 105, 109-115, which encodes an engineered AAV capsid polypeptide that results in higher expression levels transgene mRNA or another RNA transcript of interest in muscle. In other embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 111, 112, 115, 117, 118, 120, and 124, which encodes an engineered AAV capsid polypeptide that increases delivery of engineered AAV vector DNA to muscle.

[0030] In another aspect, the present disclosure provides engineered capsid expression constructs such as plasmids, e.g., helper plasmids, comprising a nucleic acid encoding the engineered AAV capsid polypeptides described herein. The plasmid comprises a nucleic acid that encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 104- 152, or a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to any one of SEQ ID NOs: 104-152. In some embodiments, the plasmid comprises a nucleic acid that encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 105, 109-115, which encodes an engineered AAV capsid polypeptide that results in higher expression levels transgene mRNA or another RNA transcript of interest in muscle. In other embodiments, the plasmid comprises a nucleic acid that encodes a polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 111, 112, 115, 117, 118, 120, and 124, which encodes an engineered AAV capsid polypeptide that increases delivery of engineered AAV vector DNA to muscle.

[0031] In another aspect, the present disclosure provides compositions comprising engineered capsid helper plasmid.

[0032] In another aspect, the present disclosure provides a cell that has been transfected with an engineered capsid helper plasmid. In some embodiments, the cell is an in vitro cell or an ex vivo cell.

[0033] In another aspect, the present disclosure provides engineered AAV vectors comprising an engineered AAV capsid polypeptide described herein. In some embodiments, the engineered AAV vector has greater tropism to a muscle cell and/or muscle tissue.

[0034] In some embodiments, the engineered AAV vector is a recombinant AAV vector and the engineered AAV capsid polypeptide is a recombinant polypeptide.

[0035] In some embodiments, the engineered AAV vector comprises a nucleic acid comprising a transgene encoding at least one polypeptide that is not the engineered AAV capsid polypeptide. For example, the nucleic acid can encode a therapeutic gene of interest, e.g., for a therapeutic antibody or peptide vaccine. The nucleic acid sequence that does not encode the engineered AAV capsid polypeptide may be donor DNA for genome editing, a coding RNA, non-coding RNA, or other protein- or RNA-encoding sequence.

[0036] In some embodiments, the engineered AAV vector comprises a nucleic acid sequence encoding a non-coding RNA, such as an siRNA, an shRNA, a guide RNA, a long non-coding RNA, a snoRNA, a piwi-interacting RNA (piRNA), snRNA, circRNA, and a microRNA. [0037] In some embodiments, the engineered AAV vector comprises a nucleic acid sequence encoding a therapeutic protein or polypeptide, such as a muscle specific protein, a neuromuscular protein, a cardiac specific protein, or a protein not specifically expressed in muscle tissues but whose expression in muscle is intended to therapeutically cross correct other tissues.

[0038] In some embodiments, the engineered AAV vector comprises an expression cassette for expressing a transgene of interest, such as a therapeutic protein or polypeptide, or another RNA transcript of interest.

[0039] In some embodiments, the engineered AAV vector comprises one or more regulatory elements, including but not limited to a promoter sequence, an enhancer, an intron, a post- transcriptional regulatory element (e.g., WPRE), and a polyA sequence.

[0040] In accordance with the present disclosure, the engineered AAV vector comprising an engineered AAV capsid polypeptide described herein has reduced reactivity to neutralizing antibodies, therefore can avoid neutralization by the transduced host, e.g., human immune globulin.

[0041] In another aspect, the present disclosure provides a cell transduced with an engineered AAV vector comprising an engineered AAV capsid polypeptide described herein.

[0042] In another aspect, the present disclosure provides compositions comprising one or more of (a) an engineered AAV capsid polypeptide, (b) an engineered AAV capsid nucleic acid, (c) an engineered capsid helper plasmid, (d) an engineered capsid helper plasmid-transfected cell, (e) an engineered AAV vector- transduced cell, and/or (f) an engineered AAV vector described herein.

[0043] In another aspect, the present disclosure provides a pharmaceutical composition comprising one or more engineered AAV capsid polypeptides, AAV capsid nucleic acids, engineered capsid helper plasmids, engineered capsid helper plasmid-transfected cells, engineered AAV vector transduced cells, and engineered AAV vectors described herein, and a pharmaceutically acceptable carrier.

[0044] In yet another aspect, the present disclosure provides methods of making and using the engineered AAV capsid polypeptides, nucleic acids encoding same, engineered capsid helper plasmids, engineered AAV vectors comprising the engineered AAV capsid polypeptides, cells transfected with the engineered capsid helper plasmids, cells transduced with engineered AAV vectors, and compositions thereof. [0045] In some embodiments, the present disclosure provides a method for transfecting a cell (e.g., an El -expressing cell) with an engineered capsid helper plasmid encoding an engineered AAV capsid polypeptide described herein. For example, one approach to creating an engineered AAV vector of the disclosure is by triple transfection of (1) an engineered capsid helper plasmid, (2) a plasmid comprising a transgene of interest flanked by 5’ and 3’ ITRs, and (3) an adenovirus helper plasmid comprising nucleic acids encoding helper proteins (e.g., E2A, E4, and VARNA). [0046] In some embodiments, the present disclosure provides a method for transducing a muscle cell with an engineered AAV vector comprising a transgene of interest and an engineered AAV capsid polypeptide described herein, or a composition thereof.

[0047] In some embodiments, the muscle cell is a human muscle cell. In some embodiments, the muscle cell is selected from the group consisting of diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue. In some embodiments, the muscle cell is a cardiac myocyte, a skeletal myocyte, or a smooth myocyte. In some embodiments, the muscle cell is a cardiac myocyte.

[0048] In some embodiments, the transgene of interest encodes one or more reprogramming factors selected from the group consisting of ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2. In some embodiments, the method reprograms the cardiac cell into a cardiomyocyte.

[0049] In some embodiments, the present disclosure provides a method for introducing a heterologous nucleic acid to a muscle cell using an engineered AAV vector comprising an engineered AAV capsid polypeptide described herein, or a composition described thereof.

[0050] In some embodiments, the present disclosure provides a method for delivering a gene product to a target cell in a subject using an engineered AAV vector comprising an engineered AAV capsid polypeptide described herein, or a composition thereof.

[0051] In some embodiments, the present disclosure provides a method for treating a muscular disease in a subject using an engineered AAV vector comprising an engineered AAV capsid polypeptide described herein, or a composition thereof. The muscular disease may be any disease that affects muscle, e.g., a cardiac pathology, a genetic muscle disorder (e.g., muscular dystrophy), and a neuromuscular disorder. [0052] In some embodiments, the present disclosure provides a method for treating a subject using an engineered AAV vector comprising an engineered AAV capsid polypeptide described herein, or a composition thereof, for the delivery of a polynucleotide encoding a therapeutic polynucleotide, peptide vaccine, or non-coding RNA.

[0053] In some embodiments, the engineered AAV vector or composition may be administered to the subject by, e.g., epicardial injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade intracoronary injection, retrograde injection, transendomyocardial injection, molecular cardiac surgery with recirculating delivery (MCARD), isolated limb perfusion, subdermal injection, or cutaneous/topical application.

[0054] The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and all combinations of features described herein are contemplated, even if the combination of features is not found together in the same sentence or paragraph, or section of this document. Other features and advantages of the disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 shows an overview of an AAV capsid screening pipeline with delineation of Examples 1 to 4. A library with over 100 million AAV capsid variants was produced (Example 1), screened in cultured human muscle and cardiac cells (Example 2) and then dosed in cynomolgus NHPs (Example 3). Based on PacBio next-generation sequencing (NGS) analysis of the AAV capsid library’s DNA at key points, a barcoded library of 49 leads along with 5 benchmark capsids was produced, dosed into 3 cynomolgus NHPs and the engineered AAV vector DNA and transgene mRNA expression in key tissues was examined by Illumina® NGS analysis (Example 4). [0056] Figure 2 shows the biodistribution the AAV library in the indicated organs of the first non-human primate (NHP) study. The number of viral genomes per diploid genome (vg/dg) was determined two weeks post-injection using droplet digital polymerase chain reaction (ddPCR) analysis. Heart, liver, and spleen contained the highest vg/dg.

[0057] Figure 3 shows the phylogenetic tree of the 24 shuffled-only (SO 1-24) or 8 shuffled with peptide (SP1-8, gray highlight) capsids along with AAV1, AAV6, AAV8 and AAV9 (black highlight). Based on the phylogenetic tree, the engineered AAV capsid polypeptides were placed into three groups: Group 1, Group 2, and Group 3.

[0058] Figures 4A and 4B show graphs of the number of reads each capsid obtained in the barcoded library. Three Illumina® reads were performed to demonstrate reproducibility of the relative proportions of each barcoded vector: the top graph (Figure 4a) values present one set of reads obtained during analysis of the DNA extractions, while the bottom graph (Figure 4b) presents the average (+/- standard deviation) of two sets of reads obtained during analysis of RNA samples.

[0059] Figures 5A, 5B, 5C, 5D, 5E, 5F, and 5G collectively show an alignment of the amino acid sequences of the Group 1 and Group 2 engineered AAV capsid polypeptides with those of AAV1, AAV6, AAV8, and AAV9 and the consensus sequence (SEQ ID NO: 103) representing the engineered AAV capsid polypeptide sequences of Group 1 and Group 2. The lightly shaded “x” positions in the consensus sequence represent variable amino acid positions among the engineered AAV capsid polypeptide sequences. Black shaded positions in the consensus sequence represent variable amino acids among AAV1, AAV6, AAV8, and AAV9 serotypes that are conserved in the Group 1 and Group 2 engineered AAV capsid polypeptides. Shading of position 24 of SO21 represents a novel amino acid mutation.

[0060] Figure 6 shows the consensus sequence (SEQ ID NO: 103) for Group 1 and Group 2 with Segments 1-5.

[0061] Figure 7 shows the stacked average normalized DNA values of each engineered AAV capsid polypeptide for each muscle (see Tables 11-21).

[0062] Figure 8 shows the stacked average normalized RNA values of each engineered AAV capsid polypeptide for each muscle (see Tables 11-21).

[0063] Figure 9 shows vector genome per diploid genome (vg/dg) for collected tissues of the 2^nd NHP study (barcoded library). [0064] Figure 10 shows the percent of 50 human plasma samples that tested negative for neutralizing antibody screen at 1: 10, 1:20 or 1:40 dilutions for SO2, SO9, SO 12, SO 14, AAV1, AAV2, AAV3b, AAV5, AAV6, AAV8, AAV9 and AAVrh74.

[0065] Figures 11 A and 11B collectively show an alignment of the amino acid sequences of a subset of Group 1 AAV capsid polypeptides with the highest stacked muscle vector DNA values and a corresponding consensus sequence (SEQ ID NO: 204). The “x” positions in the consensus sequence represent variable amino acid positions among the engineered AAV capsid polypeptide sequences of the alignment.

[0066] Figures 12A and 12B collectively show an alignment of the amino acid sequences of a subset of Group 2 AAV capsid polypeptides with the highest stacked muscle vector DNA values and a corresponding consensus sequence (SEQ ID NO: 205). The “x” positions in the consensus sequence represent variable amino acid positions among the engineered AAV capsid polypeptide sequences of the alignment.

[0067] Figures 13A, 13B, and 13C collectively show an alignment of the amino acid sequences of a subset of Group 1 and Group 2 AAV capsid polypeptides with the highest stacked muscle vector DNA values and a corresponding consensus sequence (SEQ ID NO: 206). The “x” positions in the consensus sequence represent variable amino acid positions among the engineered AAV capsid polypeptide sequences of the alignment.

DETAILED DESCRIPTION

[0068] Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0069] The terms “about” and “approximately” include being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, e.g., within 50%, within 20%, within 10%, and within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art. [0070] The use of “or” means “and/or” unless stated otherwise. The terms “comprising,” “having,” “including (as well as other forms such as “includes” or “included”),” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit unless specifically stated otherwise.

[0071] Generally, nomenclatures used in connection with genetics and protein and nucleic acid chemistry, amplification, and hybridization, cell and tissue culture, molecular biology, immunology, and microbiology described herein are those well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0072] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0073] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein.

[0074] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.

[0075] That the disclosure may be more readily understood, select terms are defined below.

Definitions [0076] AAV: The term “adeno-associated virus” or “AAV” means a small (20 nm) replicationdefective, nonenveloped virus that has a linear single-stranded DNA (ssDNA) genome of approximately 4.8 kilobases (kb) that infects humans and non-human species. AAVs belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae. AAVs are not currently known to cause disease and cause only a very mild immune response. Gene therapy vectors using AAVs can “infect” or transduce both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell or integrating the genome at a low frequency. The wt genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact to form a capsid with icosahedral symmetry. With regard to gene therapy and transgene delivery, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV), or engineered AAV, vectors containing a heterologous sequence, e.g., a reporter or nucleic acid encoding a therapeutic gene product.

[0077] AAVP. The term “adeno-associated virus 1” or “AAV1” means AAV serotype 1. The term “AAV1 capsid polypeptide” means the amino acid chain of the AAV1 capsid protein. Initially described as a contaminant of adenovirus preparations (Hoggan et al. (1966) Proc. Natl. Acad. Sci. USA 55(6): 1467-74). AAV1 binds sialic acid and AAVR (Pillay et al. (2017) J. Virol. 91(18):e00391-17; Pillay et al. (2016) Nature 530(7588): 108-12.; Wu et al. (2006) J. Virol. 80(22): 11393-7). AAV1 has been explored as a therapeutic vector in the clinic to treat various disorders, including Al AT deficiency and Pompe disease (Mueller etal. (2017) Afo/. Ther. 25(6): 1387-94; Smith etal. (2013) Hum. Gene Ther. 24(6):630-40). The amino acid sequence for AAV1 capsid polypeptide is provided in Table 1 as SEQ ID NO: 1 and an exemplary nucleic acid encoding the AAV1 capsid polypeptide is provided in Table 2 as SEQ ID NO: 6.

[0078] AAV6: The term “adeno-associated virus 6” or “AAV6” means AAV serotype 6. AAV6 has been reported to bind heparin, sialic acid EGFR and AAVR (Li and Samulski (2020) Nat. Rev. Genet. 21(4):255-72). The 6 amino acids difference of the AAV6 capsid polypeptide and the AAV1 capsid polypeptide have been examined in vitro in terms of their influence on titer, heparin binding and tropism (Wu et al. (2006) J. Virol. 80(22): 11393-7). The term “AAV6 capsid polypeptide” means the amino acid chain of the AAV6 capsid protein. The amino acid sequence for AAV6 capsid polypeptide is provided in Table 1 as SEQ ID NO: 2 and an exemplary nucleic acid encoding the AAV6 capsid polypeptide is provided in Table 2 as SEQ ID NO: 7.

[0079] AAV8 The term “adeno-associated virus 8” or “AAV8” means AAV serotype 8. AAV8 has been reported to bind LamR and AAVR ((Li and Samulski (2020) Nat. Rev. Genet. 21(4):255-72). The term “AAV8 capsid polypeptide” means the amino acid chain of the AAV8 capsid protein. The amino acid sequence for AAV8 capsid polypeptide is provided in Table 1 as SEQ ID NO: 3 and an exemplary nucleic acid encoding the AAV8 capsid polypeptide is provided in Table 2 as SEQ ID NO: 8.

[0080] AAV9 The term “adeno-associated virus 9” or “AAV9” means AAV serotype 9. Has been reported to bind Galactose, LamR and AAVR ((Li and Samulski (2020) Nat. Rev. Genet. 21(4):255-72). The term “AAV9 capsid polypeptide” means the amino acid chain of the AAV9 capsid protein. The amino acid sequence for AAV9 capsid polypeptide is provided in Table 1 as SEQ ID NO: 4 and an exemplary nucleic acid encoding the AAV9 capsid polypeptide is provided in Table 2 as SEQ ID NO: 9.

[0081] AAVMYO'. The term “adeno-associated virus MYO” or “AAVMYO” is also known as AAV9P1 (Weinmann et al. (2020) Nat. Commun. 11(1): 5432; Kunze et al. (2018) Glia 66(2): 413 -27). The amino acid sequence for AAVMYO capsid polypeptide is provided in Table 1 as SEQ ID NO: 5 and an exemplary nucleic acid encoding the AAVMYO capsid polypeptide is provided in Table 2 as SEQ ID NO: 10.

[0082] Capsid gene'. The terms “cap nucleic acid,” “cap gene,” and “capsid gene” mean a nucleic acid that encodes a capsid protein. Examples of cap nucleic acids include wild-type (wt) cap-encoding nucleic acid sequences from AAV serotype 1 (AAV1), AAV serotype 6 (AAV6), AAV serotype 8 (AAV8), and AAV serotype 9 (AAV9); a native form cap cDNA; a nucleic acid having sequences from which a cap cDNA can be transcribed; and/or allelic variants and homologs of the foregoing.

[0083] Capsid protein'. The terms “capsid protein,” “capsid polypeptide,” “cap protein,” or “cap polypeptide” refer to an expression product of a cap nucleic acid from an AAV serotype that forms a protein shell for an AAV virus, such as a wt capsid protein from serotypes 1, 6, 8, or 9; or a protein that shares at least 50% (alternatively at least 75, 80, 85, 90, 95, 96, 97, 98, 99%, or 99.5%) amino acid sequence identity with a wt capsid protein and displays a functional activity of a wt capsid protein. The capsid homology of commonly used AAV serotypes is described in, e.g., Daya and Berns (2008) Clin. Microbiol. Rev. 21 (4): 583-593, which is incorporated herein by reference in its entirety. A “functional activity” of a protein is any activity associated with the physiological function of the protein, whether in vitro, ex vivo, or in vivo. For example, functional activities of an AAV capsid protein may include its ability to form a capsid, evade host antibodies, recognize, and enter a cell, deliver DNA genome to the nucleus and transcription of its DNA genome. In some embodiments, the capsid protein is a variant of the wt capsid protein with an altered functional activity such as tissue transduction or tissue tropism, e.g., into or to muscle, respectively. The term “encapsidates” or “packaged” means encloses or surrounds a gene or virus in a protein shell or capsid.

[0084] Unless otherwise indicated, the capsid polypeptides described herein refer to the VP1 form of the capsid polypeptide. However, it will be appreciated that these VP1 sequences also define the sequences of the VP2 form, having an internal starting point of residue 138 in the consensus sequence of SEQ ID NO: 103 as indicated in Figure 6, and the VP3 form, having an internal starting point of residue 204 in the consensus sequence of SEQ ID NO: 3 as indicated in Figure 6. Evidence also suggests that VP3 species having an internal starting point of residue 212 in the consensus sequence of SEQ ID NO: 3 also exist in nature.

[0085] Accordingly, the disclosure also provides engineered AAV VP2 capsid polypeptides having sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to the residues of AAV1 (SEQ ID NO: 1), AAV6 (SEQ ID NO: 2), AAV8 (SEQ ID NO: 3), and AAV9 (SEQ ID NO: 4) corresponding to residues 138-739 of consensus sequence SEQ ID NO: 3, that contain one or more amino acid substitutions relative to the wild type AAV serotype as described herein with reference to the VP1 form. Similarly, the disclosure provides engineered AAV VP3 capsid polypeptides having sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to the residues of AAV1 (SEQ ID NO: 1), AAV6 (SEQ ID NO: 2), AAV8 (SEQ ID NO: 3), and AAV9 (SEQ ID NO: 4) corresponding to residues 204-739, or 212-739, of consensus sequence SEQ ID NO: 3, that contain one or more amino acid substitutions relative to the wild type AAV serotype as described herein with reference to the VP1 form.

[0086] Likewise, the disclosure provides engineered AAV VP2 capsid polypeptides having sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to the residues of SEQ ID NOs: 104-127, and consensus sequences thereof, corresponding to residues 138-739 of consensus sequence SEQ ID NO: 3, that contain one or more amino acid substitutions relative to the wild type AAV serotype as described herein with reference to the VP1 form. Similarly, the disclosure provides engineered AAV VP3 capsid polypeptides having sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to the residues of SEQ ID NOs: 104-127, and consensus sequences thereof, corresponding to residues 204-739, or 212-739, of consensus sequence SEQ ID NO: 3, that contain one or more amino acid substitutions relative to the wild type AAV serotype as described herein with reference to the VP1 form.

[0087] Carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington: The Science and Practice of Pharmacy,” edited by Adeboye Adejare, Elsevier, 23^rd Edition (2020).

[0088] Chimeric. The term “chimeric” as used herein refers to an AAV capsid protein with regions and/or amino acids that are derived from two or more different serotypes of AAV. For example, a AAV capsid protein may include a first region, segment, or amino acid that is derived from or having high levels of sequence similarity or identity to a first AAV serotype or known recombinant AAV capsid protein, a second region, segment, or amino acid that is derived from or having high levels of sequence similarity or identity to a second AAV serotype or known recombinant AAV capsid protein, as well third, fourth, fifth, sixth, seventh, and eighth regions, segments, or amino acids, etc. derived from or having high levels of sequence similarity or identity to another AAV serotype or known recombinant AAV capsid protein.

[0089] Consensus sequence'. The term “consensus sequences” means a sequence of nucleic acids or amino acids that represents aligned identical or related sequences, defined by the most common nucleotide or amino acid residues at each position, where positions where a residue is variable are represented by an “X.” For example, the symbol “X” at position 24 indicates that the amino acid at position 24 can be any amino acids or any one from a defined subset of amino acids. The “position” of an amino acid or nucleic acid residue within a sequence means the location of the residue as counted from the amino terminus or 5’ end of the sequence, respectively. For example, an “amino acid at position 36” means the 36^th amino acid in a sequence as counted from the amino terminus of the polypeptide. In some alignments an “X” may be assigned to a position that is present in some sequences or the alignment and not present in other sequences in the alignment and so may be described as “absent” or “omitted.”

[0090] Effective'. The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity, result, or effect upon administration to a subject in need thereof. When a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0091] Engineered AAV Capsid Polypeptide'. The term “engineered AAV capsid protein” mean a non-naturally occurring AAV capsid polypeptide that is generated or engineered to create an amino acid sequence that would not otherwise be found in a wt AAV genome such as, for example, AAV1, AAV6, AAV8, and AAV9, as disclosed herein. In some embodiments, the combination or mutation of genetic material from more than one AAV serotype is used to create an engineered chimeric AAV capsid polypeptide. In some embodiments, an engineered AAV capsid protein is generated by changing a single amino acid in the sequence of a parental AAV1, AAV6, AAV8, or AAV9 capsid polypeptide, e.g., to a corresponding amino acid present at an equivalent position in a different parental AAV serotype. [0092] As described herein, the amino acid numbering system used for the engineered AAV capsid polypeptides described herein is dependent on whether the capsid polypeptide includes a particular segment of a parental AAV serotype that contains more or less amino acids than the same segment in a different parental AAV serotype. For example, as shown by the alignment in Figure 5, the AAV8 and AAV9 serotypes include an additional amino acid, relative to the AAV1 and AAV6 serotypes, at positions 152 (AAV8), 267 (AAV8 and AAV9), and 465 (AAV9) of the alignment. Accordingly, different AAV capsid polypeptides described herein have different lengths. For example, the parental AAV1 and AAV6 capsid polypeptides have 736 amino acids, the parental AAV8 and AAV9 capsid polypeptide has 738 amino acids, and the consensus sequence SEQ ID NO: 103 has 739 amino acids. As used herein, when referring to an engineered AAV capsid polypeptide sequence relative to a particular reference AAV capsid polypeptide sequence, e.g., a parental AAV capsid polypeptide sequence or a consensus AAV capsid polypeptide sequence, the amino acid numbers refer to the numbering of the reference AAV capsid polypeptide sequence. Because some of the reference AAV capsid polypeptide sequences have different lengths, the same amino acid may be referred to with a different number depending on which AAV capsid polypeptide is used as the reference sequence. For example, when referring to the valine residue at amino acid position 598 of the parental AAV6 capsid polypeptide sequence in the context of the consensus sequence SEQ ID NO: 103, the residue is V601. However, when referring to mutation of the AAV8 capsid polypeptide to include the same valine residue, the residue is V600, e.g., representing an S600V amino acid substitution. Unless otherwise indicated, the amino acid numbering described herein refers to the corresponding amino acid in the consensus sequence SEQ ID NO: 103.

[0093] Expression'. The term “expression” with regard to nucleic acids and proteins means the ability for a cell or organism to transcribe, as determined by measuring RNA levels, or translate, as determined by measuring protein levels. The term “expression cassette” means a distinct component of an expression construct such as a plasmid or vector consisting of a heterologous nucleic acid (e.g., a transgene) that encodes a protein, polypeptide, or an RNA transcript of interest of interest and the regulatory elements to be expressed by a transfected cell. [0094] Heterologous'. The term “heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a nucleic acid introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a “heterologous nucleic acid” or, alternatively, a “transgene.” A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked, is a heterologous promoter. Thus, for example, a recombinant AAV that includes a heterologous nucleic acid encoding a heterologous gene product is a recombinant AAV that includes a nucleic acid that is not normally associated with a naturally-occurring, wt AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, WT AAV.

[0095] In vivo, Ex vivo, and In vitro'. The term “in vivo,” as used herein, means within a living organism, e.g., a procedure on a living body. The term “ex vivo,” as used herein, means outside of the living organism, e.g., a procedure using an organ, tissue, or cell that has been taken from a living body. The term “in vitro,” as used herein, means outside of the living organism or normal biological context, e.g., a procedure with microorganisms, cells, tissues, or biological molecules, which may have been previously cultured or frozen, taking place in labware such as a test tube, culture dish, or microtiter plate.

[0096] Library. The term “library” means a population of particles (e.g., AAV vectors/particles, recombinant AAV vectors/particles), or molecules (e.g., DNA or RNA) containing a complex genome or a complex mRNA population, e.g., from a virus, cell, or tissue. For example, in a virus library, the member viruses are derived from viruses of the same species that differ by specific mutations (e.g., substitutions or variable amino acids) in the viral capsid polypeptide, and their combinations, are introduced. In some embodiments, the viral vector of each virus variant in the library comprises a single mutation in the viral capsid polypeptide. In some embodiments, the viral vector of each virus variant in the library comprises one or more variable amino acids in the viral capsid polypeptide. In some embodiments, the viral vector of each virus variant in the library comprise one or more variable amino acids and a peptide inserted into the viral capsid polypeptide. In certain embodiments, each virus variant in the library comprises exactly one specific combination of amino acid substitution(s) and/or a peptide inserted into the viral capsid polypeptide.

[0097] Mammal: The term “mammal” as used herein includes, but is not limited to, humans, non-human primates (e.g., monkeys and baboons), cattle, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, and the like), etc. [0098] Nucleic acid: The terms “nucleic acid” or “polynucleotide” mean a polymer or strand of two or more nucleotides. “Nucleotides” are monomers made of three components: a 5- carbon sugar, a phosphate group, and a nitrogenous base. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). If the sugar is ribose, the polymer is RNA; if the sugar is deoxyribose, the polymer is DNA. A strand of DNA is composed of adenine (A), cytosine (C), guanine (G), and/or thymidine (T). A strand of RNA is composed of adenine (A), cytosine (C), guanine (G), and/or uracil (U). An “isolated polynucleotide” is a nucleic acid that is separate and discrete from the whole organism or other cellular components with which the nucleic acid is found in nature. The terms “nucleic acid sequence,” “nucleotide sequence,” or “polynucleotide sequence” means the succession of nucleotide nitrogenous bases that indicate the order of the nucleotides in a polynucleotide polymer of DNA (using A, C, G, and T) or RNA (using A, C, G, or U). Thus, the term nucleotide sequence is the alphabetical representation of a polynucleotide molecule.

[0099] A nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, and derivatives thereof. The terms “encoding” and “coding” refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information, e.g., to a cell or non- cellular system, from which a series of amino acids can be assembled into a specific amino acid sequence to produce a polypeptide.

[00100] Operably linked. The term “operably linked” in reference to a nucleic acid means a nucleic acid that is placed in a functional relationship with another nucleic acid. For example, if a coding nucleic acid is operably linked to a promoter nucleic acid, this generally means that the promoter may promote transcription of the coding nucleic acid. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, since enhancers may function when separated from the promoter by several kilobases and intronic sequences may be of variable length, some nucleic acids may be operably linked but not contiguous, thereby operating “zn trans.”

[00101] Patient. The terms “patient,” “subject,” and “animal” are used interchangeably herein and refer to mammals, including, without limitation, human and non-human animals such as experimental animal models (e.g., mouse, rat, and monkey) as well as other animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.). In a particular embodiment, the subject is a human.

[00102] Pharmaceutically acceptable'. The phrase “pharmaceutically acceptable” when used in connection with compositions described herein means that components or excipients of such compositions are physiologically tolerable and do not typically produce adverse reactions when administered to a subject (e.g., a human). In certain embodiments, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals (e.g., in humans).

[00103] Polypeptide'. The term “polypeptide” means any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation, unless otherwise specified herein. Typically amino acids include the following full amino acid name (as well as their 3 letter abbreviation and 1 letter code): Alanine (Ala or A), Arginine (Argo or R), Asparagine (Asn or N), Aspartic acid (Asp or D), Cysteine (Cys or C), Glutamic acid (Glu or E), Glutamine (Gin or Q), Glycine (Gly or G), Histidine (His or H), Isoleucine (He or I), Leucine (Leu or L), Lysine (Lys or K), Methionine (Met or M), Phenylalanine (Phe or F), Proline (Pro or P), Serine (Ser or S), Threonine (Thr or T), Tryptophan (Trp or W), Tyrosine (Tyr or Y), and Valine (Vai or V). The term “protein” encompasses polypeptides as well as any post-translational modifications such as glycosylation, etc.

[00104] Prophylactic compound, Prophylactic protein, or Prophylactic polypeptide'. The term “prophylactic compound,” “prophylactic protein,” or “prophylactic polypeptide” can be a compound, protein, or polypeptide, respectively, that prevents or protects against disease, such as a preventative treatment, such as an antibiotic or vaccine.

[00105] Recombinant: The term “recombinant” and the like terms as used herein means that a nucleic acid, a protein or polypeptide, or a virus (e.g., AAV) contains portions of different origins that have been joined or combined together. Typically, this can be done with the insertion, deletion, or alteration of a nucleic acid or amino acid, respectively, in a nucleic acid sequence (DNA or RNA), such that a composite nucleic acid or a protein is formed.

[00106] Regulatory element. The term “regulatory element” means any cis-acting or transacting genetic element that controls some aspect of the expression of a nucleic acid, for example, a promoter sequence, an enhancer, and other post-transcriptional regulation elements. In some embodiments, the term “promoter” comprises essentially the minimal sequences required to initiate transcription. In some embodiments, some regulatory elements can upregulate or downregulate transcription, commonly termed “enhancer elements” and “repressor elements,” respectively.

[00107] Sequence identity. The term “sequence identity” when used in reference to polynucleotide or polypeptide sequences refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Techniques for determining nucleic acid and amino acid “sequence identity” also are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotide or polypeptide) can be compared by determining their “percent identity.” The percent identity of two sequences, whether polynucleotide or polypeptide sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Percent identity may be determined manually or by the use of computer programs, for example, using a BLAST computer program, such as version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268 and as discussed in Altschul et al. (1990) J. Mol. Biol. 215:403-410; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the proteins being compared. Default parameters are provided to optimize searches with short query sequences in, e.g., blastp. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen (1993) Comput. Chem. 17:149-163. Ranges of desired degrees of sequence identity are approximately 70% to 100% and integer values therebetween. Typically, the percent identities between a disclosed sequence and a claimed sequence are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. Unless specifically stated otherwise, sequence identity is determined using the BLAST algorithm, using default parameters.

[00108] Therapeutic compound, Therapeutic protein, or Therapeutic polypeptide '. The term “therapeutic compound,” “therapeutic protein,” or “therapeutic polypeptide” can be a compound, protein, or polypeptide, respectively, that can alleviate or reduce symptoms, e.g., that result from an absence or defect in a protein in a cell or subject, or that otherwise can confer a therapeutic benefit to a subject.

[00109] Transduction'. The term “transduction” means the transfer of genetic material into a cell generally by viral methods. In the context of the present disclosure, transduction of a cell with an engineered AAV vector means entry of the engineered AAV vector comprising a nucleic acid encapsidated or packaged with a capsid protein into a cell and transfer of the nucleic acid into the cell.

[00110] Transfection: The term “transfection” means the transfer of genetic material into a cell generally by non-viral methods, e.g., the transfection of a plasmid into a cell.

[00111] Transgene: The term “transgene” means a nucleic acid encoding a gene product that is expressed in a heterologous system such as a cell system, e.g., a gene of interest in an engineered AAV vector. In some embodiments, the gene product that is expressed is a polypeptide, e.g., a therapeutic antibody, peptide vaccine, etc. In some embodiments, the gene product is a non-coding RNA, such as an siRNA, an shRNA, a guide RNA, a long non-coding RNA, a snoRNA, a piwi-interacting RNA (piRNA), snRNA, circRNA, and a microRNA.

[00112] Tropism '. The term “tropism” with regard to a virus such as an AAV virus means the tendency for the virus to infect or transduce a particular cell or tissue. For example, the term “muscle tropism” means the tendency for the virus to infect or transduce muscle cells or muscle tissue instead of other tissues or relative to other viruses. Unless otherwise specified, the tropism of an engineered AAV capsid protein is measured using vector DNA or transgene expression (mRNA or protein) analysis of tissue as described in Weinmann et al. (2020) Nat. Commun. 11 (1): 5432, the disclosure of which is incorporated herein by reference in its entirety. Another example protocol is described in Tabebordbar et al. (2021) Cell 184(19):4919-38 e22, the disclosure of which is incorporated herein by reference in its entirety.

[00113] Reactivity to Neutralizing Antibodies: The term “reactivity to neutralizing antibodies” with respect to a virus such as an AAV virus means a level of which the virus is neutralized by antibodies from blood plasma. In some embodiments, reactivity to neutralizing antibodies is determined using an in vitro assay in which cells are exposed to an AAV virus carrying a transgene for a reporter in the presence of varying concentrations of blood plasma, e.g., blood plasma from a target species, and reduction of the output for the reporter assay is indicative of reactivity. In some embodiments, an engineered AAV vector comprising an engineered capsid polypeptide as described herein has reduced reactivity to neutralizing antibodies relative to a reference AAV vector, e.g., a wild type AAV1, AAV6, AAV8, or AAV9 vector, when the engineered AAV vector has at least 5% reduced reactivity, at least 10% reduced reactivity, at least 15% reduced reactivity, at least 20% reduced reactivity, at least 25% reduced reactivity, at least 30% reduced reactivity, at least 40% reduced reactivity, at least 50% reduced reactivity, at least 60% reduced reactivity, at least 70% reduced reactivity, at least 80% reduced reactivity, at least 90% reduced reactivity, at least 100% reduced reactivity, at least

125% reduced reactivity, at least 150% reduced reactivity, at least 175% reduced reactivity, at least 200% reduced reactivity, at least 250% reduced reactivity, at least 300% reduced reactivity, at least 400% reduced reactivity, or at least 500% reduced reactivity.

[00114] Treat. The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and/or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, e.g., by prophylactic treatment such as by vaccination; or (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving or curing the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[00115] Variant. The term “variant” as used herein refers to a modified or altered form of a wt AAV sequence, such as the amino acid sequence of a wt AAV capsid protein (e.g., SEQ ID NOs: 1-4) or the nucleotide sequence encoding a wt AAV capsid protein (e.g., SEQ ID NOs: 6- 9). The variant may contain an insertion, a deletion, or a substitution of at least one amino acid residue or nucleotide. The engineered AAV capsid polypeptides disclosed herein are AAV capsid variants.

[00116] Vector. The terms “vector” and “expression vector,” with regard to non- AAV viral vectors, mean the vehicle by which a DNA or RNA sequence (e.g., a heterologous nucleic acid or transgene) can be introduced into a host cell, so as to promote expression (e.g., transcription, or transcription and translation) of the introduced sequence. Such vectors include plasmids, synthesized RNA and DNA molecules, transposons, phages, viruses, etc. In certain embodiments, the vector is a viral vector such as, but not limited to, an adenoviral, alphaviral, herpes virus, lentiviral, retroviral, or vaccinia virus vector. The term “vector” will generally be used herein with respect to the “AAV vectors”, which has a different definition in the art, as defined below.

[00117] AAV Vector. The terms “AAV vector,” “AAV virion,” “AAV particle,” and “AAV virus” as used herein mean a capsid protein or polypeptide comprising one or more of three proteins known as VP1, VP2 and VP3 that has enveloped or packaged a single-stranded DNA molecule, e.g., a gene of interest such as a nucleic acid that encodes a therapeutic protein, flanked by inverted terminal repeats ("ITRs"). The term “engineered AAV vector” means an AAV vector comprising an engineered AAV capsid polypeptide of the disclosure. The term “engineered AAV vector DNA” means the DNA encapsidated or packaged by an engineered AAV capsid polypeptide of the disclosure to form an engineered AAV vector. An AAV vector thus comprises both a DNA component and a capsid polypeptide.

Engineered AA V Capsid Polypeptides

[00118] Adeno-associated viral (AAV) vectors are utilized as a key platform for gene therapy and gene delivery for the treatment of a variety of human diseases, as well as for the production of muscle derived vaccines. An ideal AAV vector should specifically and efficiently express high levels of the therapeutic transgene product in the desired target tissue, following a peripheral delivery of a low vector dose. In particular, capsids from different AAV serotypes can affect AAV transduction, transgene delivery efficacy, and target specificity (e.g., tissue and cell specificity). To obtain superior vectors, a variety of different technologies were developed to design AAV capsids. Comprehensive capsid libraries for subsequent screening were created, positive and/or negative selection pressure was employed, and optimal capsid candidate(s) were selected for pre-clmical evaluation,

[00119] Engineered AAV capsid polypeptides were identified by screening capsid libraries created through DNA family shuffling and peptide display using four benchmark AAVs: AAV1 , AAV6, AAV8 and AAV9. AAV vector genomes (vgs) were packaged into capsid variants selected from shuffling or parent benchmarks, and then qualitatively and quantitatively tracked in transduced animals by next-generation sequencing (NGS) at both the DNA and RNA levels. Groups of engineered AAV capsid polypeptides were identified that hold significant potential for gene transfer into the musculature, including smooth muscle, skeletal muscle, heart, and diaphragm.

[00120] In summary, the present disclosure identifies optimal AAV capsid variants with greater tropism to a muscle cell and muscle tissue. The disclosure enables a direct comparison of the engineered AAV capsid polypeptides’ ability to deliver vector DNA to muscle tissues and cells and expression of RNA and protein in the target tissues and/or cells. Furthermore, the engineered AAV capsid polypeptides were selected to have reduced capacity for reaction with neutralizing antibodies in a subject (e.g., a human patient).

Design of Engineered AA V Capsid Polypeptides

[00121] The present disclosure provides engineered AAV capsid polypeptides from the AAV capsid libraries generated by DNA shuffling and peptide display of genes encoding capsid sequences of AAV serotypes 1, 6, 8 and 9 (Figure 1).

[00122] An engineered AAV capsid polypeptide of the present disclosure has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes. Such engineered AAV capsid polypeptides are engineered (1) to be chimeric, comprising two or more segments of amino acids derived from different AAV serotypes, for example, AAV1, AAV6, AAV8, and AAV9; (2) to comprise at least five segments of amino acids with at least two of the segments deriving from two different AAV serotypes; (3) to one or more variable amino acids at positions corresponding to residues 24, 163, 169, 195, 197, 198, or 601 of SEQ ID NO: 103; or (4) to comprise SEQ ID NO: 103 (Figure 5 and 6) with a specific amino acid residue at positions 24, 31, 38, 41, 42, 56, 84, 125, 129, 135, 158, 163, 169, 180, 195, 197, 198, 202, 206, 263, 264, 265, 266, 267, 269, 274, 420, 587, 601, and 645.

[00123] In one aspect, the present disclosure provides, an engineered chimeric AAV capsid polypeptide comprising two or more segments of amino acids derived from different AAV serotypes selected from AAV1, AAV6, AAV8 and AAV9, wherein the engineered chimeric AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes.

[00124] In some embodiments, the two or more segments of amino acids include segment 1 of residues 24-42. In some embodiments, the two or more segments of amino acids include segment 2 of residues 125-135. In some embodiments, the two or more segments of amino acids include segment 3 of residues 158-169. In some embodiments, the two or more segments of amino acids include segment 4 of residues 195-206. In some embodiments, the two or more segments of amino acids include segment 5 of residues 600-605. The numbering of the positions corresponds to the residue positions of SEQ ID NO: 103, unless indicated otherwise. [00125] In one aspect, the present disclosure provides, an engineered chimeric AAV capsid polypeptide comprising five segments of amino acids, wherein at least two of the segments are derived from two different AAV serotypes selected from AAV1, AAV6, AAV8 and AAV9, wherein the engineered chimeric AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes.

[00126] In some embodiments, the five segments of amino acids are segment 1 of residues 24-42, segment 2 of residues 125-135, segment 3 of residues 158-169, segment 4 of residues 195-206, and segment 5 of residues 600-605 based on SEQ ID NO: 103.

[00127] In some embodiments, an engineered chimeric AAV capsid polypeptide comprising segment 5 of residues 600-605 of SEQ ID NO: 103 derived from AAV6 (HVMGAL) provides an enhanced tropism for muscle tissues, thereby increasing the levels of delivered engineered AAV vector DNA in the target muscle tissues.

[00128] In some embodiments, segment 5 of residues 600-605 of SEQ ID NO: 103 is derived from AAV6.

[00129] In some embodiments, segment 3 is derived from AAV 1. In some embodiments, segment 3 is derived from AAV6. [00130] In some embodiments, segment 3 is derived from AAV8. In some embodiments, segment 4 is derived from AAV1. In some embodiments, segment 4 is derived from AAV6. In some embodiments, segment 4 is derived from AAV8.

[00131] In some embodiments, segment 1 is derived from AAV 1. In some embodiments, segment 1 is derived from AAV6. In some embodiments, segment 1 is derived from AAV8. In some embodiments, segment 1 is derived from AAV9.

[00132] In some embodiments, segment 2 is derived from AAV 1. In some embodiments, segment 2 is derived from AAV6. In some embodiments, segment 2 is derived from AAV8. In some embodiments, segment 2 is derived from AAV9.

[00133] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises five segments of amino acids according to:

Sl_a-S2_b-S3_c-S4d-S5e wherein SI is segment 1 of residues 24-42 and a is the AAV serotype from which SI is derived; wherein S2 is segment 2 of residues 125-135 and b is the AAV serotype from which S2 is derived; wherein S3 is segment 3 of residues 158-169 and c is the AAV serotype from which S3 is derived; wherein S4 is segment 4 of residues 195-206 and d is the AAV serotype from which S4 is derived; and wherein S5 is segment 5 of residues 600-605 and e is the AAV serotype from which S5 is derived.

[00134] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1AAV1 or AAV6-S2AAV1 or AAV8-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAVlor AAV6.

[00135] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8-S2AAV6-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV1.

[00136] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV1 or AAV6-S2AAV6-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV1.

[00137] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8-S2AAV1 or AAV8-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV1. [00138] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8-S2AAV6-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV1.

[00139] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 -S2AAV1 or AAV8-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV6 wherein SI comprises amino acid sequence of ELKPGAPKPKANQQKQDDG (SEQ ID NO: 202).

[00140] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1AAV1 or AAV6-S2AAV1 or AAV8-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV6.

[00141] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8-S2AAV6-S3AAV1 or AAV6-S4AAV1 or AAV6-S5AAV6.

[00142] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV9- S2AAV9- S 3 AAV8- S 4AAV8- S 5 AAV6.

[00143] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV9- S2AAV9- S 3 AAV8- S 4AAV8- S 5 AAV1.

[00144] In some embodiments, the engineered chimeric AAV capsid polypeptide comprises the following segments:

S 1 AAV8-S2AAV1 or AAV8-S3AAV8-S4-S5AAV6 wherein S4 comprises amino acid sequence APSGVGPTTMAS (SEQ ID NO: 203).

[00145] In one aspect, the present disclosure provides, an engineered AAV capsid polypeptide, comprising one or more variable amino acids at positions corresponding to residues 24, 163, 169, 195, 197, 198, 587, and 601 of SEQ ID NO: 103, wherein the engineered AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes.

[00146] In some embodiments, the one or more substitutions comprise an alanine (A) at position 24 of SEQ ID NO: 103. In some embodiments, the one or more variable amino acids comprise a threonine (T) at position 163 of SEQ ID NO: 103. In some embodiments, the one or more variable amino acids comprise an arginine (R) at position 169 of SEQ ID NO: 103. In some embodiments, the one or more variable amino acids comprise a serine (S) at position 197 of SEQ ID NO: 103. In some embodiments, the one or more variable amino acids comprise a glycine (G) at position 198 of SEQ ID NO: 103. In some embodiments, the one or more variable amino acids comprise a leucine (L) at position 587 of SEQ ID NO: 103. In some embodiments, the one or more variable amino acids comprise a valine (V) at position 601 of SEQ ID NO: 103.

[00147] In some embodiments, the present disclosure provides an engineered AAV capsid polypeptide with an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes comprising an amino acid sequence of SEQ ID NO: 103; the engineered AAV capsid polypeptide comprises one or more amino acid variable amino acids, wherein the amino acid at position 24 is A, D, or E; the amino acid at position 31 is Q or K; the amino acid at position 38 is H or K; the amino acid at position 41 is N or D; the amino acid at position 42 is A or G; the amino acid at position 56 is F or G; the amino acid at position 84 is K or Q; the amino acid at position 125 is L or V; the amino acid at position 129 is L or F; the amino acid at position 135 is A or G; the amino acid at position 158 is T or S; the amino acid at position 163 is K or T; the amino acid at position 169 is R or K; the amino acid at position 180 is S or T; the amino acid at position 195 is A or T; the amino acid at position 197 is S or A; the amino acid at position 198 is G or A; the amino acid at position 202 is N or T; the amino acid at position 206 is A or S; the amino acid at position 263 is S or N; the amino acid at position 264 is A or S; the amino acid at position 265 is S or T; the amino acid at position 266 is S or T; the amino acid at position 269 is A or S; the amino acid at position 274 is H or A; the amino acid at position 420 is D or E; the amino acid at position 587 is L or F; the amino acid at position 601 is V or A; and the amino acid at position 645 is N or H; the numbering of residue positions corresponding to the residue positions of SEQ ID NO: 103.

[00148] In some embodiments, the engineered AAV capsid polypeptide comprises A at position 24; K at position 84; L at position 129; K at position 163; R at position 169; S at position 180; A at position 195; S at position 197; and G at position 198; the numbering of residue positions corresponding to the residue positions of SEQ ID NO: 103. [00149] In some embodiments, the engineered AAV capsid polypeptide comprises A at position 24; F at position 56; K at position 84; L at position 129; K at position 163; R at position 169; S at position 180; A at position 195; S at position 197; G at position 198; D at position 420; L at position 587; V at position 601; and N at position 645; the numbering of residue positions corresponding to the residue positions of SEQ ID NO: 103

[00150] In some embodiment, the engineered AAV capsid polypeptide further comprises a G at position 267.

[00151] In some embodiments, the engineered AAV capsid polypeptide comprises F at position 56; S at position 180; S at position 263; A at position 264; S at position 265; T at position 266; omitted at position 267; A at position 269; H at position 274; L at position 587; and V at position 601.

[00152] In some embodiments, the engineered AAV capsid polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 104-124. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 104. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 105. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 106. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 107. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 108. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 109, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 109. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 110, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 110. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 111, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 111. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 112. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 113. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 114. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 115, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 115. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 116. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 117. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 118. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 119, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 119. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 120. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 121, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 121. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 122. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 123. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 124.

[00153] In other embodiments, the engineered AAV capsid polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 125-152. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 125, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 125. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 126, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 126. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 127. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 128, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 128. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 129. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 130. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 131. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 132. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 133. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 134. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 135, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 135. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 136. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 137. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 138. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 139. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 140. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 141. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 142. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 143. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 144. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 145. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 146. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 147. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 148, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 148. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 149. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 150, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 150. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 151. In one embodiment, the engineered AAV capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 152.

[00154] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide VP1 having a sequence selected from the group consisting of: A) a sequence having at least 97% sequence identity to SEQ ID NO: 1 including a variant segment selected from group consisting of: (i) a variant AAV1 segment 1 corresponding to residues 24- 42 of SEQ ID NO: 1, where the variant AAV1 segment 1 has at least one amino acid variation relative to residues 24-42 of SEQ ID NO: 1, (ii) a variant AAV1 segment 2 corresponding to residues 125-135 of SEQ ID NO: 1, where the variant AAV1 segment 2 has at least one amino acid variation relative to residues 125-135 of SEQ ID NO: 1, (iii) a variant AAV1 segment 3 corresponding to residues 157-168 of SEQ ID NO: 1, where the variant AAV1 segment 3 has at least one amino acid variation relative to residues 157-168 of SEQ ID NO: 1, (iv) a variant AAV1 segment 4 corresponding to residues 194-205 of SEQ ID NO: 1, where the variant AAV1 segment 4 has at least one amino acid variation relative to residues 194-205 of SEQ ID NO: 1, (v) a variant AAV1 segment 5 corresponding to residues 597-602 of SEQ ID NO: 1, where the variant AAV1 segment 5 has at least one amino acid variation relative to residues 597-602 of SEQ ID NO: 1; and B) a sequence having at least 97% sequence identity to SEQ ID NO: 2 including a variant segment selected from group consisting of: (i) a variant AAV6 segment 1 corresponding to residues 24-42 of SEQ ID NO: 2, where the variant AAV6 segment 1 has at least one amino acid variation relative to residues 24-42 of SEQ ID NO: 2, (ii) a variant AAV6 segment 2 corresponding to residues 125-135 of SEQ ID NO: 2, where the variant AAV6 segment 2 has at least one amino acid variation relative to residues 125-135 of SEQ ID NO: 2, (iii) a variant AAV6 segment 3 corresponding to residues 157-168 of SEQ ID NO: 2, where the variant AAV6 segment 3 has at least one amino acid variation relative to residues 157-168 of SEQ ID NO: 2, (iv) a variant AAV6 segment 4 corresponding to residues 194-205 of SEQ ID NO: 2, where the variant AAV6 segment 4 has at least one amino acid variation relative to residues 194-205 of SEQ ID NO: 2, and (v) a variant AAV6 segment 5 corresponding to residues 597-602 of SEQ ID NO: 2, where the variant AAV6 segment 5 has at least one amino acid variation relative to residues 597-602 of SEQ ID NO: 2.

[00155] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 1, where the engineered AAV capsid polypeptide includes a variant AAV1 segment 1 having the amino acid sequence X1LKPGAPX2PKANQQX3QDX4X5 (SEQ ID NO: 207), where XI is A, D, or E, X2 is K or Q, X3 is H or K, X4 is N or D, and X5 is A or G. In some embodiments, the variant AAV 1 segment 1 has an amino acid sequence selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208), ELKPGAPKPKANQQKQDDG (SEQ ID NO: 209), and ALKPGAPKPKANQQKQDDG (SEQ ID NO: 210).

[00156] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 1, where the engineered AAV capsid polypeptide includes a variant AAV1 segment 2 having the amino acid sequence X1LEPX2GLVEEX3 (SEQ ID NO: 211), where XI is L or V, X2 is L or F, and X3 is A or G. In some embodiments, the variant AAV1 segment 2 has an amino acid sequence of LLEPLGLVEEA (SEQ ID NO: 212) or VLEPFGLVEEG (SEQ ID NO: 213).

[00157] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 1, where the engineered AAV capsid polypeptide includes a variant AAV1 segment 3 having the amino acid sequence X1GIGKX2GQQPAX3 (SEQ ID NO: 214), where XI is T or S, X2 is K or T, and X3 is K or R. In some embodiments, the variant AAV1 segment 3 has an amino acid sequence selected from the group consisting of SGIGKTGQQPAK (SEQ ID NO: 215), TGIGKKGQQPAR (SEQ ID NO: 216), and SGIGKKGQQPAR (SEQ ID NO: 217).

[00158] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 1, where the engineered AAV capsid polypeptide includes a variant AAV1 segment 4 having the amino acid sequence X1PX2X3VGPX4TMAX5 (SEQ ID NO: 218), where XI is A or T, X2 is S or A, X3 is G or A, X4 is N or T, and X5 is A or S. In some embodiments, the variant AAV1 segment 4 has an amino acid sequence of APSGVGPNTMAA (SEQ ID NO: 219), or APSGVGPTTMAS (SEQ ID NO: 220).

[00159] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 1, where the engineered AAV capsid polypeptide includes a variant AAV1 segment 5 having the amino acid sequence HVMGAL (SEQ ID NO: 221).

[00160] In some embodiments, the engineered AAV capsid polypeptide has an F56G amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has a K84Q amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has an S179T amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has an SASTGA262-267NSTSGGS amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has an H272A amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has an E418D amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has an F584L amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has an N642H amino acid substitution relative to SEQ ID NO: 1.

[00161] In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 1. [00162] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 2, where the engineered AAV capsid polypeptide includes a variant AAV6 segment 1 having the amino acid sequence X1LKPGAPX2PKANQQX3QDX4X5 (SEQ ID NO: 207), where XI is A, D, or E, X2 is K or Q, X3 is H or K, X4 is N or D, and X5 is A or G. In some embodiments, the variant AAV6 segment 1 has an amino acid sequence selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208), ELKPGAPKPKANQQKQDDG (SEQ ID NO: 209), and ALKPGAPKPKANQQKQDDG (SEQ ID NO: 210).

[00163] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 2, where the engineered AAV capsid polypeptide includes a variant AAV6 segment 2 having the amino acid sequence X1LEPX2GLVEEX3 (SEQ ID NO: 211), where XI is L or V, X2 is L or F, and X3 is A or G. In some embodiments, the variant AAV6 segment 2 has an amino acid sequence of LLEPLGLVEEA (SEQ ID NO: 212) or VLEPLGLVEEG (SEQ ID NO: 222).

[00164] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 2, where the engineered AAV capsid polypeptide includes a variant AAV6 segment 3 having the amino acid sequence X1GIGKX2GQQPAX3 (SEQ ID NO: 214 where XI is T or S, X2 is K or T, and X3 is K or R. In some embodiments, the variant AAV6 segment 3 has an amino acid sequence of TGIGKKGQQPAR (SEQ ID NO: 216) or SGIGKKGQQPAR (SEQ ID NO: 217).

[00165] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 2, where the engineered AAV capsid polypeptide includes a variant AAV6 segment 4 having the amino acid sequence X1PX2X3VGPX4TMAX5 (SEQ ID NO: 218), where XI is A or T, X2 is S or A, X3 is G or A, X4 is N or T, and X5 is A or S. In some embodiments, the variant AAV6 segment 4 has an amino acid sequence of APSGVGPNTMAA (SEQ ID NO: 219), or APSGVGPTTMAS (SEQ ID NO: 220).

[00166] In some embodiments, the engineered AAV capsid polypeptide has at least 97% sequence identity to SEQ ID NO: 2, where the engineered AAV capsid polypeptide includes a variant AAV6 segment 5 having the amino acid sequence HAMGAL (SEQ ID NO: 223). [00167] In some embodiments, the engineered AAV capsid polypeptide has an F56G amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has a K84Q amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has an S179T amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has an SASTGA262-267NSTSGGS amino acid substitution relative to SEQ ID NO: 2.

[00168] In some embodiments, the engineered AAV capsid polypeptide has an H272A amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has an D418E amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has an K531E amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has an L584F amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has an H642N amino acid substitution relative to SEQ ID NO: 2.

[00169] In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 2.

[00170] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 1 and including an amino acid substitution selected from the group consisting of D24A, D24E, K31Q, K38H, D41N, G42A, F56G, K84Q, V125L, L129F, G135A, S157T, T162K, A166P, P167A, K168R, S179T, T194A, A196S, A197G, T201N, S205A, SASTGA262- 267NSTSGGS, H272A, E418D, F584L, A598V, and N642H. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 1.

[00171] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 2 and including an amino acid substitution selected from the group consisting of D24A, D24E, K31Q, K38H, D41N, G42A, F56G, K84Q, V125L, F129L, G135A, S157T, T162K, K168R, S179T, T194A, A196S, A197G, T201N, S205A, SASTGA262-267NSTSGGS, H272A, D417E, K531E, L584F, V598A, and H642N. In some embodiments, the engineered AAV capsid polypeptide has a glutamic acid at position 531. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 2. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 2.

[00172] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 124 including a glutamic acid at position 24. In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 124 and including (i) at least one amino acid selected from the group consisting of proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, valine at position 598, and histidine at position 642 and (ii) one or both of leucine at position 129 and glutamic acid at position 531. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 124. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 124. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 124.

[00173] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 120 and including (i) at least one amino acid selected from the group consisting of proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, and valine at position 598 and (ii) at least one amino acid selected from the group consisting of leucine at position 129, glutamic acid at position 531, and asparagine at position 642. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 120. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 120. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 120. [00174] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 118 and including one or both of an alanine at position 24 and a glutamine at position 84. In some embodiments, the disclosure provides engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 118 and including (i) at least one amino acid selected from the group consisting of phenylalanine at position 129, proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, valine at position 598, and histidine at position 642 and (ii) glutamic acid at position 531. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 118. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 118. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 118.

[00175] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 118 and including an alanine at position 24. In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 117 and including (i) at least one amino acid selected from the group consisting of phenylalanine at position 129, proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, valine at position 598, and histidine at position 642 and (ii) glutamic acid at position 531. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 117. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 117. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 117.

[00176] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 112 and including a segment from an AAV8 or AAV9 capsid protein selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) at residues 24-42, LLEPLGLVEEA (SEQ ID NO: 212) at residues 125-135, AGIGKSGAQPAK (SEQ ID NO: 224) at residues 157-168, and APSGVGSLTMAS (SEQ ID NO: 220) at residues 194-205. In some embodiments, the engineered AAV capsid polypeptide has a segment from an AAV6 capsid protein of HVMGAL (SEQ ID NO: 221) at residues 597-602. In some embodiments, the engineered AAV capsid polypeptide has at least one amino acid selected from the group consisting of a phenylalanine at position 56, a lysine at position 84, an aspartic acid at position 418, a leucine at position 584, and an asparagine at position 642. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 112. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 112. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 112.

[00177] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 111 and including a segment from an AAV8 or AAV9 capsid protein selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) at residues 24-42, LLEPLGLVEEA (SEQ ID NO: 212) at residues 125-135, AGIGKSGAQPAK (SEQ ID NO: 224) at residues 157-168, and APSGVGSLTMAS (SEQ ID NO: 220) at residues 194-205. In some embodiments, the engineered AAV capsid polypeptide has a segment from an AAV6 capsid protein of HVMGAL (SEQ ID NO: 221) at residues 597-602. In some embodiments, the engineered AAV capsid polypeptide has at least one amino acid selected from the group consisting of a phenylalanine at position 56, a lysine at position 84, an aspartic acid at position 418, a leucine at position 584, and a histidine at position 642. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 111. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 111. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 111.

[00178] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including a sequence having at least 97% sequence identity to SEQ ID NO: 115 and including a segment from an AAV8 or AAV9 capsid protein selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) at residues 24-42, LLEPLGLVEEA (SEQ ID NO: 212) at residues 125-135, AGIGKSGAQPAK (SEQ ID NO: 224) at residues 157-168, and APSGVGSLTMAS (SEQ ID NO: 220) at residues 194-205. In some embodiments, the engineered AAV capsid polypeptide has a segment from an AAV6 capsid protein of HVMGAL (SEQ ID NO: 221) at residues 597-602. In some embodiments, the engineered AAV capsid polypeptide has at least one amino acid selected from the group consisting of a phenylalanine at position 56, a lysine at position 84, a glutamic acid at position 418, a leucine at position 584, and an asparagine at position 642. In some embodiments, the engineered AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 115. In some embodiments, the engineered AAV capsid polypeptide has at least 99% sequence identity to SEQ ID NO: 115. In some embodiments, the engineered AAV capsid polypeptide has at least 99.5% sequence identity to SEQ ID NO: 115.

[00179] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including the amino acid sequence of SEQ ID NO: 204. In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 204.

[00180] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including the amino acid sequence of SEQ ID NO: 205. In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 205.

[00181] In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide including the amino acid sequence of SEQ ID NO: 206. In some embodiments, the disclosure provides an engineered adeno-associated virus (AAV) capsid polypeptide having an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the amino acid sequence of SEQ ID NO: 206.

[00182] In some embodiments, the engineered AAV capsid polypeptide has reduced reactivity to neutralizing antibodies, for example neutralizing antibodies in human plasma. [00183] Exemplary engineered AAV capsid polypeptides that can increase delivery of engineered AAV vector DNA to muscle comprise amino acid sequences from the group consisting of SEQ ID NOs: 111, 112, 115, 117, 118, 120, and 124. Exemplary engineered AAV capsid polypeptides that can enhance transgene expression in muscle comprise amino acid sequences from the group consisting of SEQ ID NOs: 105 and 110-115. In certain embodiments, the engineered AAV capsid polypeptides are recombinant.

Engineered Capsid Nucleic Acids

[00184] In some embodiments, the present disclosure further provides a polynucleotide encoding an engineered AAV capsid polypeptide described herein. The nucleic acid encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-152. In other embodiments, the nucleic acid sequence encoding the engineered AAV capsid polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the nucleic acid sequence encoding a polypeptide comprising an amino acid sequence any one of SEQ ID NOs: 104-152, wherein the nucleic acid encodes the engineered AAV capsid polypeptide that retains at least one property of the engineered AAV capsid polypeptide described herein selected from the group consisting of increased muscle tropism, increased muscle transduction, increased muscle DNA levels, and increased muscle mRNA levels as compared to a wt capsid polypeptide from any one of AAV1, AAV6, AAV8 and AAV9.

[00185] In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 104. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 105, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 105. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 106, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 106. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 107, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 107. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 108. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 109, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 109. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 110, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 110. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 111, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 111. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 112. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 113, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 113. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 114, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 114. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 115, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 115. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 116, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 116. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 117, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 117. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 118, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 118. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 119, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 119. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 120, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 120. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 121, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 121. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 122. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 123, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 123. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 124, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 124. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 125, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 125.

[00186] In other embodiments, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-152.

[00187] In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 126, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 126. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 127, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 127. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 128, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 128. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 129, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 129. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 130, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 130. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 131, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 131. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 132, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 132. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 133, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 133. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 134, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 134. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 135, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 135. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 136, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 136. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 137, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 137. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 138. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 139, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 139. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 140, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 140. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 141, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 141. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 142, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 142. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 143, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 143. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 144, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 144. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 145, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 145. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 146, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 146. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 147, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 147. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 148, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 148. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 149, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 149. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 150, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 150. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 151, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 151. In one embodiment, the polynucleotide encodes an engineered AAV capsid polypeptide that comprises the amino acid sequence of SEQ ID NO: 152, or an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to SEQ ID NO: 152.

[00188] In some embodiments, the polynucleotide encoding the engineered AAV capsid polypeptide may comprises one or more modified nucleotides. Examples of modified nucleotides include 5-fluorouracil, 5 -bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomet-hyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 '-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopenten-adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

[00189] In some embodiments, the polynucleotide encoding the engineered AAV capsid polypeptide is codon-optimized.

[00190] Other modifications to nucleic acids to improve the stability, nuclease-resistance, bioavailability, formulation characteristics and/or pharmacokinetic properties are known in the art.

Engineered Capsid Expression Constructs and Helper Plasmids [00191] In some embodiments, the present disclosure provides expression constructs such as helper plasmids (e.g., non- AAV expression constructs) comprising a nucleic acid that encodes one or more of the engineered AAV capsid polypeptides described herein. Such plasmids, referred to herein as “engineered capsid helper plasmids,” are useful as expression constructs for producing engineered AAV capsid polypeptides or proteins or to transfect cells (e.g., as part of a triple transfection) in the preparation of engineered AAV vectors. Alternatively, engineered AAV vectors could be produced using herpes virus, baculovirus, stable genetically engineered cell lines, or any other method known in the art (Dobrowsky et al. (2021) Curr. Opinion Biomed. Engin. 20:100353, the disclosure of which is hereby incorporated herein by reference in its entirety).

[00192] In other embodiments, the engineered capsid helper plasmid comprises a polynucleotide encoding an engineered AAV capsid polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-125. In other embodiments, the engineered capsid helper plasmid comprises a polynucleotide encoding an engineered AAV capsid polypeptide that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 126-152.

[00193] In some embodiments, the engineered capsid helper plasmid may comprise one or more nucleic acid sequences to regulate expression of the engineered AAV capsid polypeptide. The sequences include but are not limited to, a promoter, an enhancer, an intron, a post- transcriptional regulatory sequence, a polyadenylation (poly A) signal, or any combination thereof, which are operably linked to the nucleic acid sequences that encode the engineered AAV capsid polypeptide.

[00194] The promoter may be a heterologous promoter, a tissue-specific promoter, a cellspecific promoter, a constitutive promoter, an inducible promoter, a hybrid promoter, or any combination thereof. In an embodiment, the engineered capsid helper plasmid of the present disclosure comprises at least one promoter capable of expressing, or directed to primarily express, the nucleic acid segment in a suitable host cell (e.g., a muscle cell) into which the engineered capsid helper plasmid can be transfected. Exemplary promoters include, but are not limited to, a ubiquitous promoter, a CMV promoter, a 0-actin promoter, a muscle-specific promoter, a Desmin promoter, an SPc5-12 promoter, an MCK- based promoter an insulin promoter, an enolase promoter, a BDNF promoter, an NGF promoter, an EGF promoter, a growth factor promoter, an axon-specific promoter, a dendrite-specific promoter, a brainspecific promoter, a hippocampal-specific promoter, a kidney-specific promoter, a retinal- specific promoter, an elafin promoter, a cytokine promoter, an interferon promoter, a growth factor promoter, an al -antitrypsin promoter, a brain cell-specific promoter, a neural cellspecific promoter, a central nervous system cell-specific promoter, a peripheral nervous system cell-specific promoter, an interleukin promoter, a serpin promoter, a hybrid CMV promoter, a hybrid 0-actin promoter, an EFl promoter, a Ula promoter, a Ulb promoter, a Tet- inducible promoter, a VP1 6-Lex A promoter, or any combination thereof. In exemplary embodiments, the promoter may include a mammalian or avian 0-actin promoter.

[00195] Exemplary enhancer sequences include, but are not limited to, one or more selected from the group consisting of a CMV enhancer, a muscle-specific enhancer, a synthetic enhancer, a liver-specific enhancer, a vascular-specific enhancer, a brain-specific enhancer, a neural cell-specific enhancer, a lung-specific enhancer, a kidney-specific enhancer, a pancreasspecific enhancer, retinal- specific enhancer, and an islet cell-specific enhancer.

[00196] Exemplary post- transcriptional regulatory sequences include a woodchuck hepatitis post-transcription regulatory element (WPRE)), one or more ribosome entry sites (IRES), one or more polyadenylation (poly A) signal sequences, or any combination thereof. A polyA signal may be an artificial polyA. Examples of other suitable polyA sequences include, e.g., bovine growth hormone, SV40, rabbit beta globin, and TK polyA, amongst others.

[00197] In some embodiments, the engineered capsid helper plasmid described herein may contain other appropriate transcription initiation, termination, and efficient RNA processing signals. Such sequences include splicing, inducible expression control elements, regulatory elements that enhance expression, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequence), sequences that enhance protein stability, and when desired, sequences that enhance secretion of the encoded product. In one embodiment, a Kozak sequence is included.

[00198] In some embodiments, the engineered capsid helper plasmid described herein comprises one or more nucleic acid sequences that encode one or more transgenes of interest. The nucleic acid sequences may be any heterologous sequences that do not encode an engineered AAV capsid polypeptide. Such transgenes of interest may comprise proteins, polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, and other non-coding RNAs, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.

[00199] As understood by a person having ordinary skill in the art, the nucleic acids encoding the engineered AAV capsid polypeptide and any regulatory sequences within the engineered capsid helper plasmid can collectively be referred to as an expression cassette. In this context, the engineered capsid helper plasmid of the present disclosure comprises an expression cassette. The exact composition of the expression cassette will depend upon the intended use of the engineered capsid helper plasmid.

[00200] In some embodiments, the engineered capsid helper plasmid described herein is a recombinant engineered capsid helper plasmid.

Tropism of Engineered AA V Capsid Polypeptides

[00201] Engineered AAV capsid polypeptides of the present disclosure have an enhanced tropism for muscle and reduced immunogenicity as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes.

[00202] In some embodiment, the engineered AAV capsid polypeptide has an enhanced tropism for a human muscle cell or muscle tissue as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes. The tropism efficiency can be determined by reference to a suitable control vector or isolated control capsid protein (e.g., AAV1, AAV6, AAV8 or AAV9).

[00203] In some embodiments, the muscle tissue is heart. In some embodiments the muscle tissue is gastrocnemius. In some embodiments, the muscle tissue is biceps. In some embodiments the muscle tissue is quadriceps. In some embodiments the muscle tissue is triceps. In some embodiments the muscle tissue is soleus. In some embodiments the muscle tissue is diaphragm. In some embodiments the muscle tissue is tongue. In some embodiments the muscle tissue is skeletal muscle. In some embodiments the muscle cells are cardiac muscle cells.

[00204] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in one or more muscle tissues or muscle cells. In some embodiments, the tropism is measured by the level of transgene expression (e.g., RNA or polypeptide expression) in the muscle tissues or muscle cells. In some embodiments, the tropism is measured by the level of expression of a transgene in the one or more muscle tissues or muscle cells. In certain embodiments, the transgene is a reporter gene, a gene encoding a therapeutic protein or peptide vaccine, or a gene encoding a non-coding RNA.

[00205] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in diaphragm. In some embodiments, the tropism is measured by the level of transgene RNA expressed in diaphragm. In some embodiments, the tropism is measured by the level of transgene product expressed in diaphragm, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in diaphragm.

[00206] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in gastrocnemius. In some embodiments, the tropism is measured by the level of transgene RNA expressed in gastrocnemius. In some embodiments, the tropism is measured by the level of transgene product expressed in gastrocnemius, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in gastrocnemius.

[00207] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in triceps. In some embodiments, the tropism is measured by the level of transgene RNA expressed in triceps. In some embodiments, the tropism is measured by the level of transgene product expressed in triceps, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in triceps.

[00208] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in heart. In some embodiments, the tropism is measured by the level of transgene RNA expressed in heart. In some embodiments, the tropism is measured by the level of transgene product expressed in heart, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in heart.

[00209] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in biceps. In some embodiments, the tropism is measured by the level of transgene RNA expressed in biceps. In some embodiments, the tropism is measured by the level of transgene product expressed in biceps, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in biceps.

[00210] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in soleus. In some embodiments, the tropism is measured by the level of transgene RNA expressed in soleus. In some embodiments, the tropism is measured by the level of transgene product expressed in soleus, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in soleus.

[00211] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in tongue. In some embodiments, the tropism is measured by the level of transgene RNA expressed in tongue. In some embodiments, the tropism is measured by the level of transgene product expressed in tongue, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in tongue.

[00212] In some embodiments, the tropism is measured by the level of engineered AAV vector DNA in quadriceps. In some embodiments, the tropism is measured by the level of transgene RNA expressed in quadriceps. In some embodiments, the tropism is measured by the level of transgene product expressed in quadriceps, e.g., the level of a therapeutic protein or vaccine peptide encoded by the transgene in quadriceps.

Engineered AA V Vectors

[00213] In another aspect, the present disclosure provides engineered AAV vectors (also referred to herein as AAV particles or AAV virions) comprising an engineered AAV capsid polypeptide described herein. In some cases, the engineered AAV vector comprises an engineered AAV capsid polypeptide and a transgene of interest (e.g., encoding a therapeutic molecule, such as, e.g., an RNA, protein, or vaccine peptide). An engineered AAV vector of the present disclosure comprises an AAV vector genome packaged within an engineered AAV capsid polypeptide of the present disclosure.

[00214] In some embodiments, the engineered AAV vector is a recombinant AAV vector.

[00215] In some embodiments, the engineered AAV vector has reduced reactivity to neutralizing antibodies, for example, neutralizing antibodies in human plasma.

[00216] In some embodiments, the engineered AAV vector described herein can be used for gene therapy.

[00217] In some embodiments, the engineered AAV vectors described herein can be used to deliver transgenes, e.g., for gene transfer, to muscle cells, where the muscle is used as a production unit of therapeutic polypeptides or peptide vaccines. For a review of AAV vector- mediated gene transfer see, for example, Boisgerault and Mingozzi (2015) Curr. Gene Ther. 15(4): 381 -94, the disclosure of which is hereby incorporated by reference in its entirety. For example, AAV-mediated expression of anti-HIV antibodies has been achieved following intramuscular administration of an AAV vector encoding the antibody. See, van den Berg et al. (2019) Methods & Clin. Dev. 14: 100-12, the disclosure of which is hereby incorporated by reference in its entirety. As shown in the van den Berg et al. study, the antibody was expressed in muscle cell and released into the blood in mice.

Transsenes of Interest

[00218] In some embodiments, the engineered AAV vectors that are encapsidated by an engineered AAV capsid polypeptide also comprise one or more non-capsid coding nucleic acid sequences (e.g., one or more transgenes of interest).

[00219] In some embodiments, the one or more non-capsid coding nucleic acid sequences are flanked on each side with an inverted terminal repeat (ITR) sequence. The ITRs are the genetic elements responsible for the replication and packaging of the genome during AAV vector production and are the only viral cis elements required to generate an AAV vector. In one embodiment, the engineered AAV vectors described herein comprise an AAV 5’ ITR and an AAV 3 ’ ITR, which can be of the same AAV origin as the capsid, or which are of a different AAV origin (e.g., to produce an AAV pseudotype). The ITR sequences can be derived from any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV 10) or can be derived from more than one serotype. In some embodiments, the ITR sequences are derived from AAV2 or AAV3. In other embodiments, the ITR sequences of the first serotype are derived from AAV1, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV 10. ITR sequences and plasmids containing ITR sequences are known in the art and commercially available. Typically, an expression cassette for an AAV vector comprises an AAV 5 ' ITR, a transgene and any regulatory sequences, and an AAV 3 ' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5 ' ITR, termed AITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In other embodiments, the full-length AAV 5 ' and 3 ' ITRs are used.

[00220] As described herein, the engineered AAV vector may express one or more transgenes of interest. In some embodiments, the transgene of interest in the engineered AAV vector comprises a transgene encoding a therapeutic agent. Therapeutic agents useful in the engineered AAV vectors may include one or more agonists, antagonists, anti-apoptosis factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents, enzymes, lysosomal enzymes, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, receptor binding proteins, transport proteins or one or more inhibitors thereof, serotonin receptors, or one or more uptake inhibitors thereof, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, or any combination thereof, such as a therapeutic polypeptide.

[00221] In some cases, the transgene may encode dystrophin, a mini-dystrophin, a microdystrophin, a laminin-a2, a mini-agrin, an a-sarcoglycan, a 0-sarcoglycan, a y-sarcoglycan, a 5- sarcoglycan, a utrophin, a Fukutin-related protein, a myostatin pro-peptide, a soluble myostatin receptor, a dominant negative myostatin, a follistatin, an insulin-like growth factor 1 (IGF-1), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), a 0-adrenergic receptor kinase C terminus (0ARKct), a phospholamban, a phosphatidylinositol 3-kinase (PI3K), a proto-oncogene serine/threonine- protein kinase (Pim-1), a peroxisome proliferat or-activated receptor-gamma coactivator (PGC-la), a superoxide dismutase (SOD), an SOD-1, an SOD-2, an extracellular SOD (EC-SOD), a kallikrein, a hypoxia inducible factor (HIF), thymosin 04 (TMSB4X), a mir-1 microRNA, a mir-133 microRNA (e.g., a miR-133a-l, a miR-133a-2, and a miR-133b), a mir-206 microRNA, a mir-208 microRNA, an inhibitor 1 of protein phosphatase 1, an anti-apoptotic factor, an angiogenic factor, an insulin, a Factor IX, a Factor VIII, a glucocerebrosidase, an a-galactosidase A (GLA), an acid a glucosidase (GAA), a lipoprotein lipase, an apolipoprotein-E (ApoE), an al -antitrypsin (al AT), an apelin, a glucagon-like peptide 1, an iduronidase, an iduronate-2-sulfatase, a myotubularin (MTM1), a calpain-3 (CAPN3), a dysferlin, a lysosome-associated membrane protein 2 (LAMP2), an N- acetylgalactosamine 4-sulfatase; a cardiac troponin T, a cardiac troponin I, a troponin I type 3a, a troponin C (TNNC1), a cardiac sarcomeric protein, a 0- myosin heavy chain, a myosin ventricular essential light chain 1, a myosin light chain 2, a myosin ventricular regulatory light chain 2, a myosin light chain 3 (MYL3), an a-tropomyosin, a cardiac myosin binding protein C, a four-and-a-half LIM protein 1 (FHL1), a titin, an AMP-activated protein kinase (AMPK) subunit gamma-2, a cardiac muscle actin alpha 1 (ACTC1), a muscle LIM protein (MLP), a cardiac LIM protein (CLP), a caveolin 3 (CAV3), a mitochondrial transfer RNA glycine (MTTG), a mitochondrial transfer RNA isoleucine (MTU), a mitochondrial transfer RNA lysine (MTTK), a mitochondrial transfer RNA glutamine (MTTQ), a transthyretin (TTR), a stromal-derived factor-1 (SDF-1), an adenylate cyclase-6 (AC6), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor, a hypoxia inducible growth factor, a nitric oxide synthase-3 (NOS3), and SI 00 Al protein.

[00222] In some embodiments, the transgene may encode one or more reprogramming factors, optionally selected from ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2. The factor reprograms the non-cardiomyocyte cardiac cell or cardiac fibroblast cell into a cardiac cardiomyocyte. Such reprogramming is described, for example, in US Patent No. 11,015,211.

[00223] In some embodiments, the transgene may encode Collagen type 6(COL6A), BINI, heat shock protein family B (HSPB1), lamin A/C (LMNA), actin (ACTA1), dysferlin (DYSF), desmin (DES), troponin 12 (TNN12), troponin 3 (TNN3), troponin T2 (TNNT2), myosin heavy chain 3 (MYH3), myosin heavy chain 8 (MYH8), fibronectin 1 (FN1), transforming growth factor beta 3 (TGFB3), emerin (EMD), filamin A (FLNA), enolase 3 (ENO3), actinin alpha 2 (ACTN2), myosin light chain kinase (MYLK), sarcoglycan alpha (SGCA), dystroglycan 1 (DAG), syntrophin alpha 1 (SNTA1), crystallin alpha B(CRYAB), filamin C (FLNC), kelch like family member 40 (KLHL40), poly(A) binding protein nuclear 1 (PABPN1), and ataxin 3 (ATXN3).

[00224] In some embodiments, the engineered AAV vector comprises at least one nucleic acid sequence encoding a non-coding RNA molecule. Exemplary non-coding RNA molecules include a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a guide RNA (gRNA), a long non-coding RNA (IncRNA), a snoRNA, a pi wi- interacting RNA (piRNA), a small nuclear RNA (snRNA), a circular RNA (circRNA), a microRNA, an antisense oligonucleotide, and a ribozyme.

[00225] siRNA is an RNA duplex of nucleotides that is targeted to a gene interest which comprises a sense strand and an antisense strand. siRNA can target to a gene in that the nucleotide sequence of the duplex portion of the siRN A is complementary to a nucleotide sequence of the targeted gene. siRNA can be used to silence or repress the expression of the target gene, such as a disease-causing gene. The engineered AAV vector described herein can be used to deliver siRNA into cells, tissues, and subjects for the treatment of a disease, e.g., a muscular disease.

[00226] An “shRNA” includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA) that can silence expression of a target gene. Single-stranded interfering RNA has been found to effect mRNA silencing. The engineered AAV vector described herein can be used to deliver shRNA into cells, tissues, and subjects for the treatment of a disease. [00227] MicroRNAs (miRNA) are natural cellular RNA molecules that can regulate the expression of multiple genes by controlling the stability of the mRNA. Over-expression or diminution of a particular microRNA can be used to treat a dysfunction and has been shown to be effective in a number of disease states and animal models of disease. The engineered AAV vector described herein can be used to deliver microRNA into cells, tissues, and subjects for the treatment of genetic and acquired diseases, or to enhance functionality and promote growth of certain tissues.

[00228] Small nuclear RNAs (snRNAs) are non-coding RNA molecules ranging in size from about 150 to about 300 nucleotides. snRNAs have a conserved secondary structure that includes a 5' cap structure, a stem-loop structure called the Sm-binding site, and a 3' poly(A) tail. snRNAs help regulate splicing, nuclear transport, and gene regulation. For example, snRNAs are involved in the formation of spliceosomes, which are large RNA-protein complexes that remove introns and join exons in messenger RNA (mRNA) molecules. snRNAs also have been implicated in the regulation of alternative splicing, which leads to the production of different mRNA isoforms from a single gene. The dysregulation of small non-coding RNAs (sncRNAs), such as tRNA-derived small RNA (tsRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and PlWI-interacting RNA (piRNA), has been linked with cancer progression. See, for example, Xiao etal (2022) J. Med. Genet. 59(7):623-31, the disclosure of which is hereby incorporated herein by reference in its entirety. For a description of an example therapeutic use of snRNA see, for example, Imbert et al. (2017) Genes 8(2):51, the disclosure of which is hereby incorporated herein by reference in its entirety.

[00229] circRNA are circular single stranded RNAs with various transcriptional and translational regulatory functions in vivo, thought to be mediated through at least sequestration of proteins and micoRNA. The dysregulation of circRNAs has been implicated in the development of cancer, cardiovascular disease, and various neurological diseases. For a review of various therapeutic strategies using circRNA see, for example, He etal. (2021) Signal Transduc. Targeted Then 6: 185, the disclosure of which is hereby incorporated herein by reference in its entirety.

Methods of Preparing Engineered AA V Vectors

[00230] Various methods of producing engineered AAV vectors comprising the engineered AAV capsid polynucleotides of the present disclosure are known.

[00231] In some embodiments, a plasmid or other non- AAV vector comprising a transgene of interest may be combined with one or more helper plasmids, e.g., that contain a rep gene (e.g., encoding Rep78, Rep68, Rep52 and Rep40) and a cap gene encoding an engineered AAV capsid polynucleotide described herein, and transfected into recombinant cells, called helper or producer cells, such that the plasmid or non- AAV vector is packaged or encapsidated inside the capsid protein and subsequently purified such that the rAAV vector is packaged.

[00232] In some embodiments, the one or more helper plasmids include a first helper plasmid, referred to herein as an engineered capsid helper plasmid, comprising a rep gene and a cap gene encoding an engineered AAV capsid polypeptide, and a second helper plasmid comprising one or more of the following helper genes: Ela gene, Elb gene, E4 gene, E2a gene, and VA gene. For clarity, helper genes are genes that encode helper proteins Ela, Elb, E4, E2a, and VA. Helper plasmids, and methods of making such plasmids, are known in the art and commercially available (see, e.g., pDF6, pRep, pDM, pDG, pDPlrs, pDP2rs, pDP3rs, pDP4rs, pDP5rs, pDP6rs, pDG(R484E/R585E), and pDP8.ape plasmids).

[00233] Non-limiting examples of mammalian producer cells include HEK293 cells, COS cells, HeLa cells, BHK cells, or CHO cells (see, e.g., ATCC® CRL-1573™, ATCC® CRL- 1651™, ATCC® CRL-1650™, ATCC® CCL-2, ATCC® CCL-10™, or ATCC® CCL- 61™). A non-limiting example of an insect producer cells is Sf9 cells (see, e.g., ATCC® CRL- 1711™). A producer cell may comprise rep and/or cap genes that encode the Rep protein and/or engineered AAV capsid polypeptide. In some embodiments, the packaging is performed in vitro.

[00234] In one embodiment, the present disclosure provides a method of producing an engineered AAV vector comprising an engineered AAV capsid, the method comprising: providing a cell in vitro with a nucleic acid encoding an engineered AAV capsid of the disclosure, an AAV rep coding sequence, an AAV vector genome (engineered AAV vector DNA) comprising a transgene of interest, and helper functions for generating a productive AAV transduction; and allowing assembly of the engineered AAV vectors comprising the engineered AAV capsid and encapsidating the engineered AAV vector genome. In an embodiment, the cell is transfected with plasmids comprising (1) a transgene of interest (e.g., the recombinant AAV genome); (2) a nucleic acid encoding the engineered AAV capsid polypeptide disclosed herein; and (3) AAV replication proteins (Rep 78, 68, 52, and 40) are transfected into a producer cell. Because AAVs are inherently replication deficient, the producer cell is also infected with a helper virus, generally adenovirus (Ad) or herpes simplex virus type 1 (HSV). The helper virus provides additional factors, e.g., replication factors E4, E2a and VA, to mediate AAV production.

[00235] In some embodiments, rather than co-transfecting plasmids encoding (1) the recombinant AAV genome, (2) the engineered AAV capsid protein, and (3) the Rep proteins, nucleic acids encoding one or more of these factors are stably incorporated into the producer cell. For example, in some embodiments, the present disclosure provides a cell that is transfected to produce an engineered AAV capsid polypeptide. In some embodiments, the cell is a packaging cell stably expressing an engineered AAV capsid polypeptide of the present disclosure. For example, the polynucleotide encoding the engineered AAV capsid polypeptide can be stably incorporated into the genome of the cell or can be stably maintained in an episomal form. In some embodiments, the additional replication factors otherwise provided by a helper virus are introduced into the producer cells via plasmid and/or stably integrated into the producer cell.

[00236] In some embodiments, an HSV-based AAV production system is used to produce the engineered AAV vectors described herein. For example, a recombinant HSV carrying the AAV rep genes and/or an engineered cap gene as described herein can be used to infect a producer cell transfected or stably integrating the recombinant AAV genome, eliminating the need for cotransfection of multiple plasmids. For further description of such HSV-based AAV production systems see, for example, Clement etal. (2009) Human Gene Ther., 20(8):796-806, the disclosure of which is hereby incorporated herein by reference in its entirety.

[00237] It has also been found that replication factors expressed by baculovirus promote AAV replication. Advantageously, insect cells targeted by baculovirus can be easily grown in suspension culture. Accordingly, in some embodiments, insect cells, e.g., Spodoptera frugiperda (sf9) cells, in suspension culture are infected with a recombinant baculovirus encoding a recombinant AAV genome, an engineered AAV capsid polypeptide as described herein, and AAV Rep genes, as well as the endogenous baculovirus replication machinery sufficient for helper function. For further description of the baculovirus / sf9 system for producing recombinant AAV particles see, for example, Negrete et al., (2007) J. Gene Med. 9:938-48 and Kotin (2011) Hum. Mol. Genet. 20(Rl):R2-6, the disclosure of which are hereby incorporated herein by reference in their entireties.

Compositions and Formulations

[00238] In another aspect, the present disclosure provides compositions comprising one or more of the disclosed engineered AAV capsid polypeptides, engineered AAV vectors, transfected or transduced cells, or compositions thereof. In some embodiments, the compositions of engineered AAV capsid polypeptides, engineered AAV capsid nucleic acids, helper plasmids, engineered AAV vectors, or host cells further comprise a pharmaceutically acceptable carrier for use in therapy, and for use in the manufacture of medicaments for the prophylaxis or treatment of one or more mammalian diseases. Such pharmaceutical compositions may optionally comprise one or more diluents, buffers, liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof. [00239] In some embodiments, the pharmaceutical compositions disclosed herein are formulated for administration by suitable means such as pulmonary, intranasal, oral, inhalation, parenteral such as intravenous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraocular, intraorbital, intravitreal, intracardiac, intrathecal, intracerebro-ventricular, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection or by direct injection to one or more cells, tissues, or organs, e.g., muscle tissue.

[00240] The pharmaceutical forms of the compositions suitable for injectable use include sterile aqueous solutions or dispersions. In some embodiments, the form is sterile and fluid to provide easy syringability. In some embodiments, the form is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms, such as bacteria, yeast, and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, saline, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. [00241] Injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. For example, an injectable, stable, sterile composition of this disclosure in a unit dosage form in a sealed container can be provided. The composition can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject.

[00242] The compositions can be presented in unit dose or multi-dose containers, for example, in sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water- for-inj ection immediately prior to use.

[00243] In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁵ vg to about 1 x 10¹⁷ vg. In certain embodiments, for the methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10¹¹ vg to about 1 x 10¹⁷ vg. In certain embodiments, the vg is total vector genome per subject. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁵ vg to about 1 x 10¹⁵ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 107⁷ vg to about 1 x 10¹⁵ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁸ vg to about 1 x 10¹⁵ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁹ vg to about 1 x 10¹⁵ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁵ vg to about 1 x 10¹³ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 107⁷ vg to about 1 x 10¹³ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁸ vg to about 1 x 10¹³ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁹ vg to about 1 x 10¹³ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁵ vg to about 1 x 10¹¹ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 107⁷ vg to about 1 x 10¹¹ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁸ vg to about 1 x 10¹¹ vg. In some embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁹ vg to about 1 x 10¹¹ vg.

[00244] In certain embodiments, for the therapeutic methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10⁵ vg/kg to about 1 x 10¹⁴ vg/kg. In certain embodiments, for the methods disclosed herein, the dose of the engineered AAV vectors administered to the subject is between about 1 x 10¹³ vg/kg to about 1 x 10¹⁵ vg/kg. In certain embodiments, the vg/kg is total vector genome per kg of the subject.

Methods of Use

[00245] In accordance with the present disclosure, the engineered AAV capsid polypeptides, engineered capsid helper plasmids, engineered AAV vectors, host cells, and compositions thereof may find use in both veterinary and medical applications. Suitable subjects include mammals, e.g., humans or non-human primates, and other animals in need of treatment. Human subjects include neonates, infants, juveniles, and adults. Optionally, the subject is “in need of’ the methods of the present disclosure, e.g., because the subject has, or is believed to be at risk for, a disorder including those described herein or that would benefit from delivery of a transgene of interest (e.g., encoding a therapeutic polypeptide, peptide vaccine, or non-coding RNA), as disclosed herein. For example, in particular embodiments, the subject has, or has had, or is at risk for a muscular dystrophy or heart disease (e.g., a myocardial infarct, peripheral arterial disease (PAD), or congestive heart failure). The subject can also be a laboratory animal and/or an animal model of disease.

[00246] In some embodiments, the engineered AAV capsid polypeptides, engineered capsid helper plasmids, engineered AAV vectors, host cells, and compositions thereof are used for transducing muscle cells and muscle tissues. Accordingly, the engineered AAV capsid polypeptides, engineered capsid helper plasmids, engineered AAV vectors, host cells, and compositions thereof can be used for transducing one or more transgenes of interest into muscle cells and tissues, for example, encoding one or more therapeutic polypeptides, peptide vaccine, or non-coding RNA.

[00247] In some embodiments, the present disclosure provides a method of introducing a heterologous nucleic acid (e.g., a transgene of interest as described herein) to a cell comprising contacting the cell with an engineered AAV vector described herein. In some embodiments, the transduced cell can be used for treating a subject in need. Accordingly, the engineered AAV vector may be introduced to cells in vitro for the purpose of administering the modified cells to a subject. In particular embodiments, the cells have been removed from a subject, the engineered AAV vector is introduced therein, and the cells are then replaced back into the subject. Alternatively, the engineered AAV vector is introduced into cells from another subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof.

[00248] In some embodiments, the present disclosure provides a method of producing therapeutic or prophylactic compounds in muscle cells using engineered AAV vector described herein. In some embodiments, the therapeutic or prophylactic compound is a peptide vaccine. In some embodiments, the therapeutic or prophylactic compound is a therapeutic polypeptide. In some embodiments, the therapeutic or prophylactic compound is a non-coding RNA.

[00249] In some embodiments, the present disclosure provides a method of delivering a gene therapy product to a target cell in a subject comprising administering to the subject an engineered AAV vector described herein. The gene therapy product may comprise a transgene of interest as described herein.

[00250] In some embodiments, the heterologous nucleic acid or gene therapy product is delivered to a muscle tissue, such as a skeletal muscle, a cardiac muscle, and a diaphragm muscle. In some examples, the heterologous nucleic acid or gene therapy product delivered to a skeletal muscle include those that are beneficial in the treatment of damaged, degenerated and/or atrophied skeletal muscle.

[00251] In some examples, the heterologous nucleic acid or gene therapy product delivered to cardiac muscle include those that are beneficial in the treatment of damaged, degenerated or atrophied cardiac muscle and/or congenital cardiac defects.

[00252] In some embodiments, the present disclosure provides a method of treating a muscular disorder in a subject in need thereof comprising administering to the subject an engineered AAV vector described herein. In some embodiments, the disease is a cardiac pathology. In some embodiments, the disease is a muscular dystrophy. In some embodiments, the disease is a neuromuscular dystrophy. In some embodiments, the disease is a disease of skeletal muscle, diaphragm muscle and/or cardiac muscle.

[00253] In some embodiments, the muscle cell and/or muscle tissue is a human muscle cell and/or muscle tissue. In some examples, the muscle cell is a cardiac myocyte, a skeletal myocyte, or a smooth myocyte. As a non-limiting example, the muscle cell is a cardiac myocyte.

[00254] The engineered AAV vectors and/or pharmaceutical compositions of the present disclosure can be administered to the subject being treated by standard routes including, but not limited to, pulmonary, intranasal, oral, inhalation, parenteral such as intravenous, topical, transdermal, intradermal, transmucosal, intraperitoneal, intramuscular, intracapsular, intraocular, intraorbital, intravitreal, intracardiac, intrathecal, intracerebro-ventricular, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection or by direct injection to one or more cells, tissues, or organs. [00255] In some embodiments, the engineered AAV vectors and compositions thereof may be administered by epicardial injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade intracoronary injection, retrograde injection, transendomyocardial injection, or molecular cardiac surgery with recirculating delivery (MCARD).

[00256] Administration to cardiac muscle includes without limitation administration to the left atrium, right atrium, left ventricle, right ventricle and/or septum. The present engineered AAV vectors can be delivered to the cardiac muscle by any method known in the art including, e.g., intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into left atrium, right atrium, left ventricle, right ventricle), and/or coronary artery perfusion. As a non-limiting example, the engineered AAV vectors and compositions thereof described herein are administered by epicardial injection.

[00257] In another example, the engineered AAV vectors and compositions thereof described herein are administered intramuscularly, including administration to skeletal, diaphragm, and/or cardiac muscle.

[00258] Administration to skeletal muscle according to the present disclosure includes but is not limited to administration to skeletal muscles in the limbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back, neck, head (e.g., tongue), thorax, abdomen, pelvis/perineum, and/or digits. Suitable skeletal muscle tissues include but are not limited to abductor digiti minimi of the hand, abductor digiti minimi of the foot, abductor hallucis, abductor ossis metatarsi quinti, abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, articularis genus, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferioris, digastric, dorsal interossei of the hand, dorsal interossei of the foot, extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digiti minimi, extensor digitorum, extensor digitorum brevis, extensor digitorum longus, extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis of the hand, flexor digiti minimi brevis of the foot, flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicis, iliocostalis lumborum, iliocostalis thoracis, illiacus, inferior gemellus, inferior oblique, inferior rectus, infraspinatus, interspinalis, intertransversi, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superioris, levator labii superioris alaeque nasi, levator palpebrae superioris, levator scapulae, long rotators, longissimus capitis, longissimus cervicis, longissimus thoracis, longus capitis, longus colli, lumbricals of the hand, lumbricals of the foot, masseter, medial pterygoid, medial rectus, middle scalene, multifidus, mylohyoid, obliquus capitis inferior, obliquus capitis superior, obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris, palmar interossei, palmaris brevis, palmaris longus, pectineus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus tertius, piriformis, plantar interossei, plantaris, platysma, popliteus, posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus femoris, quadratus plantae, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor, risorius, sartorius, scalenus minimus, semimembranosus, semispinalis capitis, semispinalis cervicis, semispinalis thoracis, semitendinosus, serratus anterior, short rotators, soleus, spinalis capitis, spinalis cervicis, spinalis thoracis, splenius capitis, splenius cervicis, sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius, subscapularis, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fascia lata, teres major, teres minor, thoracis, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor and any other suitable skeletal muscle as known in the art.

[00259] Administration to diaphragm muscle can be by any suitable method including intravenous administration, intra-arterial administration, and/or intra-peritoneal administration.

Disorders

[00260] The engineered AAV capsid polypeptides, engineered AAV capsid nucleic acids, plasmids, engineered AAV vectors, transfected or transduced cells, and compositions thereof, and methods described herein can be used for treating a muscle disease (e.g., a genetic muscle disorder), neuromuscular disease and/or a heart disease.

[00261] The muscle disorders may include but are not limited to Pompe disease, muscular dystrophies, dystrophinopathies and neuromuscular dystrophies. Exemplary muscular dystrophies include Anderson-Tawil syndrome, Bethlem myopathy, centronuclear myopathy, Charcot-Marie-Tooth disease, congenital muscular dystrophy, desmin- related myopathy, dilated cardiomyopathy, Distal arthrogryposis, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, glycogen storage disease, hypertrophic cardiomyopathy, limb-girdle muscular dystrophy, long QT syndrome, Miyoshi myopathy, muscular dystrophy- dystroglycanopathy, myofibrillar myopathy, myotonic muscular dystrophy, nemaline myopathy, oculopharyngeal muscular dystrophy, Friedreich's ataxia, and spinocerebellar ataxia.

[00262] In some embodiments, the disorder is cardiomyopathy.

[00263] Other common heart disorders include, but are not limited to, congenital heart diseases (e.g., bicuspid aortic valve, Eisenmenger's syndrome, patent ductus arteriosus, pulmonary stenosis, coarctation of the aorta, transposition of the great arteries, tricuspid atresia, univentricular heart, Ebstein's anomaly, and double-outlet right ventricle), Gaucher-like disease, Fabry disease, Lowry-Maclean syndrome, Rett syndrome, Opitz syndrome, Marfan syndrome, Miller-Dieker lissencephaly syndrome, Danon Disease, and mucopolysaccharidosis.

[00264] In some embodiments, the engineered AAV capsid polypeptides, engineered AAV capsid nucleic acids, plasmids, engineered AAV vectors, transfected or transduced cells, and compositions thereof, and methods described herein can be used for treating non-muscular disorders. In some embodiments, the disorder is a systemic disorder. In some embodiments, the disorder is an infection, e.g., a bacterial, viral, or fungal infection.

EXEMPLIFICATION

[00265] The aim of this research was to identify novel adeno-associated virus (AAV) variants with enhanced transduction into muscle of mice and/or cynomolgus non-human primates (NHPs) and reduced immunogenicity. As outlined in Figure 1, a library with over 100 million AAV capsid variants was produced (Example 1), screened in cultured human muscle and cardiac cells (Example 2) and then dosed in cynomolgus NHPs (Example 3). Based on PacBio next-generation sequencing (NGS) analysis of the AAV capsid library’s DNA at key points, a barcoded library of 49 leads along with 5 benchmark capsids was produced, dosed into 3 cynomolgus NHPs and the engineered vector DNA and transgene mRNA expression in key tissues was examined by Illumina® NGS analysis (Example 4).

EXAMPLE 1: Generation And Validation Of An AAV Library With >100 Million Variants

[00266] An AAV capsid polypeptide library was prepared using fragments of four natural AAV serotypes AAV1, AAV6, AAV8 and AAV9 (Tables 1 and 2). The AAV cap open reading frame (ORF), the determinant of tissue tropism, was diversified to over 10⁸ capsid variants using two described technologies: DNA shuffling and peptide display (Borner et al. (2020) Afo/. Ther. 28(4):1016-32; Kienle et al. (2012) J. Vis. Exp. (62):3819, the disclosures of which are hereby incorporated herein by reference in their entireties). For DNA shuffling, the wild type cap ORFs of AAV1, AAV6, AAV8 and AAV9, as well as modified ORFs for peptide insertion, were fragmented by controlled DNase I digestion, followed by a series of polymerase chain reactions (PCRs) that reassembled full-length capsid genes due to partial homologies between the AAV fragments. AAV1, AAV6, AAV8, and AAV9, as well as AAVMYO, a muscle trophic capsid (Weinmann et al. (2020) Nat. Commun. 11(1): 5432, the disclosure of which is hereby incorporated herein by reference in its entirety), served as benchmark controls.

Table 1: Amino Acid Sequence of AAV1, AAV6, AAV8, AAV9, and AAVMYO

Table 2: Nucleotide Sequence of AAV1, AAV6, AAV8, AAV9, and AAVMYO

[00267] For peptide display, four peptide libraries composed of 7 amino acid residues were cloned into comparable locations in the variable region (VR)-VIII (Table 3): Random;

NxxRxxx (random with fixed N and R at positions 1 and 4, respectively); Proline at position 1 and 7; and RGD at position 3.

Table 3: Design of Four Peptide Libraries

[00268] The VR-VIII peptide insertion site is a surface exposed capsid region at the threefold symmetry axis of the AAV capsid. As this region is the site of AAV's natural receptor binding, peptides displayed at this position can interact with cellular receptors and interfere with the natural AAV-receptor interaction. Thus, the displayed peptide was predicted to not only trigger or enhance the transduction of a desired cell type, but also to disrupt the inherent tropism of the underlying AAV capsid to yield a combined increase in specificity and efficiency. See, for example, US Patent Publication No. 20220340929, the disclosure of which is hereby incorporated by reference in its entirety.

[00269] AAV libraries were produced in adherent HEK293T cells by a double transfection of (i) the Adenovirus helper plasmid (Adh) that encodes the essential Adh functions (i.e., E2A, E4 and VA), and (ii) the replication-competent AAV cap plasmid library that encodes AAV2 Rep, AAV2 ITRs, and Cap (pWH.Rep2). Polyethylenimine was used as the transfection reagent (27 pg DNA/ 150 cm² dish; N/P ratio = 30; N/P = ratio between molar units of PEI nitrogen atoms to units of DNA phosphate atoms). Cells were harvested 3 days post-transfection and subjected to freeze-thaw cycles to release viral particles. The cell lysates were treated with benzonase to digest input plasmid DNA that interferes with the quantification of viral DNA. Next, iodixanol gradient centrifugation was performed according to standard methods to purify virus particles from the cell lysate and to separate full from empty particles (i.e., particles devoid of viral genomes). The virus-containing iodixanol phases were combined and dialyzed against phosphate buffered saline (PBS), sterile 0.22 pm filtrated, and concentrated using Amicon columns (Pall, Portsmouth, UK). The final concentration (viral genomes/mL) was determined using TaqMan qPCR analysis (SensiMix™ II Probe No-ROX Kit, Bioline, Herentals, Belgium). Endotoxin levels were determined using a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript, Rijswijk, Netherlands) according to the manufacturer’s instructions.

[00270] The composition of the library was examined by extracting DNA from the virus library, cloning it into pWH.Rep2, transforming bacteria, and sequencing 19 clones by Sanger sequencing according to standard methods. Two clones were found to not have a peptide insert i.e., were shuffled-only), while 17 clones had peptide inserts with the following motifs: seven were Random (xxxxxxx) (SEQ ID NO: 12); three were NxxRxxx (SEQ ID NO: 14); five were PxxxxxP (SEQ ID NO: 16); and 2 were xxRGDxx (SEQ ID NO: 18).

EXAMPLE 2: Screening Of AA V Capsids In Cultured Human Muscle Cells And Mice

[00271] The shuffled AAV capsids were screened for their transduction and expression in various muscle tissues. Cultured primary human cardiac cells (PromoCell, C- 12810) and skeletal muscle cells (PromoCell, C-12530/C-12580) were transduced at a multiplicity of infection (MOI) of 10,000 and cultured for 24 hours. Three six-week-old mice were dosed at 2.5E1 Ivg/kg. After one week, eight on-target tissues were collected: heart, quadriceps femoris, triceps, biceps, gastrocnemius, soleus, and diaphragm.

[00272] DNA extractions from cultured cells and from mouse organs in RNAlater® solution (ThermoFisher Scientific Baltics, Vilnius, Lithuania) were performed using the QIAGEN DNeasy Blood & Tissue kit (Hilden, Germany) according to the manufacturer’s instructions. Primers and probes (both 100 pM stock) were diluted 2.2X and 40X, respectively. To determine the number of viral genomes per diploid genome (vg/dg), two reaction mixes were needed: (i) a 20X rep2 target mix and (ii) a 20X housekeeper mix (albumin). Both mixes contained per reaction: 0.4 pL forward primer, 0.4 pL reverse primer and 0.2 pL probe. A ddPCR master mix was prepared, each reaction contained: 1.1 pL 20 x transgene mix, 1.1 pL 20 x housekeeper mix, 1.1 pL digestion enzyme (Hind III, 1:4 diluted in diluent B), 11 pL BioRad ddPCR™ Supermix for probes (no dUTP) and 2.2 pL H2O. Finally, 5.5 pL of each sample (~5 ng/pL) or negative control (H2O) were added (final volume 22 pL). Droplet generation was performed in the QX200™ Droplet Generator (Bio-Rad). The generated droplets were transferred into a 96-well Eppendorf PCR plate and PCR amplification was performed in the Cl 000 Touch thermal cycler (Bio-Rad, California, United States). Analysis of the wells for an absolute quantification of negative and positive events was performed in the QX200™ Droplet Reader (Bio-Rad, California, United States).

[00273] The composition of the DNA extractions from cultured cells and mouse tissues was examined by cloning into pWH.Rep2 and transforming bacteria, followed by Sanger sequencing. As shown in Table 4, a shift towards peptide inserts with NxxRxxx (SEQ ID NO: 19) and more specifically NxTRxxx (SEQ ID NO: 20) was found in mouse tissues. Table 5 shows the prevalence of the various peptide inserts in the engineered AAV capsid polypeptides after each selection procedure.

Table 4: Peptide Insert Motifs in Initial Library and Following In Vitro and Mouse Selection

Table 5: Prevalence Of Peptide Inserts In The Engineered AAV Capsid Polypeptides

EXAMPLE 3: PacBio NGS Analysis Of the Engineered AA V Capsid Polypeptides In NonHuman Primate (XI IP) Tissues And After IVIg Depletion

[00274] The Cap sequences from primary cells and on-target mouse tissues of Example 2 were cloned into an AAV production plasmid that encodes the AAV2 Rep proteins. An AAV virus library was produced as described in Example 1. Three cynomolgus monkeys (Macaca fascicularis,' NHP) were confirmed negative for neutralizing antibodies (NAbs) to AAV 1 , AAV6, AAV8, and AAV9 (1:5 serum dilution) from serum samples taken approximately 4 weeks before dosing with lE13vg/kg of the AAV library into the saphenous vein. Two weeks after dosing, the animals were euthanized and the following tissues collected in RNAlater®: diaphragm, heart, liver (middle lobe), quadriceps, gastrocnemius, biceps, triceps, soleus, spleen, subcutaneous white fat (abdominal) and tongue.

[00275] DNA extraction and PCR analysis of NHP tissues were performed as described in Example 2. Liver, spleen, and heart contained the highest number of vector genomes per diploid genome (vg/dg) with 35, 5 and 1.2vg/dg, respectively (Figure 2). Between 0.2 and 1.2vg/dg were found in the skeletal muscle tissues, and the lowest values were found in white fate (0.1 vg/dg).

[00276] DNA extractions from on-target NHP tissues (diaphragm, heart, quadriceps, gastrocnemius, biceps, triceps, soleus, and tongue) were combined and a virus library produced as described in Example 1. This virus library was then incubated overnight at 4°C with between 25 and 500pg of human antibodies (IVIg). AAV particles bound to human antibodies were removed by incubation with protein A/G agarose beads. This step removes AAV clones that are most susceptible to binding by antibodies found in the general human population. The supernatants were analyzed by PCR and the percent depletion was calculated of each condition (Table 6) The condition that was incubated with 25 pg, which was depleted by 71.42% was selected for analysis by PacBio NGS sequencing.

Table 6: Confirmation of IVIg Depletion

[00277] PacBio NGS analysis was performed on the four libraries as shown in Figure 1.

The analysis included: (1) DNA extraction of off-target NHP tissues (liver, spleen & white fat);

(2) DNA extraction of on-target NHP tissues (diaphragm, heart, quadriceps, gastrocnemius, biceps, triceps, soleus, and tongue); (3) reconstitution of the AAV library from on-target NHP tissues; and (4) reconstitution of the AAV library from on-target NHP tissues, depleted with 25 pg IVIg.

[00278] Table 7 shows the ranking (based on the number of PacBio reads corresponding to the polypeptide sequence) of AAV particles in the libraries before and after depletion with human antibodies (IVIg). The ranking of nine capsids (SO6, 8, 9, 15, 16, 17, 19, 21 & 22) was improved after IVIg depletion, suggesting those nine capsids are less susceptible to binding by antibodies found in the general human population.

Table 7: Ranking of Clones Before and After IVIg Depletion

[00279] In silico MHCI and MHCII analysis was performed to predict whether the novel engineered AAV capsid polypeptides were more likely to trigger cell-mediated immune responses. Table 8 outlines the individual MHCI and MHCII scores, as well the total MHCI and MHCII scores. A lower score of an engineered AAV capsid polypeptide suggests lower cell-mediated immunogenicity.

Table 8: In Silico MHC Predictions Sorted by Lowest Total MHCI and MHCII Score

[00280] Based on the distribution of Cap DNA sequences in the four libraries, 24 shuffled- only AAV capsids (SO1-SO24) and 8 shuffled AAV capsids containing peptide inserts (SP1- SP8) were selected for further analysis. All the peptide inserts of the 8 SP capsids contained the ‘NxxRxxx’ motif (SEQ ID NO: 14), and three had the identical peptide insert ‘NSARDHS’ (SEQ ID NO: 89) (Table 9).

[00281] To create novel engineered AAV capsid polypeptides with potentially synergistic tropism properties, the top two shuffled-only capsid backbones, i.e., capsid polypeptide lacking peptide insert, and the top capsid polypeptides with peptide inserts were independently identified and combined. The top two capsid backbones, regardless of peptide insert, were the backbones found in SP1 (Backbone ‘A’) and SP2 (Backbone ‘B’). When peptide inserts were ranked, 14 peptide inserts were selected: one was a random peptide sequence, while the remaining 13 had NxxRxxx (SEQ ID NO: 12) motifs, including two with NxTRxxx motifs (SEQ ID NO: 20) (which were found to be enriched after screening in mice, see Table 9 and Figure 2).

[00282] A barcoded capsid library was produced in adherent HEK293T cells by triple transfection of (i) the Adenovirus helper plasmid (Adh) that encodes the essential Adh functions, i.e., E2A, E4 and VA, (ii) AAV RepCap plasmid containing AAV2 Rep along with the Cap sequence one of the 49 AAV capsid leads or one of 5 benchmark capsids (AAV1, AAV6, AAV8, AAV9, and AAVMYO), and (iii) and AAV2 ITR-containing transgene plasmid with a ubiquitous promoter, a yellow fluorescent protein (YFP) transgene followed by unique stretch of nucleotides before a polyA sequence. Polyethylenimine (PEI; company, city, state) was used as the transfection reagent (27 pg DNA/ 150 cm² dish; N/P ratio = 30; N/P = ratio between molar units of PEI nitrogen atoms to units of DNA phosphate atoms). Cells were harvested 3 days post-transfection and subjected to freeze-thaw cycles to release viral particles. The cell lysates were treated with benzonase to digest input plasmid DNA that interferes with the quantification of viral DNA. Next, iodixanol gradient centrifugation was performed to purify virus particles from the cell lysate and to separate the full from the empty (i.e., devoid of viral genomes) particles (also referred to herein as vectors). The virus-containing iodixanol phases were combined and dialyzed against PBS, sterile filtrated (0.22 pm filters), and concentrated using Amicon columns. The concentration (viral genomes/mL) was determined using TaqMan qPCR analysis (SensiMix™ II Probe No-ROX Kit, Bioline). [00283] Table 9 shows the total vector genomes (VGs) obtained for each of the 58 barcoded AAV capsid leads and the 5 benchmarks. Production of nine candidate capsids did not reach 1E+12VG (bold) and were not included in the final barcoded library, which therefore contained 49 engineered AAV capsid polypeptide leads. All of the 9 engineered AAV vectors that were not successfully produced to a 1E+12VG yield contained a peptide insert: One was shuffled capsid polypeptide with a peptide that was detected in the PacBio analysis (SP5), while the other 8 were combined backbone with peptide inserts (5 with backbone ‘A’ and 3 with backbone ‘B’). Notably, the random peptide insert PNQCWIF (SEQ ID NO: 100) did not produce sufficiently well in either backbone A (CAI 1) or backbone B (CB11).

Table 9: Summary of Production Yields and Peptide Inserts of the 58 Candidates

[00284] A phylogenic tree was created for the 24 SO and 8 SP engineered AAV capsid polypeptides (Figure 3). With the exception of SP5, the capsid polypeptides containing peptide inserts (SP1, SP2, SP3, SP4, SP6, SP7, and SP8) are the most homologous to AAV8, relative to AAV1, AAV6, and AAV9. Whereas the shuffled-only capsids (SO1-24) are most homologous to AAV1 and AAV6, with three exceptions (SOI, SO23 and SO24) that were most homologous to AAV8. Based on the phylogenetic tree, the engineered AAV capsid polypeptides were placed into three groups: Group 1, Group 2, and Group 3.

EXAMPLE 4: Evaluation Of The DNA And RNA Tissue Distribution From A Barcoded Library With 49 Leads And 5 Benchmarks In Cynomolgus NHPs

[00285] The final barcoded library was generated by combining 2E+12vg of each of the 49 individually produced engineered AAV capsid polypeptide leads and benchmarks. The final concentration (viral genomes/mL) was determined using a TaqMan qPCR analysis (SensiMi™ II Probe No-ROX Kit, Bioline). Endotoxin levels were determined using a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript) according to the manufacturer’s instructions.

[00286] Three cynomolgus monkeys (Macaca fascicularis,' NHP) were confirmed negative for neutralizing antibodies (NAbs) to AAV1, AAV6, AAV8, and AAV9 (1 :5 serum dilution) approximately 4 weeks before dosing with lE13vg/kg of the barcoded AAV library into the saphenous vein. Two weeks after dosing, the animals were euthanized and the following tissues collected in RNAlater®: diaphragm, heart, liver (middle lobe), quadriceps, gastrocnemius, biceps, triceps, soleus, spleen, subcutaneous white fat (abdominal), tongue, cervical spinal cord, and dorsal root ganglion.

[00287] DNA extractions from tissues were performed using a QIAGEN DNeasy Blood & Tissue kit (Hilden, Germany) according to the manufacturer’s instructions. RNA extractions from tissues were performed using a QIAGEN RNeasy Fibrous Tissue kit (Hilden, Germany) according to the manufacturer’s instructions including on-column DNase I digestion. RNA samples were subjected to additional off-column DNase I digestion in order to remove residual DNA contaminants. In total, 424 ng of extracted RNA was incubated with 1 pl DNase I (2.7 Kunitz units) (Qiagen, Cat#79254) in 40 pL of IxRDD buffer, for 30 minutes at RT and heat inactivated for 10 minutes at 75 °C. First strand cDNA synthesis was performed using a High- Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Cat#4368813, Waltham, MA) on 300 ng of off-column DNase I treated RNA according to the manufacturer’s instructions. Random primers were annealed for 10 minutes at 25 °C and a reverse transcription reaction was performed for 120 minutes at 37 °C, followed by heat inactivation for 5 minutes at 85 °C. Next, capsid DNA barcode regions were amplified from DNA and cDNA samples by PCR amplification. For each PCR reaction 10 u 1 5X Phusion HF buffer, 1 u l dNTPs, 0.25 u 1 forward primer (100 u M), 0.25 u 1 reverse primer (100 u M), 0.5 u 1 Phusion Hot Start II Polymerase and 25 ng of cDNA or DNA template in a total of 50 pl with DEPC-treated H2O were used. Amplified 112 bp long capsid DNA barcode regions were cleaned up using a QIAGEN QiAquick PCR purification kit (Hilden, Germany) and used for Illumina NGS library preparation. To prepare Illumina NGS libraries an Ovation Library System for Low Complexity Samples (Nugen, Cat#9092) was used according to manufacturer’s instructions.

[00288] Illumina® NGS was performed on the final library as well as the DNA and RNA (cDNA) from each tissue of each of the three NHPs. Figure 4 presents graphs of the number of reads each capsid in the final library during either the analysis of tissue DNA (Figure 4a) or tissue RNA (Figure 4b). The similar relative differences between the barcoded variants demonstrates the reproducibility of this analysis.

[00289] Table 10 provided the amino acid sequences for the consensus sequence that encompasses all Group 1 (SO3, SO4, SO5, SO13, SO14, SO15, SO16, SO17, SO18, SO19, SO20, SO21, and SO22) and Group 2 (SO2, SO6, SO7, SO8, SO9, SO , SOU SO12) ammo acid sequences for the engineered AAV capsid polypeptides, as well as the amino acid sequences for each engineered AAV capsid polypeptide.

Table 10: Amino Acid Sequence of Engineered AAV Capsid Polypeptides

[00290] Figure 5 shows an alignment of the Group 1 and Group 2 capsid amino acid sequences with AAV1, AAV6, AAV8, and AAV9 as well as the consensus sequence (SEQ ID NO: 103). The lightly shaded “x” positions in the consensus sequence represent variable amino acid positions among the engineered AAV capsid polypeptide sequences. Black shaded positions in the consensus sequence represent variable amino acids among AAV1, AAV6, AAV8, and AAV9 serotypes that are conserved in the Group 1 and Group 2 engineered AAV capsid polypeptides. Shading of position 24 of SO21 represents a novel amino acid mutation.

[00291] Figure 6 shows the consensus sequence (SEQ ID NO: 103) and Segments 1-5.

[00292] A normalized read for each engineered AAV capsid polypeptide was obtained by dividing its number of reads in the final library and by the maximum value obtained for that tissue.

[00293] Figure 7 shows the stacked (i.e., combined) average normalized amount of engineered vector DNA in NHP diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue for the 49 clones compared to AAV1, AAV6, AAV8, AAV9, and AAVMYO controls. Group 1 and 2 had higher values than Group 3, suggesting better delivery of DNA to muscle tissues. Seven engineered AAV vectors (comprising engineered AAV capsid polypeptide SO8, SO9, SO12, SO14, SO15, SO17, or SO21) outperformed all 5 benchmarks and all had conserved amino acids L587 and V601, relative to SEQ ID NO: 103, suggesting these amino acids are important for increasing DNA delivery to these muscle tissues.

[00294] Figure 8 shows the stacked average normalized amount of transgene (YFP-barcode) RNA in NHP diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue for the 49 clones compared to AAV1, AAV6, AAV8, AAV9, and AAVMYO controls. In contrast to DNA analysis, all Group 2 engineered AAV vectors had higher transgene RNA values than Group 1 suggesting a positive influence of these engineered AAV vectors on the translation of their DNA genome.

[00295] Figure 9 shows vector genome per diploid genome of all variants in collected tissues. When compared to the distribution of the non-barcoded library (Figure 2), higher values were observed in skeletal muscles (diaphragm, biceps, triceps, gastrocnemius, quadriceps, and tongue) relative to heart. [00296] Tables 11-21 show the ranking by normalized RNA levels in the three NHPs for the 40 clones in various muscle and non- muscle tissues: Table 11 (diaphragm); Table 12 (biceps); Table 13 (heart); Table 14 (triceps); Table 15 (quadriceps); Table 16 (tongue); Table 17 (liver); Table 18 (spleen); Table 19 (white fat); Table 20 (dorsal root ganglion, DRG); and Table 21 (cervical spinal cord). Benchmark capsids are bolded, and the engineered AAV vectors are sorted by their mean RNA normalized reads. Two observations further shows the difficulty in transferring data on muscle tropism from mice to old world NHPs:

(1) AAV6 had higher vector DNA and transgene RNA than AAV9 in skeletal muscle tissues (biceps, diaphragm, quadriceps, tongue, and triceps), which contradicts what has been reported in mice (Zincarelli etal. (2008) Afo/. Ther. 16(6): 1073-80; Yang et al. (2009) Proc. Natl. Acad. Sci. USA 106(10): 3946-51; Weber- Adrian et a/. (2021) Set. Rep. 11(1): 1934), the disclosures of which are hereby incorporated herein by reference in their entireties)

(2) AAVMYO had lower vector DNA and transgene mRNA in several muscle tissues (biceps, heart, quadriceps, and triceps) than AAV9 which is in contrast to what has been found in mice (Weinmann et al. (2020) Nat. Commun. 11(1): 5432, the disclosure of which is hereby incorporated herein by reference in its entirety).

[00297] Many of the engineered AAV vectors, in particular Group 1 and Group 2, ranked above one or more of the benchmarks. On an RNA level, SO2 and SO9 ranked above all benchmarks in diaphragm, biceps, heart, quadriceps, and tongue. SO2 and SO9 also ranked lower than AAV6 (the top muscle-tropic benchmark) on both DNA and RNA level in liver. Tropism to liver is thought to adversely contribute to the safety profile of muscle directed AAV therapies resulting in high-dose AAV gene therapy deaths ((2020) Nat. Biotechnol. 38:910, the disclosure of which is hereby incorporated herein by reference in its entirety).

Table 11: Ranking of Normalized DNA and RNA NGS Reads in Diaphragm

Table 12: Ranking of Normalized DNA and RNA NGS Reads in Biceps

Table 13: Ranking of Normalized DNA and RNA NGS Reads in Heart

Table 14: Ranking of Normalized DNA and RNA NGS Reads in Triceps

Table 15: Ranking of Normalized DNA and RNA NGS Reads in Quadriceps

Table 16: Ranking of Normalized DNA and RNA NGS Reads in Tongue

Table 17: Ranking of Normalized DNA and RNA NGS Reads in Liver

Table 18: Ranking of Normalized DNA and RNA NGS Reads in Spleen

Table 19: Ranking of Normalized DNA and RNA NGS Reads in White Fat

Table 20: Ranking of Normalized DNA and RNA NGS Reads in Dorsal Root Ganglion (DRG)

Table 21: Ranking of Normalized DNA and RNA NGS Reads in Cervical Spinal Cord

EXAMPLE 5: Evaluation Of Reactivity of AA V Capsid Variants to Neutralizing Antibodies from Human Plasma Samples

[00298] Engineered AAV vectors with an engineered AAV capsid polypeptide SO2, SO8, SO9, SO12, or SO14) or a wt serotype benchmark (AAV1, AAV2, AAV3b, AAV5, AAV6, AAV8, AAV9 and AAVrh74) were produced with a transgene encoding the LacZ protein under the control of the ubiquitous cytomegalovirus enhancer-chicken beta-actin promoter and flanked by AAV2 inverted terminal repeats. HEK293 cells were triple transfected with a trans-plasmid containing the rep and either a AAV capsid variant or wt benchmark capsid encoding cap genes, and a helper plasmid containing adenoviral genes E2A, E4 and VA followed by purification of cell lysate and culture media with two rounds of cesium chloride sedimentation and dialysis. The genome titer was determined by ddPCR, the purity assessed by SDS-PAGE followed by silver staining to visualize VP1, VP2 and VP3. Naive mouse serum (Merck, cat # M5905, Darmstadt, Germany) and 50 human plasma samples were heat-inactivated at 56°C for 35 minutes and diluted 1 :10, 1 :20, or 1 :40 in Dulbecco's Modified Eagle's Medium (DMEM). lOOpL of the diluted serum or plasma were added to triplicate wells with 5E+4 HuH-7.5 cells cultured at 37°C and 5% CO2. The cells were transduced with wt adenovirus type 5 at an MOI of 100 (virus particles to plated cells) for 4 hours, then lOOpL of the AAV-LacZ vectors at an MOI of 10,000 (vector genomes to plated cell) for one hour. The media was then changed to DMEM with 5% fetal bovine serum. Cell lysates were obtained the next day and 0- galactosidase activity in cell lysate was measured using the Galacto-Star One-Step 0- galactosidase Reporter Gene Assay System (Thermo Fisher Scientific, cat. #T1014, Waltham, United States), in which the b-galactosidase activity correlates with luminescence readings. Reduction of the luminescence by 50% or less was considered negative. Results for each plasma donor are presented in Table 22 and summarized in Figure 10.

Table 22: Neutralizing Antibody Screen Of Human Plasma

[00299] As shown in Figure 10, the results suggest that the engineered AAV capsid polypeptides have similar negativity rates (combined 1: 10 and 1:20 percentage) as AAV1 and AAV6.

[00300] Figures 11A and 11B collectively show an alignment of the amino acid sequences of a subset of Group 1 AAV capsid polypeptides with the highest stacked muscle vector DNA values and a corresponding consensus sequence (SEQ ID NO: 204). Figures 12A and 12B collectively show an alignment of the amino acid sequences of a subset of Group 2 AAV capsid polypeptides with the highest stacked muscle vector DNA values and a corresponding consensus sequence (SEQ ID NO: 205). Figures 13A, 13B, and 13C collectively show an alignment of the amino acid sequences of a subset of Group 1 and Group 2 AAV capsid polypeptides with the highest stacked muscle vector DNA values and a corresponding consensus sequence (SEQ ID NO: 206). The “x” positions in the consensus sequence represent variable amino acid positions among the engineered AAV capsid polypeptide sequences of the alignment.

INCORPORATION BY REFERENCE

[00301] The present disclosure incorporates by reference in their entirety techniques well known in the field of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications: Ausubel et al. (eds.) (1993) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY; Ausubel et al. (eds.) (1999) SHORT PROTOCOLS IN MOLECULAR BIOLOGY (4th Ed.) John Wiley & Sons, NY; Smolen and Ball (eds.) (1984) CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Wiley & Sons, NY; Giege et al. (1999) CRYSTALLIZATION OF NUCLEIC ACIDS AND PROTEINS, a Practical Approach (2nd Ed.) Oxford University Press, NY; Hammerling etal. (1981) MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS, Elsevier, NY; Harlow et al. (1988) ANTIBODIES: A LABORATORY MANUAL (2^nd Ed.) Cold Spring Harbor Laboratory Press, NY; Kabat et al. (1987 and 1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health, Bethesda, MD; Kabat et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (5^th Ed.) U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Kontermann and Dubel (eds.) (2001) ANTIBODY ENGINEERING, Springer- Verlag, NY; Knegler (1990) GENE TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY; Lu and Weiner (eds.) (2001) CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS BioTechniques Press, MA; Old and Primrose (1985) PRINCIPLES OF GENE MANIPULATION: AN INTRODUCTION TO GENETIC ENGINEERING (3rd Ed.) Blackwell Scientific Publications, MA; Sambrook etal. (eds.) (1989) MOLECULAR CLONING: A LABORATORY MANUAL (2nd Ed.) Cold Spring Harbor Laboratory Press, NY; Winnacker (1987) FROM GENES TO CLONES: INTRODUCTION TO GENE TECHNOLOGY, VCH Publishers, NY. ADENO-ASSOCIATED VIRUS VECTORS Humana New York, NY. Michael J. Castle (2019)

[00302] The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein. The disclosure will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, and cell biology, which are well known in the art.

EQUIVALENTS AND SCOPE

[00303] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims

CLAIMS We claim:

1. An engineered chimeric adeno-associated virus (AAV) capsid polypeptide comprising two or more unique segments of at least six consecutive amino acids from different AAV serotypes selected from the group consisting of AAV1, AAV6, AAV8, and AAV9, wherein the engineered chimeric AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wild-type (wt) AAV capsid polypeptide from any one of the AAV1, AAV6, AAV8, and AAV9 serotypes.

2. The engineered chimeric AAV capsid polypeptide of claim 1, wherein the two or more segments of amino acids include segment 1 of residues 24-42, segment 2 of residues 125-135, segment 3 of residues 158-169, segment 4 of residues 195-206, or segment 5 of residues 600- 605, numbered relative to SEQ ID NO: 103.

3. An engineered chimeric AAV capsid polypeptide comprising five segments of at least six consecutive amino acids, wherein at least two of the segments are from different AAV serotypes selected from the group consisting of AAV1, AAV6, AAV8 and AAV9, wherein the engineered chimeric AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from any one of AAV1, AAV6, AAV8, and AAV9 serotypes.

4. The engineered chimeric AAV capsid polypeptide of claim 3, wherein the five segments of amino acids are segment 1 of residues 24-42, segment 2 of residues 125-135, segment 3 of residues 158-169, segment 4 of residues 195-206, and segment 5 of residues 600-605, numbered relative to SEQ ID NO: 103.

5. The engineered chimeric AAV capsid polypeptide of claim 4, wherein segment 5 comprises amino acids HVMGAL (SEQ ID NO: 221) from AAV6.

6. The engineered chimeric AAV capsid polypeptide of claim 5, wherein segment 3 or segment 4 is from AAV1, AAV6, or AAV8.

7. The engineered chimeric AAV capsid polypeptide of claim 6, wherein segment 3 comprises amino acids TGIGKKGQQPAR (SEQ ID NO: 216) from AAV8.

8. The engineered chimeric AAV capsid polypeptide of claim 6 or 7, wherein segment 4 comprises amino acids APSGVGPNTMAA (SEQ ID NO: 219) from AAV8.

9. The engineered chimeric AAV capsid polypeptide of any one of claims 4-8, wherein segment 1 or segment 2 is from AAV1, AAV6, AAV8, or AAV9.

10. The engineered chimeric AAV capsid polypeptide of any one of claims 4-9, wherein segment 1 comprises amino acids ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) from AAV9.

11. The engineered chimeric AAV capsid polypeptide of any one of claims 4-9, wherein segment 2 comprises amino acids LLEPLGLVEEA (SEQ ID NO: 212) from AAV9.

12. The engineered chimeric AAV capsid polypeptide of any one of claims 4-9, wherein segment 2 comprises amino acids VLEPFGLVEEG (SEQ ID NO: 213) from AAV6.

13. The engineered chimeric AAV capsid polypeptide of any one of claims 2-4, wherein the engineered chimeric AAV capsid polypeptide comprises five segments of amino acids:

SI a, S2b, S3_C, S4_d, and S5_e wherein SI is segment 1 of residues 24-42 and a is the AAV serotype from which SI is derived; wherein S2 is segment 2 of residues 125-135 and b is the AAV serotype from which S2 is derived; wherein S3 is segment 3 of residues 158-169 and c is the AAV serotype from which S3 is derived; wherein S4 is segment 4 of residues 195-206 and d is the AAV serotype from which S4 is derived; and wherein S5 is segment 5 of residues 600-605 and e is the AAV serotype from which S5 is derived.

14. The engineered chimeric AAV capsid polypeptide of claims 13, comprising:

SI a, S2AAVI, S3AAVI, S4AAVI, and S5AAV6 wherein SI comprises the amino acid sequence of ELKPGAPKPKANQQKQDDG (SEQ ID NO: 202).

15. The engineered chimeric AAV capsid polypeptide of claim 13, comprising:

SIAAVI, S2AAVI, S3AAVI, S4AAVI, and S5AAV6.

16. The engineered chimeric AAV capsid polypeptide of claim 13, comprising:

SIAAVS, S2AAV6, S3AAVI, S4AAVI, and S5AAV6.

17. The engineered chimeric AAV capsid polypeptide of claim 13, comprising:

S1AAV9, S2AAV9, S3AAV8, S4AAV8, and S5AAV6.

18. The engineered chimeric AAV capsid polypeptide of claim 13, comprising: S1AAV9, S2AAV9, S3AAV8, S4AAV8, and S5AAVI.

19. The engineered chimeric AAV capsid polypeptide of claim 13, comprising:

SIAAVS, S2AAVI, S3AAV8, S4a, and S5AAV6, wherein S4 comprises the amino acid sequence of APSGVGPTTMAS (SEQ ID NO: 203).

20. An engineered AAV capsid polypeptide having a greatest sequence identity to a first AAV serotype selected from the group consisting of AAV1, AAV6, AAV8, and AAV9, wherein the AAV capsid protein comprises an amino acid substitution relative to the first AAV serotype at one or more positions selected from the group consisting of residues 24, 163, 169, 195, 197, 198, 587, and 601 of SEQ ID NO: 103, and wherein the engineered AAV capsid polypeptide has an enhanced tropism for muscle as compared to a wt AAV capsid polypeptide from the first AAV serotype.

21. The engineered AAV capsid polypeptide of claim 20, comprising an alanine at position 24.

22. The engineered AAV capsid polypeptide of claim 20 or 21, comprising a threonine at position 163.

23. The engineered AAV capsid polypeptide of any one of claims 20-22, comprising an arginine at position 169.

24. The engineered AAV capsid polypeptide of any one of claims 20-23, comprising an alanine at position 195.

25. The engineered AAV capsid polypeptide of any one of claims 20-24, comprising a serine at position 197.

26. The engineered AAV capsid polypeptide of any one of claims 20-25, comprising a glycine at position 198.

27. The engineered AAV capsid polynucleotide of any one of claims 20-26, comprising a threonine at position 163 and an arginine at position 169.

28. The engineered AAV capsid polypeptide of any one of claims 20-27, comprising a leucine at position 587.

29. The engineered AAV capsid polypeptide of any one of claims 20-28, comprising a valine at position 601.

30. An engineered AAV capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 103, wherein the amino acid at position 24 is A, D, or E; the amino acid residue at position 31 is Q or K; the amino acid residue at position 38 is H or K; the amino acid residue at position 41 is N or D; the amino acid residue at position 42 is A or G; the amino acid residue at position 56 is F or G; the amino acid residue at position 84 is K or Q; the amino acid residue at position 125 is L or V; the amino acid residue at position 129 is L or F; the amino acid residue at position 135 is A or G; the amino acid residue at position 152 is absent; the amino acid residue at position 158 is T or S; the amino acid residue at position 163 is K or T; the amino acid residue at position 169 is R or K; the amino acid residue at position 180 is S or T; the amino acid residue at position 195 is A or T; the amino acid residue at position 197 is S or A; the amino acid residue at position 198 is G or A; the amino acid residue at position 202 is N or T; the amino acid residue at position 206 is A or S; the amino acid residue at position 263 is S or N; at the amino acid residue at position 264 is A or S; the amino acid residue at position 265 is S or T; the amino acid residue at position 266 is S or T; the amino acid residue at position 267 is G or absent; the amino acid residue at position 269 is A or S; the amino acid residue at position 274 is H or A; the amino acid residue at position 420 is D or E; the amino acid residue at position 465 is absent; the amino acid residue at position 587 is L or F; the amino acid residue at position 601 is V or A; and the amino acid residue at position 645 is N or H, and wherein the engineered AAV capsid protein does not comprise the amino acid sequence of any one of SEQ ID NOS: 1-4.

31. The engineered AAV capsid polypeptide of claim 30, wherein the amino acid at position 24 is A; at position 84 is K; at position 129 is L; at position 163 is K; at position 169 is R; at position 180 is S; at position 195 is A; at position 197 is S; and at position 198 is G; and at position 267 is G or is absent.

32. The engineered AAV capsid polypeptide of claim 30, wherein the amino acid at position 24 is A; at position 56 is F; at position 84 is K; at position 129 is L; at position 163 is K; at position 169 is R; position 180 is S; at position 195 is A; at position 197 is S; at position 198 is G; at position 267 is G or absent; at position 420 is D; at position 587 is L; at position 601 is V; and at position 645 is N.

33. The engineered AAV capsid polypeptide of claim 31, wherein the amino acid at position 267 is G.

34. The engineered AAV capsid polypeptide of claim 30, wherein the amino acid at position 56 is F; at position 180 is S; at position 263 is S; at position 264 is A; at position 265 is S; at position 266 is T; at position 267 is absent; at position 269 is A; at position 274 is H; at position 587 is L; and at position 601 is V.

35. An engineered AAV capsid polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 104-152, or a variant that comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 104-152 wherein the variant maintains at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70, at least 80%, at least 90%, or at least 95% of the level of muscle tropism of the AAV capsid polypeptide having the sequence selected from the group consisting of SEQ ID NOs: 104- 152 that has the greatest sequence identity to the variant .

36. An engineered AAV capsid polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 105 and 109-115.

37. The engineered AAV capsid polypeptide of claim 36, comprising the amino acid sequence of SEQ ID NO: 105.

38. The engineered AAV capsid polypeptide of claim 36, comprising the amino acid sequence of SEQ ID NO: 112.

39. The engineered AAV capsid polypeptide of any one of the preceding claims, wherein an engineered AAV vector comprising the engineered chimeric AAV capsid polypeptide results in higher expression levels of transgene RNA in muscle as compared to an engineered AAV vector comprising a wt capsid polypeptide of at least one of AAV1, AAV6, AAV8 and AAV9.

40. The engineered AAV capsid polypeptide of claim 39, wherein the higher expression levels of transgene RNA in muscle occur in vivo.

41. The engineered AAV capsid polypeptide of claim 39 or 40, wherein the higher mRNA levels occur in at least one muscle tissue selected from the group consisting of diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue.

42. The engineered AAV capsid polypeptide of claim 41, wherein the higher mRNA levels occur in at least two muscle tissues selected from the group consisting of diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue.

43. The engineered AAV capsid polypeptide of any one of claims 40-42, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 105, and 109-115.

44. The engineered AAV capsid polypeptide of any one of the preceding claims, wherein an engineered AAV vector comprising the engineered chimeric AAV capsid polypeptide results in increased delivery of engineered AAV vector DNA into muscle tissues of a transduced host as compared to an AAV vector comprising a wt capsid polypeptide of at least one of AAV1, AAV6, AAV8 and AAV9.

45. The engineered AAV capsid polypeptide of claim 44, wherein the host muscle tissue is selected from the group consisting of diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue.

46. The engineered AAV capsid polypeptide of claim 45, wherein the engineered AAV vector comprising the engineered chimeric AAV capsid polypeptide results in increased delivery of engineered AAV vector DNA to at least two muscle tissues of a transduced host, the muscle tissue selected from the group consisting of diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue.

47. The engineered AAV capsid polypeptide of any one of claims 44-46, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 111, 112, 115, 117, 118, 120 and 124.

48. The engineered AAV capsid polypeptide of any one of claims 39-43, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 105, 111, 112, and 115.

49. The engineered AAV capsid polypeptide of any one of claims 39-43, comprising the amino acid sequence of SEQ ID NO: 112.

50. The engineered chimeric AAV capsid polypeptide of any one of the preceding claims, wherein the muscle tissue is human muscle tissue.

51. The engineered chimeric AAV capsid polypeptide of claim 50, wherein the human muscle tissue is selected from the group consisting of heart, gastrocnemius, biceps, quadriceps, triceps, soleus, diaphragm, and tongue.

52. An engineered adeno-associated virus (AAV) capsid polypeptide VP1 having a sequence selected from the group consisting of:

A) a sequence having at least 97% sequence identity to SEQ ID NO: 1 comprising a variant segment selected from group consisting of:

(i) a variant AAV1 segment 1 corresponding to residues 24-42 of SEQ ID NO: 1, wherein the variant AAV1 segment 1 has at least one amino acid variation relative to residues 24-42 of SEQ ID NO: 1,

(ii) a variant AAV1 segment 2 corresponding to residues 125-135 of SEQ ID NO: 1, wherein the variant AAV1 segment 2 has at least one amino acid variation relative to residues 125-135 of SEQ ID NO: 1,

(iii) a variant AAV1 segment 3 corresponding to residues 157-168 of SEQ ID NO: 1, wherein the variant AAV1 segment 3 has at least one amino acid variation relative to residues 157-168 of SEQ ID NO: 1, (iv) a variant AAV1 segment 4 corresponding to residues 194-205 of SEQ ID

NO: 1, wherein the variant AAV1 segment 4 has at least one amino acid variation relative to residues 194-205 of SEQ ID NO: 1,

(v) a variant AAV1 segment 5 corresponding to residues 597-602 of SEQ ID NO:

1, wherein the variant AAV1 segment 5 has at least one amino acid variation relative to residues 597-602 of SEQ ID NO: 1 ; and

B) a sequence having at least 97% sequence identity to SEQ ID NO: 2 comprising a variant segment selected from group consisting of:

(i) a variant AAV6 segment 1 corresponding to residues 24-42 of SEQ ID NO: 2, wherein the variant AAV6 segment 1 has at least one amino acid variation relative to residues 24-42 of SEQ ID NO: 2,

(ii) a variant AAV6 segment 2 corresponding to residues 125-135 of SEQ ID NO:

2, wherein the variant AAV6 segment 2 has at least one amino acid variation relative to residues 125-135 of SEQ ID NO: 2,

(iii) a variant AAV6 segment 3 corresponding to residues 157-168 of SEQ ID NO: 2, wherein the variant AAV6 segment 3 has at least one amino acid variation relative to residues 157-168 of SEQ ID NO: 2,

(iv) a variant AAV6 segment 4 corresponding to residues 194-205 of SEQ ID NO: 2, wherein the variant AAV6 segment 4 has at least one amino acid variation relative to residues 194-205 of SEQ ID NO: 2, and

(v) a variant AAV6 segment 5 corresponding to residues 597-602 of SEQ ID NO:

2, wherein the variant AAV6 segment 5 has at least one amino acid variation relative to residues 597-602 of SEQ ID NO: 2.

53. The engineered AAV capsid polypeptide of claim 52, having at least 97% sequence identity to SEQ ID NO: 1, wherein the engineered AAV capsid polypeptide comprises a variant AAV1 segment 1 having the amino acid sequence X1LKPGAPX2PKANQQX3QDX4X5 (SEQ ID NO: 207), wherein Xi is A, D, or E, X2 is K or Q, X3 is H or K, X4 is N or D, and X5 is A or G.

54. The engineered AAV capsid polypeptide of claim 53, wherein the variant AAV1 segment 1 has an amino acid sequence selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208), ELKPGAPKPKANQQKQDDG (SEQ ID NO: 209), and ALKPGAPKPKANQQKQDDG (SEQ ID NO: 210).

55. The engineered AAV capsid polypeptide of any one of claims 52-54, having at least 97% sequence identity to SEQ ID NO: 1, wherein the engineered AAV capsid polypeptide comprises a variant AAV1 segment 2 having the amino acid sequence X1LEPX2GLVEEX3 (SEQ ID NO: 211), wherein Xi is L or V, X2 is L or F, and X3 is A or G.

56. The engineered AAV capsid polypeptide of claim 55, wherein the variant AAV1 segment 2 has an amino acid sequence of LLEPLGLVEEA (SEQ ID NO: 212) or VLEPFGLVEEG (SEQ ID NO: 213).

57. The engineered AAV capsid polypeptide of any one of claims 52-56, having at least 97% sequence identity to SEQ ID NO: 1, wherein the engineered AAV capsid polypeptide comprises a variant AAV1 segment 3 having the amino acid sequence X1GIGKX2GQQPAX3 (SEQ ID NO: 214), wherein Xi is T or S, X2 is K or T, and X3 is K or R.

58. The engineered AAV capsid polypeptide of claim 57, wherein the variant AAV1 segment 3 has an amino acid sequence selected from the group consisting of SGIGKTGQQPAK (SEQ ID NO: 215), TGIGKKGQQPAR (SEQ ID NO: 216), and SGIGKKGQQPAR (SEQ ID NO: 217).

59. The engineered AAV capsid polypeptide of any one of claims 52-58, having at least 97% sequence identity to SEQ ID NO: 1, wherein the engineered AAV capsid polypeptide comprises a variant AAV1 segment 4 having the amino acid sequence X1PX2X3VGPX4TMAX5 (SEQ ID NO: 218), wherein Xi is A or T, X2 is S or A, X3 is G or A, X4 is N or T, and X5 is A or S.

60. The engineered AAV capsid polypeptide of claim 59, wherein the variant AAV1 segment 4 has an amino acid sequence of APSGVGPNTMAA (SEQ ID NO: 219), or APSGVGPTTMAS (SEQ ID NO: 220).

61. The engineered AAV capsid polypeptide of any one of claims 52-60, having at least 97% sequence identity to SEQ ID NO: 1, wherein the engineered AAV capsid polypeptide comprises a variant AAV1 segment 5 having the amino acid sequence HVMGAL (SEQ ID NO: 221).

62. The engineered AAV capsid polypeptide of any one of claims 53-61, comprising an F56G amino acid substitution relative to SEQ ID NO: 1.

63. The engineered AAV capsid polypeptide of any one of claims 53-62, comprising a K84Q amino acid substitution relative to SEQ ID NO: 1.

64. The engineered AAV capsid polypeptide of any one of claims 53-63, comprising an S179T amino acid substitution relative to SEQ ID NO: 1.

65. The engineered AAV capsid polypeptide of any one of claims 53-64, comprising an SASTGA262-267NSTSGGS amino acid substitution relative to SEQ ID NO: 1.

66. The engineered AAV capsid polypeptide of any one of claims 53-65, comprising an H272A amino acid substitution relative to SEQ ID NO: 1.

67. The engineered AAV capsid polypeptide of any one of claims 53-66, comprising an E418D amino acid substitution relative to SEQ ID NO: 1.

68. The engineered AAV capsid polypeptide of any one of claims 53-67, comprising an F584L amino acid substitution relative to SEQ ID NO: 1.

69. The engineered AAV capsid polypeptide of any one of claims 53-68, comprising an N642H amino acid substitution relative to SEQ ID NO: 1.

70. The engineered AAV capsid polypeptide of any one of claims 53-69, having at least 98% sequence identity to SEQ ID NO: 1.

71. The engineered AAV capsid polypeptide of any one of claims 53-69, having at least 99% sequence identity to SEQ ID NO: 1.

72. The engineered AAV capsid polypeptide of any one of claims 53-69, having at least 99.5% sequence identity to SEQ ID NO: 1.

73. The engineered AAV capsid polypeptide of claim 52, having at least 97% sequence identity to SEQ ID NO: 2, wherein the engineered AAV capsid polypeptide comprises a variant AAV6 segment 1 having the amino acid sequence X1LKPGAPX2PKANQQX3QDX4X5 (SEQ ID NO: 207), wherein Xi is A, D, or E, X2 is K or Q, X3 is H or K, X4 is N or D, and X5 is A or G.

74. The engineered AAV capsid polypeptide of claim 73, wherein the variant AAV6 segment 1 has an amino acid sequence selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208), ELKPGAPKPKANQQKQDDG (SEQ ID NO: 209), and ALKPGAPKPKANQQKQDDG (SEQ ID NO: 210).

75. The engineered AAV capsid polypeptide of any one of claims 52, 73, and 74, having at least 97% sequence identity to SEQ ID NO: 2, wherein the engineered AAV capsid polypeptide comprises a variant AAV6 segment 2 having the amino acid sequence X1LEPX2GLVEEX3 (SEQ ID NO: 211), wherein Xi is L or V, X2 is L or F, and X3 is A or G.

76. The engineered AAV capsid polypeptide of claim 75, wherein the variant AAV6 segment 2 has an amino acid sequence of LLEPLGLVEEA (SEQ ID NO: 212) or VLEPLGLVEEG (SEQ ID NO: 222).

77. The engineered AAV capsid polypeptide of any one of claims 52 and 73-76, having at least 97% sequence identity to SEQ ID NO: 2, wherein the engineered AAV capsid polypeptide comprises a variant AAV6 segment 3 having the amino acid sequence X1GIGKX2GQQPAX3 (SEQ ID NO: 214 wherein Xi is T or S, X2 is K or T, and X3 is K or R.

78. The engineered AAV capsid polypeptide of claim 77, wherein the variant AAV6 segment 3 has an amino acid sequence of TGIGKKGQQPAR (SEQ ID NO: 216) or SGIGKKGQQPAR (SEQ ID NO: 217).

79. The engineered AAV capsid polypeptide of any one of claims 52 and 73-78, having at least 97% sequence identity to SEQ ID NO: 2, wherein the engineered AAV capsid polypeptide comprises a variant AAV6 segment 4 having the amino acid sequence X1PX2X3VGPX4TMAX5 (SEQ ID NO: 218), wherein Xi is A or T, X2 is S or A, X3 is G or A, X4 is N or T, and X5 is A or S.

80. The engineered AAV capsid polypeptide of claim 79, wherein the variant AAV6 segment 4 has an amino acid sequence of APSGVGPNTMAA (SEQ ID NO: 219), or APSGVGPTTMAS (SEQ ID NO: 220).

81. The engineered AAV capsid polypeptide of any one of claims 52 and 73-80, having at least 97% sequence identity to SEQ ID NO: 2, wherein the engineered AAV capsid polypeptide comprises a variant AAV6 segment 5 having the amino acid sequence HAMGAL (SEQ ID NO: 223).

82. The engineered AAV capsid polypeptide of any one of claims 73-81, comprising an F56G amino acid substitution relative to SEQ ID NO: 2.

83. The engineered AAV capsid polypeptide of any one of claims 73-82, comprising a K84Q amino acid substitution relative to SEQ ID NO: 2.

84. The engineered AAV capsid polypeptide of any one of claims 73-83, comprising an S179T amino acid substitution relative to SEQ ID NO: 2.

85. The engineered AAV capsid polypeptide of any one of claims 73-84, comprising an SASTGA262-267NSTSGGS amino acid substitution relative to SEQ ID NO: 2.

86. The engineered AAV capsid polypeptide of any one of claims 73-85, comprising an H272A amino acid substitution relative to SEQ ID NO: 2.

87. The engineered AAV capsid polypeptide of any one of claims 73-86, comprising an D418E amino acid substitution relative to SEQ ID NO: 2.

88. The engineered AAV capsid polypeptide of any one of claims 73-87, comprising an K531E amino acid substitution relative to SEQ ID NO: 2.

89. The engineered AAV capsid polypeptide of any one of claims 73-88, comprising an L584F amino acid substitution relative to SEQ ID NO: 2.

90. The engineered AAV capsid polypeptide of any one of claims 73-89, comprising an H642N amino acid substitution relative to SEQ ID NO: 2.

91. The engineered AAV capsid polypeptide of any one of claims 73-90, having at least 98% sequence identity to SEQ ID NO: 2.

92. The engineered AAV capsid polypeptide of any one of claims 73-90, having at least 99% sequence identity to SEQ ID NO: 2.

93. The engineered AAV capsid polypeptide of any one of claims 73-90, having at least 99.5% sequence identity to SEQ ID NO: 2.

94. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 1 and comprising an amino acid substitution selected from the group consisting of D24A, D24E, K31Q, K38H, D41N, G42A, F56G, K84Q, V125L, L129F, G135A, S157T, T162K, A166P, P167A, K168R, S179T, T194A, A196S, A197G, T201N, S205A, SASTGA262-267NSTSGGS, H272A, E418D, F584L, A598V, and N642H.

95. The engineered AAV capsid polypeptide of claim 94, having at least 98% sequence identity to SEQ ID NO: 1.

96. The engineered AAV capsid polypeptide of claim 94, having at least 99% sequence identity to SEQ ID NO: 1.

97. The engineered AAV capsid polypeptide of claim 94, having at least 99.5% sequence identity to SEQ ID NO: 1.

98. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 2 and comprising an amino acid substitution selected from the group consisting of D24A, D24E, K31Q, K38H, D41N, G42A, F56G, K84Q, V125L, F129L, G135A, S157T, T162K, K168R, S179T, T194A, A196S, A197G, T201N, S205A, SASTGA262-267NSTSGGS, H272A, D417E, K531E, L584F, V598A, and H642N.

99. The engineered AAV capsid polypeptide of claim 98, comprising a glutamic acid at position 531.

100. The engineered AAV capsid polypeptide of claim 98 or 99, having at least 98% sequence identity to SEQ ID NO: 2.

101. The engineered AAV capsid polypeptide of claim 98 or 99, having at least 99% sequence identity to SEQ ID NO: 2.

102. The engineered AAV capsid polypeptide of claim 98 or 99, having at least 99.5% sequence identity to SEQ ID NO: 2.

103. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 124 comprising a glutamic acid at position 24.

104. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 124 and comprising (i) at least one amino acid selected from the group consisting of proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, valine at position 598, and histidine at position 642 and (ii) one or both of leucine at position 129 and glutamic acid at position 531.

105. The engineered AAV capsid polypeptide of claim 103 or 104, having at least 98% sequence identity to SEQ ID NO: 124.

106. The engineered AAV capsid polypeptide of claim 103 or 104, having at least 99% sequence identity to SEQ ID NO: 124.

107. The engineered AAV capsid polypeptide of claim 103 or 104, having at least 99.5% sequence identity to SEQ ID NO: 124.

108. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 120 and comprising (i) at least one amino acid selected from the group consisting of proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, and valine at position 598 and (ii) at least one amino acid selected from the group consisting of leucine at position 129, glutamic acid at position 531, and asparagine at position 642.

109. The engineered AAV capsid polypeptide of claim 108, having at least 98% sequence identity to SEQ ID NO: 120.

110. The engineered AAV capsid polypeptide of claim 108, having at least 99% sequence identity to SEQ ID NO: 120.

111. The engineered AAV capsid polypeptide of claim 108, having at least 99.5% sequence identity to SEQ ID NO: 120.

112. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 118 and comprising one or both of an alanine at position 24 and a glutamine at position 84.

113. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 118 and comprising (i) at least one amino acid selected from the group consisting of phenylalanine at position 129, proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, valine at position 598, and histidine at position 642 and (ii) glutamic acid at position 531.

114. The engineered AAV capsid polypeptide of claim 112 or 113, having at least 98% sequence identity to SEQ ID NO: 118.

115. The engineered AAV capsid polypeptide of claim 112 or 113, having at least 99% sequence identity to SEQ ID NO: 118.

116. The engineered AAV capsid polypeptide of claim 112 or 113, having at least 99.5% sequence identity to SEQ ID NO: 118.

117. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 118 and comprising an alanine at position 24.

118. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 117 and comprising (i) at least one amino acid selected from the group consisting of phenylalanine at position 129, proline at position 166, alanine at position 167, aspartic acid at position 418, leucine at position 584, valine at position 598, and histidine at position 642 and (ii) glutamic acid at position 531.

119. The engineered AAV capsid polypeptide of claim 117 or 118, having at least 98% sequence identity to SEQ ID NO: 117.

120. The engineered AAV capsid polypeptide of claim 117 or 118, having at least 99% sequence identity to SEQ ID NO: 117.

121. The engineered AAV capsid polypeptide of claim 117 or 118, having at least 99.5% sequence identity to SEQ ID NO: 117.

122. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 112 and comprising a segment from an AAV8 or AAV9 capsid protein selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) at residues 24-42, LLEPLGLVEEA (SEQ ID NO: 212) at residues 125-135, AGIGKSGAQPAK (SEQ ID NO: 224) at residues 157-168, and APSGVGSLTMAS (SEQ ID NO: 220) at residues 194-205.

123. The engineered AAV capsid polypeptide of claim 122, comprising a segment from an AAV6 capsid protein of HVMGAL (SEQ ID NO: 221) at residues 597-602.

124. The engineered AAV capsid polypeptide of claim 122 or 123, comprising at least one amino acid selected from the group consisting of a phenylalanine at position 56, a lysine at position 84, an aspartic acid at position 418, a leucine at position 584, and an asparagine at position 642.

125. The engineered AAV capsid polypeptide of any one of claims 122-124, having at least 98% sequence identity to SEQ ID NO: 112.

126. The engineered AAV capsid polypeptide of any one of claims 122-124, having at least 99% sequence identity to SEQ ID NO: 112.

127. The engineered AAV capsid polypeptide of any one of claims 122-124, having at least 99.5% sequence identity to SEQ ID NO: 112.

128. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 111 and comprising a segment from an AAV8 or AAV9 capsid protein selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) at residues 24-42, LLEPLGLVEEA (SEQ ID NO: 212) at residues 125-135, AGIGKSGAQPAK (SEQ ID NO: 224) at residues 157-168, and APSGVGSLTMAS (SEQ ID NO: 220) at residues 194-205.

129. The engineered AAV capsid polypeptide of claim 128, comprising a segment from an AAV6 capsid protein of HVMGAL (SEQ ID NO: 221) at residues 597-602.

130. The engineered AAV capsid polypeptide of claim 128 or 129, comprising at least one amino acid selected from the group consisting of a phenylalanine at position 56, a lysine at position 84, an aspartic acid at position 418, a leucine at position 584, and a histidine at position 642.

131. The engineered AAV capsid polypeptide of any one of claims 128-130, having at least 98% sequence identity to SEQ ID NO: 111.

132. The engineered AAV capsid polypeptide of any one of claims 128-130, having at least 99% sequence identity to SEQ ID NO: 111.

133. The engineered AAV capsid polypeptide of any one of claims 128-130, having at least 99.5% sequence identity to SEQ ID NO: 111.

134. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a sequence having at least 97% sequence identity to SEQ ID NO: 115 and comprising a segment from an AAV8 or AAV9 capsid protein selected from the group consisting of ALKPGAPQPKANQQHQDNA (SEQ ID NO: 208) at residues 24-42, LLEPLGLVEEA (SEQ ID NO: 212) at residues 125-135, AGIGKSGAQPAK (SEQ ID NO: 224) at residues 157-168, and APSGVGSLTMAS (SEQ ID NO: 220) at residues 194-205.

135. The engineered AAV capsid polypeptide of claim 134, comprising a segment from an AAV6 capsid protein of HVMGAL (SEQ ID NO: 221) at residues 597-602.

136. The engineered AAV capsid polypeptide of claim 134 or 135, comprising at least one amino acid selected from the group consisting of a phenylalanine at position 56, a lysine at position 84, a glutamic acid at position 418, a leucine at position 584, and an asparagine at position 642.

137. The engineered AAV capsid polypeptide of any one of claims 134-136, having at least 98% sequence identity to SEQ ID NO: 115.

138. The engineered AAV capsid polypeptide of any one of claims 134-136, having at least 99% sequence identity to SEQ ID NO: 115.

139. The engineered AAV capsid polypeptide of any one of claims 134-136, having at least 99.5% sequence identity to SEQ ID NO: 115.

140. An engineered adeno-associated virus (AAV) capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 204.

141. An engineered adeno-associated virus (AAV) capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 205.

142. An engineered adeno-associated virus (AAV) capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 206.

143. The engineered AAV capsid polypeptide of any one of the preceding claims for use in preparation of an engineered AAV vector.

144. A polynucleotide encoding the engineered AAV capsid polypeptide of any one of claims 1-142.

145. The polynucleotide of claim 144, wherein the polynucleotide comprising a nucleic acid sequence selected from the group consisting of: (a) the nucleic acid sequence encoding an engineered AAV capsid polypeptide of any one of claims 1-52; or (b) a nucleic acid sequence that is at least 85%, 90%, or 95% identical to a nucleic acid sequence of (a) and that retains at least one property of the engineered AAV capsid polypeptide having the nucleic acid sequence of (a), the at least one property selected from the group consisting of (i) increased muscle tropism, (ii) increased muscle transduction, (iii) increased muscle transgene DNA levels, and (iv) increased muscle transgene mRNA levels, compared to a wt capsid polypeptide from any one of AAV1, AAV6, AAV8 and AAV9.

146. The polynucleotide of claim 144 or 145, wherein the polynucleotide comprises one or more modified nucleotides.

147. An engineered capsid helper plasmid comprising a polynucleotide of any one of claims 144-146.

148. A composition comprising the engineered capsid helper plasmid of claim 147.

149. An in vitro cell that has been transfected with the engineered capsid helper plasmid of claim 147.

150. An ex vivo cell or tissue that has been transfected with the engineered capsid helper plasmid of claim 147.

151. An engineered AAV vector comprising an engineered AAV capsid polypeptide of any one of claims 1-142, wherein the engineered AAV capsid polypeptide encapsidates a nucleic acid comprising a transgene encoding a gene product of interest.

152. An engineered AAV vector comprising an engineered AAV capsid polypeptide of any one of claims 1-142, wherein the engineered AAV capsid polypeptide encapsidates a nucleic acid comprising an expression cassette comprising a transgene for the expression of a gene product of interest.

153. The engineered AAV vector of claim 151 or 152, wherein the gene product of interest is selected from the group consisting of a coding RNA, non-coding RNA, and a protein.

154. The engineered AAV vector of claim 153, wherein the non-coding RNA is selected from the group consisting of an siRNA, an shRNA, a guide RNA, a long non-coding RNA, an snoRNA, a pi wi- interacting RNA (piRNA), an antisense RNA, or a microRNA.

155. The engineered AAV vector of claim 153, wherein the gene product of interest is a therapeutic protein.

156. The engineered AAV vector of claim 153, wherein the gene product of interest is a peptide vaccine.

157. The engineered AAV vector of claim 152, wherein the nucleic acid or expression cassette comprises at least one regulatory nucleic acid sequence that regulates the expression of the transgene.

158. The engineered AAV vector of claim 157, wherein the at least one regulatory nucleic acid sequence is selected from the group consisting of a promoter, an enhancer, an intron, a post- transcriptional regulatory element, an inverted terminal repeat (ITR), a polyadenylation (poly A) sequence, or any combination thereof.

159. The engineered AAV vector of any of claims 151-158, wherein the transgene encodes a dystrophin, a mini-dystrophin, a micro-dystrophin, a laminin-a2, a mini-agrin, an a-sarcoglycan, a P-sarcoglycan, a y-sarcoglycan, a 5-sarcoglycan, a utrophin, a Fukutin-related protein, a myostatin pro-peptide, a soluble myostatin receptor, a dominant negative myostatin, a follistatin, an insulin-like growth factor 1 (IGF-1), a sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a), a P-adrenergic receptor kinase C terminus (PARKct), a phospholamban, a phosphatidylinositol 3-kinase (PI3K), a proto-oncogene serine/threonine-protein kinase (Pim-1), a peroxisome proliferator-activated receptor-gamma coactivator (PGC-la), a superoxide dismutase (SOD), an SOD-1, an SOD-2, an extracellular SOD (EC-SOD), a kallikrein, a hypoxia inducible factor (HIF), thymosin P4 (TMSB4X), a mir-1 microRNA, a mir-133 microRNA (e.g., a miR-133a-l, a miR-133a-2, and a miR-133b), a mir-206 microRNA, a mir-208 microRNA, an inhibitor 1 of protein phosphatase 1 , an anti-apoptotic factor, an angiogenic factor, an insulin, a Factor IX, a Factor VIII, a glucocerebrosidase, an a-galactosidase A (GLA), an acid a glucosidase (GAA), a lipoprotein lipase, an apolipoprotein-E (ApoE), an al -antitrypsin (al AT), an apelin, a glucagon-like peptide 1 , an iduronidase, an iduronate-2-sulfatase, a myotubularin (MTM1), a calpain-3 (CAPN3), a dysferlin (DYSF), a lysosome-associated membrane protein 2 (LAMP2), an N-acetylgalactosamine 4-sulfatase; a cardiac troponin T, a cardiac troponin I, a troponin I type 3a, a troponin C (TNNC1), a cardiac sarcomeric protein, a P-myosin heavy chain, a myosin ventricular essential light chain 1, a myosin light chain 2, a myosin ventricular regulatory light chain 2, a myosin light chain 3 (MYL3), an a-tropomyosin, a cardiac myosin binding protein C, a four-and-a-half LIM protein 1 (FHL1), a titin, an AMP-activated protein kinase (AMPK) subunit gamma-2, a cardiac muscle actin alpha 1 (ACTC1), a muscle LIM protein (MLP), a cardiac LIM protein (CLP), a caveolin 3 (CAV3), a mitochondrial transfer RNA glycine (MTTG), a mitochondrial transfer RNA isoleucine (MTTI), a mitochondrial transfer RNA lysine (MTTK), a mitochondrial transfer RNA glutamine (MTTQ), a transthyretin (TTR), a stromal-derived factor- 1 (SDF-1), an adenylate cyclase-6 (AC6), a fibroblast growth factor (FGF), a platelet-derived growth factor (PDGF), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor, a hypoxia inducible growth factor, a nitric oxide synthase-3 (NOS3), S100A1 protein, collagen type 6 (C0L6A), BINI, heat shock protein family B (HSPB1), lamin A/C (LMNA), actin (ACTA1), dysferlin (DYSF), troponin 12 (TNN12), troponin 3 (TNN3), troponin T2 (TNNT2), myosin heavy chain 3 (MYH3), myosin heavy chain 8 (MYH8), fibronectin 1 (FN1), transforming growth factor beta 3 (TGFB3), emerin (EMD), filamin A (FLNA), enolase 3 (EN03), actinin alpha 2 (ACTN2), myosin light chain kinase (MYLK), sarcoglycan alpha (SGCA), dystroglycan 1 (DAG), syntrophin alpha 1 (SNTA1), crystallin alpha B (CRY AB), filamin C (FLNC), kelch like family member 40 (KLHL40), poly(A) binding protein nuclear 1 (PABPN1), and ataxin 3 (ATXN3).

160. The engineered AAV vector of any one of claims 151-159, wherein the engineered AAV vector has reduced reactivity to neutralizing antibodies.

161. The engineered AAV vector of any one of claims 151-160, wherein the engineered AAV vector exhibits greater tropism for a muscle tissue compared to a wt AAV vector selected from the group consisting of AAV1, AAV6, AAV8, and AAV9.

162. The engineered AAV vector of claim 161, wherein the muscle tissue is selected from the group consisting of heart, gastrocnemius, biceps, quadriceps, triceps, soleus, diaphragm, and tongue.

163. The engineered AAV vector of any one of claims 151-162, wherein the engineered AAV vector is a recombinant AAV vector.

164. An ex vivo or in vitro cell that has been transduced with the engineered AAV vector of any one of claims 151-163.

165. A composition comprising the engineered AAV vector of any one of claims 151-163, or the cell of claim 164.

166. A pharmaceutical composition comprising the engineered AAV vector of any one of claims 151-163, or the cell of claim 164, and a pharmaceutically acceptable carrier.

167. A method for transducing a muscle cell with a transgene of interest, the method comprising the step of contacting the muscle cell with the engineered AAV vector of any one of claims 151-163, wherein the engineered AAV vector transduces the muscle cell.

168. The method of claim 167, wherein the muscle cell is a human muscle cell.

169. The method of claim 167, wherein the muscle cell is selected from the group consisting of diaphragm, gastrocnemius, triceps, heart, biceps, quadriceps, and tongue.

170. The method of claim 167, wherein the muscle cell is a cardiac myocyte, a skeletal myocyte, or a smooth myocyte.

171. The method of claim 170, wherein the muscle cell is a cardiac myocyte.

172. The method of any one of claims 167-171, wherein the transgene of interest encodes one or more reprogramming factors selected from the group consisting of ASCL1, MYOCD, MEF2C, TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, 0CT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, and ZFPM2.

173. The method of claim 172, wherein the method reprograms the cardiac cell into a cardiac cardiomyocyte.

174. A method of treating a cardiac pathology in a subject in need thereof comprising administering to the subject a composition comprising the engineered AAV vector of any one of claims 151-163, wherein the engineered AAV vector transduces cardiac muscle.

175. The method of any one of claims 167-174, wherein the engineered AAV vector is administered by epicardial injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracardiac injection, intracardiac catheterization, direct intramyocardial injection, transvascular administration, antegrade intracoronary injection, retrograde injection, transendomyocardial injection, or molecular cardiac surgery with recirculating delivery (MCARD).

176. The method of claim 175, wherein the engineered AAV vector is administered by epicardial injection.

177. A method of introducing a heterologous nucleic acid to a cell comprising the step of transducing the cell with an engineered AAV vector comprising an engineered AAV capsid polypeptide of any one of claims 1-142.

178. A method of delivering a gene of interest to a cell in a subject, the method comprising the step of administering to the subject an engineered AAV vector of any one of claims 151-163.