Liver de-targeted muscle tropic capsids

Abstract

The present invention provides novel capsid variants comprising a first amino acid sequence comprising the amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) and a second amino acid sequence comprising the amino acid sequence RGD. These novel capsid variants de-target liver tissue and target muscle and heart tissue.

Description

[0001] Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0002] LIVER DE-TARGETED MUSCLE TROPIC CAPSIDS

[0003] CLAIM OF PRIORITY

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 688.379, filed August 29. 2024; which is incorporated by reference herein in its entirety.

[0005] FIELD OF DISCLOSURE

[0006] This disclosure relates to viral capsids.

[0007] BACKGROUND

[0008] Recombinant AAVs (rAAVs) are the most commonly used delivery vehicles for gene therapy and gene editing. Nonetheless. rAAVs that contain natural capsid variants have limited cell tropism. Indeed, rAAVs used today mainly infect the liver after systemic delivery. Further, the transduction efficiency of conventional rAAVs in other cell-types, tissues, and organs by these conventional rAAVs with natural capsid variants is limited. Therefore, AAV -mediated polynucleotide delivery for diseases that affect cells, tissues, and organs other than the liver, such as the central nervous system) typically requires an injection of a large dose of virus (typically about 2 x 1014vg / kg), which often results in liver toxicity. Furthermore, because large doses are required when using conventional rAAVs, manufacturing sufficient amounts of a therapeutic rAAV needed to dose adult patients is extremely challenging. Additionally, due to differences in gene expression and physiology, mouse and primate models respond differently to viral capsids. As such there exists a need for improved rAAVs for use in the treatment of various genetic diseases.

[0009] SUMMARY

[0010] The present invention provides novel capsid protein variants for viral vectors that de-target liver tissue and target muscle and heart tissue. The present invention provides adeno-associated virus (AAV) vectors comprising an engineered capsid protein comprising two separate amino acid sequences, a first amino acid sequence comprising the ammo acids RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) and a second amino acid sequence comprising the amino acids RGD.

[0011] In some embodiments, RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) may be inserted after amino acid 455 in reference to an AAV9 capsid or equivalent position in another AAV capsid. AAV vectors may comprise the amino acid sequence Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0012] X 1NX2X3X4RGDR / YX5X6L (SEQ ID NO: 4288) (X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or X1NX2X3X4RGDYX5X6L) (SEQ ID NO: 4290), wherein Xi,X2,X3, X4, X5, and Xe may be any amino acid.

[0013] Accordingly, aspects of the invention provide an AAV vector comprising a capsid protein comprising a first amino acid sequence comprising the amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) and a second amino acid sequence comprising the amino acid sequence RGD.

[0014] The amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) may be inserted after amino acid 455 in reference to an AAV9 capsid and the amino acid sequence comprising RGD may be inserted after amino acid 588 in reference to an AAV9 capsid.

[0015] For example, with regard to the first amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287), the capsid protein may comprise the amino acid sequence X1NX2X3X4RGDR / YX5X6L (SEQ ID NO: 4288) (X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or X1NX2X3X4RGDYX5X6L) (SEQ ID NO: 4290), which comprises the RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) sequence. Xi, X2, X3, X4, X5, and Xe, may be selected from among any amino acid.

[0016] Xi may be located at amino acid 451, X2 may be located at amino acid 453, X3 may be located at amino acid 454, X4 may be located amino acid 455, and RGDR / YXsXeL (SEQ ID NO: 4291) (RGDRXsXeL (SEQ ID NO: 4292) or RGDYXsXeL (SEQ ID NO: 4293)) may be inserted after amino acid 455 in reference to an AAV9 capsid or the equivalent position in another AAV capsid.

[0017] Xi may be an amino acid selected from the group consisting of: A, L, M, S, T, V.

[0018] X2 may be an amino acid selected from the group consisting of A. G, S. T, Y.

[0019] X3 may be an amino acid selected from the group consisting of A, D, G, N, S, T In aspects of the invention, X3 may be S.

[0020] X4 may be an amino acid selected from the group consisting of A, G, H, I, M, S, T, V.

[0021] X5 may be an amino acid selected from the group consisting of G. Q, S. In aspects of the invention, X5 may be an amino acid selected from the group consisting of G or Q.

[0022] Xe may be an amino acid selected from the group consisting of A, G, L, S, T, Y. In aspects of the invention, Xe may be an amino acid selected from the group consisting of A and Y. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0023] With regard to the second amino acid sequence RGD, the capsid protein may comprise the amino acid sequence X7QX8X9X10RGDX11X12X13L (SEQ ID NO: 4294). which comprises the RGD sequence. X7, Xs, X9, X10, X11, X12, and X13 may be selected from among any amino acid.

[0024] X7 may be located at amino acid 584, Xs may be located at amino acid 586, X9 may be located at amino acid 587, X10 may be located at amino acid 588, and RGDX11X12X13L (SEQ ID NO: 4295) may be inserted after amino acid 588 in reference to an AAV9 capsid or the equivalent position in another AAV capsid.

[0025] X7 may be an amino acid selected from the group consisting of: I, L, N, R, S. In aspects of the invention, X7 may be the amino acid I.

[0026] Xs may be an amino acid selected from the group consisting of: A, E. G, N. S, T. In aspects of the invention. Xs may be the amino acid G.

[0027] X9 may be an amino acid selected from the group consisting of: A, D, G, N, S, T.

[0028] X10 may be an amino acid selected from the group consisting of: A, G, N, S, T. In aspects of the invention, X10 is the amino acid T.

[0029] X11 may be an ammo acid selected from the group consisting of: H, Q, R, Y. In aspects of the invention, Xu is the amino acid H.

[0030] X12 may be an amino acid selected from the group consisting of: G, N, R, S. In aspects of the invention, X12 is the amino acid G or S.

[0031] X13 may be an amino acid selected from the group consisting of: A. G, S. T. In aspects of the invention, X13 is the amino acid T.

[0032] In aspects of the invention, the capsid protein may comprise an amino acid sequence selected from Table la, which comprises first amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287). The amino acid sequence selected from Table la may be in hypervariable region IV (HVR IV) relative to wild-type AAV9. The capsid protein may comprise substitutions at amino acids 451-455 relative to a wildtype AAV9 vector capsid. The substitutions at amino acids 451-455 relative to a wildtype AAV9 vector capsid may be substituted with an amino acid sequence selected from column 1 of Table lb. The capsid protein may further comprise a 7-mer insert selected from column 2 of Table lb. The 7-mer insert may inserted after amino acid 455 relative to a wild-ty pe AAV9 vector.

[0033] The capsid protein may further comprise an amino acid sequence selected from Table 2a. which comprises the second amino acid sequence comprising RGD. The amino acid sequence selected from Table 2a is in hypervariable region VIII (HVR VIII) relative Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) to wild-ty pe AAV9. The capsid protein may comprise substitutions at amino acids 584- 588 relative to a wild-type AAV9 vector capsid. The substitutions at amino acids 584-588 relative to a wild-type AAV9 vector capsid may be substituted with an amino acid sequence selected from column 1 of Table 2b. The capsid protein may further comprise a 7-mer insert selected from column 2 of Table 2b. The 7-mer insert may be inserted after amino acid 588 relative to a wild-type AAV9 vector.

[0034] For example, aspects of the invent on provide a capsid protein comprising a first amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) in the hypen ariable region IV (HVR IV) relative to wild-type AAV9 and further comprises substitutions at amino acids 451-455 relative to a wild-ty pe AAV9 vector capsid. The capsid protein comprises a 7-mer insert comprising the RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) motif inserted after amino acid 455 relative to a wild- type AAV9 vector.

[0035] The capsid protein further comprises a second amino acid sequence comprising RGD in the hypervariable region VIII (HVR VIII) relative to wild-type AAV9 and further comprises substitutions at amino acids 584-588 relative to a wild-type AAV9 vector capsid. The capsid protein comprises a 7-mer insert comprising the RGD motif inserted after amino acid 588 relative to a wild-type AAV9 vector.

[0036] In preferred aspects of the invention, the first amino acid sequence and second amino acid sequence are paired as shown in Table 3.

[0037] Advantageously, viral vectors comprising an amino acid sequence of the invention exhibit increased muscle and heart tropism as compared to a wild-type AAV vector.

[0038] In aspects of the invention, the capsid protein further comprises a deletion of G267 in reference to an AAV9 capsid or equivalent position in another AAV capsid. Without being limited to a mechanism of action, the deletion of glycine at position 267 promotes reduced liver tropism. Aspects of the invention provide a viral vector comprising a deletion of glycine at position 267 (G267) without causing regional destabilization within the VR1 loop of the AAV vector.

[0039] Aspects of the present disclosure provide an engineered adeno-associated virus (AAV) capsid protein comprising a first amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287); and a second amino acid sequence comprising RGD.

[0040] In some embodiments, the first amino acid sequence is in a hypervariable region IV (HVR IV) relative to a wild type AAV9 capsid protein. In some embodiments, the first Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) is inserted after amino acid 455 in reference to a wild ty pe AAV9 capsid protein.

[0041] In some embodiments, the second amino acid sequence is in a hypervariable region VIII (HVR VIII) relative to a wild type AAV9 capsid protein. In some embodiments, the second amino acid sequence comprising RGD is inserted after amino acid 588 in reference to a wild type AAV9 capsid.

[0042] In some embodiments, the first amino acid sequence comprises the amino acid sequence X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or the amino acid sequence X1NX2X3X4RGDYX5X6L (SEQ ID NO: 4290), and wherein each of Xi-X6is any amino acid. In some embodiments, relative to a wild type AAV9 capsid protein, Xi is located at amino acid 451, X2 is located at amino acid 453, X3 is located at amino acid 454. X4 is located amino acid 455, and RGDRX5X6L (SEQ ID NO: 4292) or RGDYX5X6L (SEQ ID NO: 4293) is inserted after amino acid 455. In some embodiments, Xi is A, L, M, S, T, or V; X2 is A, G, S, T, or Y; X3 is A, D, G, N, S, or T; X4is A. G, H, I, M, S, T, or V; X5is G, Q. or S; and Xe is A, G. L, S, T, or Y.

[0043] In some embodiments, the second amino acid sequence comprises the amino acid sequence X7QX8X9X10RGDX11X12X13L (SEQ ID NO: 4294), and wherein each of X7-X13 is any amino acid. In some embodiments, relative to a wild ty pe AAV9 capsid protein, X7 is located at amino acid 584, Xs is located at amino acid 586, X9 is located at amino acid 587, X10 is located at amino acid 588, and RGDX11X12X13L (SEQ ID NO: 4295) is inserted after amino acid 588. In some embodiments, X7 is I, L, N, R, or S; Xs is A, E, G, N, S, or T; X9 is A, D, G, N, S, or T; X10 is A, G, N, S, or T; X11 is H, Q, R, or Y; X12 is G, N, R, or S; and X13 is A, G, S, or T.

[0044] In some embodiments, the first amino acid sequence comprises any one of SEQ ID NOs: 2-715.

[0045] In some embodiments, the second amino acid sequence comprises any one of SEQ ID NOs: 716-1429.

[0046] In some embodiments, the first amino acid sequence and the second amino acid sequence each correspond to the HVR IV sequence and the HVR VIII sequence, respectively, set forth in Table 3 for a single capsid protein variant.

[0047] In some embodiments, the engineered AAV capsid protein further comprises a deletion of amino acid G267 relative to a wild ty pe AAV9 vector.

[0048] In some embodiments, the engineered AAV capsid protein exhibits increased muscle and heart tropism compared to a wild type AAV capsid protein. In some Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) embodiments, the engineered capsid protein exhibits reduced liver tropism as compared to a wild type AAV capsid protein.

[0049] Aspects of the present disclosure provide an AAV particle comprising any one or more of the engineered capsid proteins described herein.

[0050] Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0051] Table la: HVRIV 12-mer motifs Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0052] Table lb: Split HVR IV motifs from Table la Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0053] Table 2a: HVR VIII 12-mer motifs Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0054] Table 2b: Split HVR VIII motifs from

[0055] Table 2a Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0056] Table 3: Paired HVR IV and HVR VIII 12-mer motifs Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) Atorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0057] Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0058] BRIEF DESCRIPTION OF THE DRAWINGS

[0059] FIG. 1 shows a graph of AAV capsid transduction of human primary myotubes.

[0060] FIG. 2 shows a graph of AAV capsid transduction of non-human primate (NHP) skeletal muscles.

[0061] FIG. 3 shows a graph of AAV capsid transduction of NHP cardiac muscle.

[0062] FIG. 4 shows a graph of AAV capsid transduction of NHP liver.

[0063] DETAILED DESCRIPTION

[0064] The present invention provides novel capsid variants comprising a first amino acid sequence comprising the amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) and a second amino acid sequence comprising the amino acid sequence RGD. Such novel capsid variants de-target liver tissue and target muscle and heart tissue.

[0065] Aspects of the invention provide a capsid protein comprising (i) a first amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) in the hypervariable region IV (HVR IV) relative to a wild-type AAV9 capsid protein, and (ii) one or more substitutions at amino acids 451-455 relative to the wild-type AAV9 capsid protein. In such instances, the capsid protein can comprise a 7-mer insert comprising the RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) motif inserted after amino acid 455 relative to a wild-type AAV9 capsid protein.

[0066] The capsid protein can further comprise (hi) a second amino acid sequence comprising RGD in the hypervariable region VIII (HVR VIII) relative to a wild-type AAV9 capsid protein, and (iv) one or more substitutions at amino acids 584-588 relative to the wildtype AAV9 capsid protein. In such instances, the capsid protein comprises a 7-mer insert comprising the RGD motif inserted after amino acid 588 relative to the wild-type AAV9 capsid protein.

[0067] I. Adeno Associated Virus Vectors

[0068] AAVs are particularly appropriate viral vectors for delivery of genetic material into mammalian cells. AAVs are not known to cause disease in mammals and cause a very mild immune response. Additionally, AAVs are able to infect cells in multiple stages whether at rest or in a phase of the cell replication cycle. Advantageously, AAV DNA is not regularly inserted into the hosf s genome at random sites, reducing the oncogenic properties of this vector. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0069] AAVs have been engineered to deliver a variety of treatments, especially for genetic disorders caused by single nucleotide polymorphisms (SNPs). Genetic diseases that have been studied in conjunction with AAV vectors include cystic fibrosis, hemophilia, arthritis, macular degeneration, muscular dystrophy, Parkinson’s disease, congestive heart failure, and Alzheimer's disease. AAV can be used as a vector to deliver an engineered nucleic acid to a host and utilize the host's own ribosomes to transcribe the engineered nucleic acid into the desired proteins. See, e.g.. West et al.. Virology 160:38-47 (1987); U.S. Pat. No. 4.797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94: 1351 (1994). AAVs have some deficiency in their replication and / or pathogenicity and thus can be safer than adenoviral vectors. In some embodiments, AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In some embodiments, the capacity of the AAV vector, system thereof, and / or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more engineered capsid polynucleotides described herein.

[0070] AAVs are small, replication-defective, nonenveloped viruses that infect humans and other primate species and have a linear single-stranded DNA genome. Naturally occurring AAV serotypes exhibit liver tropism. As a result, transfection of non-liver tissue with traditional AAV vectors is impeded by the virus’s natural liver tropism. Moreover, because the liver acts to break down substances delivered to a subject, transfection of non-liver tissue with unmodified AAV vectors requires higher dosing to provide sufficient viral load to overcome the liver and reach non-liver tissue. More than 30 naturally occurring serotypes of AAV are available. Many natural variants in the AAV capsid exist. AAV serotypes include, but are not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6. AAV7. AAV8, AAV9, AAV10. AAV11, AAV 12, and AAV13. AAVs may be engineered using conventional molecular biology techniques, making it possible to optimize these particles, for example, for cell specific delivery, for minimizing immunogenicity, for tuning stability and particle lifetime, for efficient degradation, and for accurate delivery' to the nucleus. AAV vectors can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0071] Previous approaches to identify AAV sequences correlated with tropism have relied upon the comparison of highly related extant serotypes w ith distinct characteristics, random domain swaps betw een unrelated serotypes, or consideration of higher-order structure, to identity’ motifs that define liver tropism. For example, mapping determinants of AAV tropism have been carried out by comparing highly related serotypes. One such example is the single- Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) amino acid change (E531K) between AAV1 and AAV6 that improves murine liver transduction in AAV1. See Wu et al. (2006) J. Virol., 80(22): 11393-7, incorporated by reference herein. Another example is a reciprocal domain swap between AAV2 and AAV8 that alters tropism, but fails to define any robust specific tissue-targeting motifs. See Raupp et al. (201) J. Virol., 86(17):9396-408, incorporated by reference herein. Further, global consideration of structure has only highlighted gross differences between better- or worseliver-transducers that are more observational than useful in practice. Nam et al (2007) J. Virol., 81(22): 12260-71.

[0072] AAVs exhibiting modified tissue tropism that may be used with the present invention are described in U.S. Patent No. 9.695,220, U.S. Patent No. 9,719,070; U.S. Patent No. 10,119.125; U.S. Patent No. 10,526.584; U.S. Patent Application Publication No. 2018- 0369414; U.S. Patent Application Publication No. 2020-0123504; U.S. Patent Application Publication No. 2020-0318082; PCT International Patent Application Publication No. WO 2015 / 054653; PCT International Patent Application Publication No. WO 2016 / 179496; PCT International Patent Application Publication No. WO 2017 / 100791; and PCT International Patent Application Publication No. WO 2019 / 217911, the entirety of the contents of each of which are incorporated by reference herein.

[0073] The AAV vector or system thereof may include one or more regulatory' molecules, such as promoters, enhancers, repressors and the like. In some embodiments, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In some embodiments, the one or more regulatory proteins can be selected from Rep78, Rep68, Rep52, Rep40, variants thereof, and combinations thereof. In some embodiments, the muscle specific promoter can drive expression of an engineered AAV capsid polynucleotide.

[0074] The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein. The engineered capsid proteins can be capable of assembling into a protein shell (an engineered capsid) of the AAV virus particle. The engineered capsid can have a cell-, tissue-, and / or organ-specific tropism.

[0075] The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In some embodiments, the serotype can be AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-8, AAV-9, or any combinations thereof. In some embodiments, the AAV can be AAV1, AAV-2, AAV-5, AAV-9, or any combination thereof. One can select the AAV with regard to the cells to be targeted, e.g., one can select AAV Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) seroty pes 1, 2, 5, 9 or a hybrid capsid AAV-1, AAV-2, AAV-5. AAV-9 or any combination thereof for targeting brain and / or neuronal cells: and one can select AAV-4 for targeting cardiac tissue; and one can select AAV-8 for delivery to the liver. Thus, in some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV-1, AAV-2, AAV-5 or any combination thereof. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV-4 serotype. In some embodiments, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV-8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.

[0076] It will be appreciated that while the different serotypes can provide some level of cell, tissue, and / or organ specificity', each serotype still is multi-tropic and thus can result in tissuetoxicity if using that serotype to target a tissue that the serotype is less efficient in transducing. Thus, in addition to achieving some tissue targeting capacity via selecting an AAV of a particular serotype, it will be appreciated that the tropism of the AAV serotype can be modified by an engineered AAV capsid described herein. As described elsewhere herein, variants of wild-type AAV of any serotype can be generated via a method described herein and determined to have a particular cell-specific tropism, which can be the same or different as that of the reference wild-type AAV serotype. In some embodiments, the cell, tissue, and / or specificity' of the wild-type serotype can be enhanced (e.g., made more selective or specific for a particular cell type that the serotype is already biased towards). For example, wild-type AAV-9 is biased towards muscle and brain in humans (see. e.g., Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.) By including an engineered AAV capsid and / or capsid protein variant of wild-type AAV-9 as described herein, the tropism for nervous cells might be reduced or eliminated and / or the muscle specificity increased such that the nervous specificity' appears reduced in comparison, thus enhancing the specificity for muscle as compared to the wild-type AAV-9. As previously mentioned, inclusion of an engineered capsid and / or capsid protein variant of a wild-type AAV serotype can have a different tropism than the wild-type reference AAV serotype. For example, an engineered AAV capsid and / or capsid protein variant of AAV-9 can have specificity for a tissue other than muscle or brain in humans.

[0077] In some embodiments, the AAV vector is a hybrid AAV vector or system thereof.

[0078] Hybrid AAVs are AAVs that include genomes with elements from one serotype that are Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) packaged into a capsid derived from at least one different seroty pe. For example, if it is the rAAV2 / 5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed above, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the 2nd plasmid, the pRepCap will be different. In this plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2 / 5, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed by this AAV2 / 5 hybrid virus should be the same as that of AAV5. It will be appreciated that wild-type hybrid AAV particles suffer the same specificity issues as with the non-hybrid wild-type serotypes previously discussed.

[0079] Advantages achieved by the wild-type based hybrid AAV systems can be combined with the increased and customizable cell-specificity that can be achieved with the engineered AAV capsids can be combined by generating a hybrid AAV that can include an engineered AAV capsid described elsewhere herein. It will be appreciated that hybrid AAVs can contain an engineered AAV capsid containing a genome with elements from a different serotype than the reference wild-ty pe serot pe that the engineered AAV capsid is a variant of. For example, a hybrid AAV can be produced that includes an engineered AAV capsid that is a variant of an AAV-9 serotype that is used to package a genome that contains components (e.g, rep elements) from an AAV-2 serotype. As with wild-type based hybrid AAVs previously discussed, the tropism of the resulting AAV particle will be that of the engineered AAV capsid.

[0080] In some embodiments, the AAV vector or system thereof is configured as a ’'gutless" vector, similar to that described in connection with a retroviral vector. In some embodiments, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g., the engineered AAV capsid polynucleotide(s)).

[0081] The vectors described herein can be constructed using any suitable process or technique. In some embodiments, one or more suitable recombination and / or cloning methods or techniques can be used to the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but not limited to, those described in U.S. Application publication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0082] Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al.. Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81 :6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any ofthe techniques and / or methods can be used and / or adapted for constructing an AAV or other vector described herein. AAV vectors are discussed elsewhere herein.

[0083] In some embodiments, the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors.

[0084] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a engineered AAV capsid system described herein are as used in the foregoing documents, such as International Patent Application Publications WO 2021 / 050974, WO 2021 / 077000, and WO 2022 / 020616, the contents of which are incorporated by reference herein.

[0085] Additional AAV vectors are described in International Patent Application Publication WO 2019 / 2071632, the contents of which are incorporated by reference herein.

[0086] Further AAV vectors are described in International Patent Application Publications WO 2020 / 086881 and WO 2020 / 235543, the contents of each of which are incorporated by reference herein.

[0087] Further AAV vectors are described in International Patent Application Publications WO 2005 / 033321; WO 2006 / 110689; WO 2007 / 127264; WO 2008 / 027084; WO 2009 / 073103; WO 2009 / 073104; WO 2009 / 105084; WO 2009 / 134681; WO 2009 / 136977; WO 2010 / 051367; WO 2010 / 138675; WO 2001 / 038187; WO 2012 / 112832; WO 2015 / 054653; WO 2016 / 179496; WO 2017 / 100791; WO 2017 / 019994; WO 2018 / 209154; WO 2019 / 067982; WO 2019 / 195701; WO 2019 / 217911; WO 2020 / 041498; WO 2020 / 210839; U.S. Patent No. 7.906,111; U.S. Patent No. 9,737,618; U.S. Patent No. 10,265.417; U.S. Patent No. 10,485.883; U.S. Patent No. 10,695.441; U.S. Patent No. 10,722,598; U.S. Patent No. 8,999,678; U.S. Patent No. 10,301,648; U.S. Patent No. 10,626,415; U.S. Patent No. 9,198,984; U.S. Patent No. 10,155,931; U.S. Patent No. 8,524,219; U.S. PatentNo. 9,206,238; U.S. Patent No. 8,685,387; U.S. Patent No. 9,359,618; U.S. Patent No. 8,231,880; U.S. Patent No. 8,470.310; U.S. Patent No. 9.597,363; U.S. Patent No. 8,940,290; U.S. Patent No. 9,593,346; U.S. Patent No. 10,501,757; U.S. Patent Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0088] No. 10,786,568; U.S. Patent No. 10,973,928; U.S. Patent No. 10,519,198; U.S. Patent No. 8,846,031; U.S. Patent No. 9,617,561; U.S. Patent No. 9,884.071; U.S. Patent No. 10,406, 173; U.S. Patent No. 9,596,220; U.S. Patent No. 9,719,010; U.S. Patent No. 10,117,125; U.S. Patent No. 10,526,584; U.S. Patent No. 10,881,548; U.S. Patent No. 10,738,087; U.S. Patent Publication No. 2011-023353; U.S. Patent Publication No. 2019- 0015527; U.S. Patent Publication No. 2020-155704; U.S. Patent Publication No 2017- 0191079; U.S. Patent Publication No. 2019-0218574; U.S. Patent Publication No. 2020- 0208176; U.S. Patent Publication No. 2020-0325491; U.S. Patent Publication No. 2019- 0055523; U.S. Patent Publication No. 2020-0385689; U.S. Patent Publication No. 2009- 0317417; U.S. Patent Publication No. 2016-0051603; U.S. Patent Publication No. 2016- 00244783; U.S. Patent Publication No. 2017-0183636; U.S. Patent Publication No. 2020- 0263201; U.S. Patent Publication No. 2020-0101099; U.S. Patent Publication No. 2020- 0318082; U.S. Patent Publication No. 2018-0369414; U.S. Patent Publication No. 2019- 0330278; U.S. Patent Publication No. 2020-0231986, the contents of each of which are incorporated by reference herein.

[0089] II. Capsid Proteins

[0090] The capsid protein is the shell or coating of the virus that enables its delivery7into the host. Without the protein, the nucleic acids would be destroyed by the host without entering into the host cells and beginning transcription and translation. The capsid protein may be in the natural conformation of a naturally occurring AAV, or it may be modified.

[0091] In some embodiments, the AAV capsid protein is an engineered AAV capsid protein having reduced or eliminated uptake in a non-muscle cell as compared to a corresponding wild-type AAV capsid polypeptide.

[0092] In some embodiments, the engineered AAV capsid encoding polynucleotide can be included in a polynucleotide that is configured to be an AAV genome donor in an AAV vector system that can be used to generate engineered AAV particles described elsewhere herein. In some embodiments, the engineered AAV capsid encoding polynucleotide can be operably coupled to a poly adenylation tail. In some embodiments, the poly adenylation tail can be an SV40 poly adenylation tail. In some embodiments, the AAV capsid encoding polynucleotide can be operably coupled to a promoter. In some embodiments, the promoter can be a tissue specific promoter. In some embodiments, the tissue specific promoter is specific for muscle (e.g. cardiac, skeletal, and / or smooth muscle), neurons and supporting cells (e.g, astrocytes, glial cells, Schwann cells, etc.), fat, spleen, liver, kidney, immune cells, Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) spinal fluid cells, synovial fluid cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal gland, blood cell, bone marrow cells, placenta, endothelial cells, and combinations thereof. In some embodiments, the promoter can be a constitutive promoter. Suitable tissue specific promoters and constitutive promoters are discussed elsewhere herein and are generally known in the art and can be commercially available. Suitable muscle specific promoters include, but are not limited to CK8, MHCK7, Myoglobin promoter (Mb). Desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoter.

[0093] Described herein are various embodiments of engineered viral capsids, such as adeno- associated virus (AAV) capsids, that can be engineered to confer cell-specific tropism, such as muscle specific tropism, to an engineered viral particle. Engineered viral capsids can be lentiviral, retroviral, adenoviral, or AAV capsids. The engineered capsids can be included in an engineered virus particle (e.g., an engineered lentiviral, retroviral, adenoviral, or AAV virus particle), and can confer cell-specific tropism, reduced immunogenicity , or both to the engineered viral particle. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein that can contain a muscle-specific targeting moiety containing or composed of an n-mer motif described elsewhere herein.

[0094] The engineered viral capsid and / or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides. Tn some embodiments, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide. In some embodiments, an engineered viral capsid polynucleotide (e.g.. an engineered AAV capsid polynucleotide, engineered lentiviral capsid polynucleotide, engineered retroviral capsid polynucleotide, or engineered adenovirus capsid polynucleotide) can include a 3’ polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal.

[0095] The engineered viral capsids can be variants of wild-type viral capsid. For example, in some embodiments, the engineered AAV capsids can be variants of wild-type AAV capsids. In some embodiments, the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins or a combination thereof. In other words, the engineered AAV capsids can include one or more variants of a wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. In some embodiments, the serotype of the reference wild-type AAV capsid Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) can be AAV-1. AAV-2, AAV-3. AAV-4, AAV-5. AAV-6, AAV-8, AAV-9 or any combination thereof. In some embodiments, the serotype of the wild-type AAV capsid can be AAV-9. The engineered AAV capsids can have a different tropism than that of the reference wild-type AAV capsid.

[0096] The engineered viral capsid can contain 1-60 engineered capsid proteins. In some embodiments, the engineered viral capsids can contain 1, 2. 3, 4, 5, 6, 7, 8, 9. 10. 11, 12, 13, 14. 15. 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26. 27. 28. 29. 30. 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, the engineered viral capsid can contain 0- 59 wild-type viral capsid proteins. In some embodiments, the engineered viral capsid can contain 0, 1, 2. 3, 4, 5, 6. 7, 8, 9. 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23. 24. 25. 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type viral capsid proteins.

[0097] In some embodiments, the engineered AAV capsid can contain 1 -60 engineered capsid proteins. In some embodiments, the engineered AAV capsids can contain 1. 2, 3, 4, 5,

[0098] 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In some embodiments, the engineered AAV capsid can contain 0-59 wild-type AAV capsid proteins. In some embodiments, the engineered AAV capsid can contain 0, 1, 2, 3. 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wild-type AAV capsid proteins.

[0099] In some embodiments, the engineered viral capsid protein can have an n-mer amino acid motif, where n can be at least 3 amino acids. In some embodiments, n can be 3, 4, 5, 6,

[0100] 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, an engineered AAV capsid can have a 6-mer or 7-mer amino acid motif. In some embodiments, the n-mer amino acid motif can be inserted between two amino acids in the wild-type viral protein (VP) (or capsid protein). In some embodiments, the n-mer motif can be inserted between two amino acids in a variable amino acid region in a viral capsid protein.

[0101] In some embodiments, the n-mer motif can be inserted between two amino acids in a variable amino acid region in an AAV capsid protein. The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parvovirus capsids (see, e.g., DiMattia et al. 2012. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0102] J. Virol. 86(12):6947-6958). Structural variable regions (VRs) occur in the surface loops that connect the beta-strands, which cluster to produce local variations in the capsid surface. AAVs have 12 variable regions (also referred to as hypervariable regions) (see, e.g., Weitzman and Linden. 2011. “Adeno- Associated Virus Biology.” In Snyder, R.O., Moullier, P. (eds.) Totowa, NJ: Humana Press). In some embodiments, one or more / 7-mer motifs can be inserted between two amino acids in one or more of the 12 variable regions in the wildtype AVV capsid proteins. In some embodiments, the one or more n-mer motifs can be each be inserted between two ammo acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR-XI, VR-XII, or a combination thereof. In some embodiments, the / 7-mer can be inserted between two amino acids in the VR-III of a capsid protein.

[0103] In some embodiments, the engineered capsid can have an / 7-mer inserted between any two contiguous amino acids between amino acids 262 and 269, between any two contiguous amino acids between amino acids 327 and 332, between any two contiguous amino acids between amino acids 382 and 386, between any two contiguous amino acids between amino acids 452 and 460, between any two contiguous amino acids between amino acids 488 and 505, between any two contiguous amino acids between amino acids 545 and 558, between any two contiguous amino acids between amino acids 581 and 593, between any two contiguous amino acids between amino acids 704 and 714 of an AAV9 viral protein. In some embodiments, the engineered capsid can have an / 7-nier inserted between amino acids 588 and 589 of an AAV9 viral protein. In some embodiments, the engineered capsid can have a 7- mer motif inserted between amino acids 588 and 589 of an AAV9 viral protein. In other embodiments, the motif inserted is a 10-mer motif, with replacement of amino acids 586-88 and an insertion before 589. SEQ ID NO: 1 is a reference AAV9 capsid sequence for at least referencing the insertion sites discussed above. It will be appreciated that v-mers can be inserted in analogous positions in AAV viral proteins of other serotypes. In some embodiments as previously discussed, the w-mer(s) can be inserted between any two contiguous amino acids within the AAV viral protein and in some embodiments the insertion is made in a variable region.

[0104] In some embodiments, the first 1, 2, 3. or 4 amino acids of an n-mer motif can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted and preceding the insertion site. In some embodiments, the amino acids of the n-mer motif that replace 1 or more amino acids of the polypeptide into which the n-mer motif is inserted come before or immediately before an ’RGD ” in an n-mer motif. For example, in one or more of the 10-mer inserts, the first three amino acids shown can replace 1-3 amino acids into a polypeptide to which they Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) may be inserted. Using an AAV as another non-limiting example, one or more of the n-mer motifs can be inserted into, e. , and AAV9 capsid prolylpeptide between amino acids 588 and 589 and the insert can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the n-mer motif after insertion is residue 585. It will be appreciated that this principle can apply in any other insertion context and is not necessarily limited to insertion between residues 588 and 589 of an AAV9 capsid or equivalent position in another AAV capsid. It will further be appreciated that in some embodiments, no amino acids in the polypeptide into which the n-mer motif is inserted are replaced by the n-mer motif.

[0105] In some embodiments, the AAV capsids or other viral capsids or compositions can be muscle-specific. In some embodiments, muscle-specificity' of the engineered AAV or other viral capsid or other composition is conferred by a muscle specific n-mer motif incorporated in the engineered AAV or other viral capsid or other composition described herein. While not intending to be bound by theory, it is believed that the n-mer motif confers a 3D structure to or within a domain or region of the engineered AAV capsid or other viral capsid or other composition such that the interaction of the viral particle or other composition containing the engineered AAV capsid or other viral capsid or other composition described herein has increased or improved interactions (e.g., increased affinity) with a cell surface receptor and / or other molecule on the surface of a muscle cell. In some embodiments, the cell surface receptor is AAV receptor (AAVR). In some embodiments, the cell surface receptor is a muscle cell specific AAV receptor. In some embodiments, the cell surface receptor or other molecule is a cell surface receptor or other molecule selectively expressed on the surface of a muscle cell. In some embodiments, the cell surface receptor or molecule is an integrin or dimer thereof. In some embodiments, the cell surface receptor or molecule is an Vb6 integrin heterodimer.

[0106] In some embodiments, a muscle specific engineered viral particle or other composition described herein containing the muscle-specific capsid, n-mer motif, or musclespecific targeting moiety described herein can have an increased uptake, delivery rate, transduction rate, efficiency, amount, or a combination thereof in a muscle cell as compared to other cells types and / or other virus particles (including but not limited to AAVs) and other compositions that do not contain the muscle-specific n-mer motif of the present invention.

[0107] First-and second-generation muscle specific AAV capsids were developed using a muscle specific promoter and the resulting capsid libraries were screened in mice and nonhuman primates as described elsewhere herein and / or in, e.g.. WO 2021 / 050974 and WO Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0108] 2021 / 077000. First and second generation myoAAV capsids were further optimized in mice and non-human primates as previously described to generate enhanced myoAAV capsids.

[0109] Further MyoAAV capsids are described in PCT / US2024 / 010859 and PCT / US2024 / 015200, each of which is incorporated by reference in their entirety herein.

[0110] Dual Motifs

[0111] Aspects of the present disclosure provide engineered capsid proteins comprising dual motifs, which are also referred to as a first sequence and a second sequence. In some embodiments, the first sequence and the second sequence are included in the HVR IV and HVR VIII, respectively, of the engineered capsid protein.

[0112] Aspects of the invention provide a capsid protein comprising a first amino acid sequence comprising RGDR or RGDY in the hypervariable region IV (HVR IV) relative to wild- type AAV9 and further comprising one or more substitutions at amino acids 451-455 relative to a wild-ty pe AAV9 vector capsid. In such instances, the capsid protein comprises a 7-mer insert comprising the RGDR or RGDY motif inserted after amino acid 455 relative to a wild type AAV9 vector.

[0113] The capsid protein further comprises a second amino acid sequence comprising RGD in the hypervariable region VIII (HVR VIII) relative to wild ty pe AAV9 and further comprises substitutions at amino acids 584-588 relative to a wild type AAV9 vector capsid. In such instances, the capsid protein comprises a 7-mer insert comprising the RGD motif inserted after amino acid 588 relative to a wild-type AAV9 vector.

[0114] In some embodiments, the capsid protein comprises an amino acid sequence X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or an amino acid sequence X1NX2X3X4RGDYX5X6L (SEQ ID NO: 4290), and an amino acid sequence X7QX8X9X10RGDX11X12X13L (SEQ ID NO: 4294), wherein each of X1-X13 is any amino acid. In some embodiments, Xi is A, L, M, S, T, or V. In some embodiments, X2 is A, G, S, T, or Y. In some embodiments, X3 is A, D, G, N, S, or T. In some embodiments, X4 is A, G, H, I, M, S, T, or V. In some embodiments, X5 is G, Q, or S. In some embodiments, Xe is A, G, L. S, T, or Y. In some embodiments, X7 is I, L. N, R, or S. In some embodiments. Xs is A, E, G, N, S, or T. In some embodiments, X9 is A, D, G, N, S, or T. In some embodiments, X10 is A, G, N, S, or T. In some embodiments, X11 is H, Q, R, or Y. In some embodiments, X12 is G, N. R, or S. In some embodiments, X13 is A, G, S, or T.

[0115] The amino acid sequence X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or an amino acid sequence X1NX2X3X4RGDYX5X6L (SEQ ID NO: 4290) can be in a hypervariable Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) region IV (HVR IV) relative to a wild type AAV9 capsid protein. For example, relative to a wild type AAV9 capsid, Xi is a substitution at amino acid 451, X2 is a substitution at amino acid 453, X3 is a substitution at amino acid 454, X4 is a substitution at amino acid 455, and RGDRX5X6L (SEQ ID NO: 4292) or RGDYXsXeL (SEQ ID NO: 4293) is inserted after amino acid 455.

[0116] The amino acid sequence X7QX8X9X10RGDX11X12X13L (SEQ ID NO: 4294) can be in a hypervariable region VIII (HVR VIII) relative to a wild type AAV9 capsid. For example, relative to a wild type AAV9 capsid, X7 is a substitution at amino acid 584, Xs is a substitution at amino acid 586, X9 is a substitution at amino acid 587, X10 is a substitution at amino acid 588, and RGDX11X12X13L (SEQ ID NO: 4295) is inserted after amino acid 588.

[0117] The capsid protein can comprise one or more amino acid sequences provided in Table la, Table 2a, and Table 3. For example, the capsid protein can comprise a first amino acid sequence selected from any one of SEQ ID NOs: 2-715, optionally wherein the amino acid sequence is in HVR IV relative to a wild ty pe AAV9 capsid protein, and a second amino acid sequence selected from any one of SEQ ID NOs: 716-1429, optionally wherein the amino acid sequence is in HVR VIII relative to a wild type AAV9 capsid protein. Non-limiting examples of a first sequence and a second sequence that can be included in the HVR IV and HVR VIII, respectively, of an engineered capsid protein are provided in Table 3.

[0118] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNGST (SEQ ID NO: 1667) at positions 451-455, the amino acid sequence RGDRQYE (SEQ ID NO: 2381) inserted after amino acid 455, the amino acid sequence IQGAS (SEQ ID NO: 2869) at positions 584-588, the amino acid sequence RGDHGAL (SEQ ID NO: 3583) inserted after amino acid 588, and a deletion of the amino acid G267.

[0119] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence VNGSA (SEQ ID NO: 1941) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2655) inserted after amino acid 455, the amino acid sequence LQGGS (SEQ ID NO: 3284) at positions 584-588, the amino acid sequence RGDHSSL (SEQ ID NO: 3998) inserted after amino acid 588, and a deletion of the amino acid G267.

[0120] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNGSA (SEQ ID NO: 1632) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2346) inserted after amino acid 455. the amino acid sequence LQGGT (SEQ ID NO: 3289) at positions 584-588, the amino acid Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) sequence RGDHSAL (SEQ ID NO: 4003) inserted after amino acid 588, and a deletion of the amino acid G267.

[0121] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNGGT (SEQ ID NO: 1618) at positions 451-455, the amino acid sequence RGDRGSL (SEQ ID NO: 2332) inserted after amino acid 455, the amino acid sequence IQSNT (SEQ ID NO: 3171) at positions 584-588. the amino acid sequence RGDHGTL (SEQ ID NO: 3885) inserted after amino acid 588. and a deletion of the amino acid G267.

[0122] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNGST (SEQ ID NO: 1667) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2381) inserted after amino acid 455. the amino acid sequence SQGDS (SEQ ID NO: 3473) at positions 584-588, the amino acid sequence RGDRGGL (SEQ ID NO: 4187) inserted after amino acid 588, and a deletion of the amino acid G267.

[0123] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNANT (SEQ ID NO: 1520) at positions 451-455, the amino acid sequence RGDRGTL (SEQ ID NO: 2234) inserted after amino acid 455, the amino acid sequence IQGNT (SEQ ID NO: 3048) at positions 584-588, the amino acid sequence RGDHGTL (SEQ ID NO: 3762) inserted after amino acid 588, and a deletion of the amino acid G267.

[0124] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNGSG (SEQ ID NO: 1656) at positions 451-455, the amino acid sequence RGDRGAL (SEQ ID NO: 2370) inserted after amino acid 455, the amino acid sequence IQGSG (SEQ ID NO: 3082) at positions 584-588, the amino acid sequence RGDHGAL (SEQ ID NO: 3796) inserted after amino acid 588, and a deletion of the amino acid G267.

[0125] In some embodiments, the capsid protein comprises, relative to the wild ty pe AAV9 capsid protein, the amino acid sequence LNASI (SEQ ID NO: 1526) at positions 451-455, the amino acid sequence RGDRQLL (SEQ ID NO: 2240) inserted after amino acid 455. the amino acid sequence IQGGS (SEQ ID NO: 2985) at positions 584-588, the amino acid sequence RGDHGGL (SEQ ID NO: 3699) inserted after amino acid 588, and a deletion of the amino acid G267.

[0126] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence VNGSS (SEQ ID NO: 1998) at positions 451-455, Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) the amino acid sequence RGDRGAL (SEQ ID NO: 2712) inserted after amino acid 455, the amino acid sequence IQSGS (SEQ ID NO: 3159) at positions 584-588. the amino acid sequence RGDHGTL (SEQ ID NO: 3873) inserted after amino acid 588, and a deletion of the amino acid G267.

[0127] In some embodiments, the capsid protein comprises, relative to the wild ty pe AAV9 capsid protein, the amino acid sequence LNSGT (SEQ ID NO: 1720) at positions 451-455, the amino acid sequence RGDRGTL (SEQ ID NO: 2434) inserted after amino acid 455, the amino acid sequence IQTST (SEQ ID NO: 3226) at positions 584-588, the amino acid sequence RGDHGTL (SEQ ID NO: 3940) inserted after amino acid 588, and a deletion of the amino acid G267.

[0128] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNASI (SEQ ID NO: 1526) at positions 451-455, the amino acid sequence RGDRQLL (SEQ ID NO: 2240) inserted after amino acid 455, the amino acid sequence IQGDT (SEQ ID NO: 2966) at positions 584-588, the amino acid sequence RGDRSTL (SEQ ID NO: 3680) inserted after amino acid 588, and a deletion of the amino acid G267.

[0129] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence VNGSA (SEQ ID NO: 1941) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2655) inserted after amino acid 455, the amino acid sequence NQGGA (SEQ ID NO: 3374) at positions 584-588. the amino acid sequence RGDHGTL (SEQ ID NO: 4088) inserted after amino acid 588, and a deletion of the amino acid G267.

[0130] In some embodiments, the capsid protein comprises, relative to the wild ty pe AAV9 capsid protein, the amino acid sequence LNGSA (SEQ ID NO: 1632) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2346) inserted after amino acid 455, the amino acid sequence NQGAT (SEQ ID NO: 3339) at positions 584-588, the amino acid sequence RGDHGAL (SEQ ID NO: 4053) inserted after amino acid 588, and a deletion of the amino acid G267.

[0131] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence SNAST (SEQ ID NO: 1850) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2564) inserted after amino acid 455, the amino acid sequence LQGDG (SEQ ID NO: 3263) at positions 584-588, the amino acid sequence RGDRGGL (SEQ ID NO: 3977) inserted after amino acid 588. and a deletion of the amino acid G267. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0132] In some embodiments, the capsid protein comprises, relative to the wild ty pe AAV9 capsid protein, the amino acid sequence VNGSA (SEQ ID NO: 1941) at positions 451-455, the amino acid sequence RGDRQYL (SEQ ID NO: 2655) inserted after amino acid 455, the amino acid sequence IQGDA (SEQ ID NO: 2912) at positions 584-588, the amino acid sequence RGDRGAL (SEQ ID NO: 3626) inserted after amino acid 588, and a deletion of the amino acid G267.

[0133] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNSSM (SEQ ID NO: 1747) at positions 451-455, the amino acid sequence RGDRQSL (SEQ ID NO: 2461) inserted after amino acid 455, the amino acid sequence LQGGS (SEQ ID NO: 3282) at positions 584-588, the amino acid sequence RGDHGGL (SEQ ID NO: 3996) inserted after amino acid 588. and a deletion of the amino acid G267.

[0134] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNSSM (SEQ ID NO: 1730) at positions 451-455, the amino acid sequence RGDRQAL (SEQ ID NO: 2444) inserted after amino acid 455, the amino acid sequence IQGAT (SEQ ID NO: 2884) at positions 584-588, the amino acid sequence RGDHGTL (SEQ ID NO: 3598) inserted after amino acid 588, and a deletion of the amino acid G267.

[0135] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence TNSSS (SEQ ID NO: 1906) at positions 451-455. the amino acid sequence RGDRGSL (SEQ ID NO: 2620) inserted after amino acid 455, the amino acid sequence NQGAT (SEQ ID NO: 3339) at positions 584-588, the amino acid sequence RGDHGAL (SEQ ID NO: 4053) inserted after amino acid 588, and a deletion of the amino acid G267.

[0136] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence LNASM (SEQ ID NO: 1542) at positions 451-455, the amino acid sequence RGDRQAL (SEQ ID NO: 2256) inserted after amino acid 455, the amino acid sequence NQGGA (SEQ ID NO: 3372) at positions 584-588, the amino acid sequence RGDHGGL (SEQ ID NO: 4086) inserted after ammo acid 588. and a deletion of the amino acid G267.

[0137] In some embodiments, the capsid protein comprises, relative to the wild type AAV9 capsid protein, the amino acid sequence VNSSG (SEQ ID NO: 2040) at positions 451-455, the amino acid sequence RGDRGAL (SEQ ID NO: 2754) inserted after amino acid 455. the amino acid sequence IQGTT (SEQ ID NO: 3118) at positions 584-588, the amino acid Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) sequence RGDHGTL (SEQ ID NO: 3832) inserted after amino acid 588, and a deletion of the amino acid G267.

[0138] Any of the capsid proteins described herein can further include a deletion. For example, the capsid protein can further comprise a deletion of amino acid G267 relative to a wild type AAV9 vector.

[0139] III. Other Embodiments

[0140] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The specific embodiments herein are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0141] In any of the aspects and embodiments described herein, the nucleic acid sequences contemplated can be DNA, RNA, or modified versions thereof. Modified nucleic acids can be distinguished from naturally occurring nucleic acids by modifications to the backbone of the polynucleotide chain, for example, peptide nucleic acids (PNA), morpholinos, locked nucleic acids (LNA). glycol nucleic acids (GNA) and threose nucleic acid (TNA). Modified nucleic acids can also include analogs with modifications to the four nucleobases. In some embodiments, the nucleic acids are PNAs. In some embodiments, the nucleic acids are LNAs. In some embodiments, the nucleic acids are morpholinos. In some embodiments, the nucleic acids are in a single-stranded form. In some embodiments, the nucleic acids are in doublestranded form. In some embodiments, the nucleic acids are linear. In some embodiments, the nucleic acids are circular. In some embodiments, the nucleic acids are plasmids.

[0142] Non-limiting embodiments of the present disclosure include:

[0143] Embodiment 1 is an adeno-associated virus (AAV) vector comprising a capsid protein comprising a first amino acid sequence comprising the ammo acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287): and a second amino acid sequence comprising the amino acid sequence RGD.

[0144] Embodiment 2 is the AAV vector of Embodiment 1, wherein the amino acid sequence RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) is inserted after amino acid 455 in reference to an AAV9 capsid. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0145] Embodiment 3 is the AAV vector of Embodiment 2, wherein the amino acid sequence RGD is inserted after amino acid 588 in reference to an AAV9 capsid.

[0146] Embodiment 4 is the AAV vector of Embodiment 1 , wherein the capsid protein comprises the amino acid sequence X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or X1NX2X3X4RGDYX5X6L (SEQ ID NO: 4290), wherein Xi is selected from among any amino acid; X2 is selected from among any amino acid; X3 is selected from among any amino acid; X4 is selected from among any amino acid; Xs is selected from among any amino acid; and Xe is selected from among any amino acid.

[0147] Embodiment 5 is the AAV vector of Embodiment 4, wherein Xi is located at amino acid 451, X2 is located at amino acid 453, X3 is located at amino acid 454, X4 is located amino acid 455, and RGDRXsXsL or RGDYXsXeL is inserted after amino acid 455 in reference to an AAV9 capsid or the equivalent position in another AAV capsid.

[0148] Embodiment 6 is the AAV vector of Embodiment 5, wherein Xi is an amino acid selected from the group consisting of A, L, M, S, T, and V. Embodiment 7 is the AAV vector of Embodiment 5, wherein X2 is an amino acid selected from the group consisting of A, G, S, T, and Y. Embodiment 8 is the AAV vector of Embodiment 5, wherein X3 is an amino acid selected from the group consisting of A, D, G, N, S, and T. Embodiment 9 is the AAV vector of Embodiment 8, wherein X3 is S. Embodiment 10 is the AAV vector of Embodiment 5, wherein X4 is an amino acid selected from the group consisting of A, G, H. I, M, S, T, and V. Embodiment 11 is the AAV vector of Embodiment 5. wherein X5 is an amino acid selected from the group consisting of G, Q, and S. Embodiment 12 is the AAV vector of Embodiment 5, wherein X5 is an amino acid selected from the group consisting of G and Q. Embodiment 13 is the AAV vector of Embodiment 5, wherein Xe is an amino acid selected from the group consisting of A, G. L, S, T, and Y. Embodiment 14 is the AAV vector of Embodiment 13, wherein Xe is an amino acid selected from the group consisting of A and Y.

[0149] Embodiment 15 is the AAV vector of Embodiment 4, wherein the capsid protein further comprises the amino acid sequence X7QX8X9X10RGDX11X12X13L, wherein X7 is selected from among any amino acid; Xs is selected from among any amino acid; X9 is selected from among any amino acid; X10 is selected from among any amino acid; X11 is selected from among any amino acid; X is selected from among any amino acid; and X13 is selected from among any amino acid.

[0150] Embodiment 16 is the AAV vector of Embodiment 15, wherein Xv is located at amino acid 584. Xs is located at amino acid 586, X9 is located at amino acid 587, X10 is located at Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) amino acid 588, and RGDX11X12X13 is inserted after amino acid 588 in reference to an AAV9 capsid or the equivalent position in another AAV capsid.

[0151] Embodiment 17 is the AAV vector of Embodiment 15, wherein X7 is an amino acid selected from the group consisting of I, L, N, R, and S. Embodiment 18 is the AAV vector of Embodiment 17, wherein X7 is the amino acid I. Embodiment 19 is the AAV vector of Embodiment 15, wherein Xs is an amino acid selected from the group consisting of A, E, G, N, S. and T. Embodiment 20 is the AAV vector of Embodiment 19, wherein Xs is the amino acid G. Embodiment 21 is the AAV vector of Embodiment 15, wherein X9 is an amino acid selected from the group consisting of A, D, G, N, S, and T. Embodiment 22 is the AAV vector of Embodiment 15, wherein X10 is an amino acid selected from the group consisting of A, G. N, S. and T. Embodiment 23 is the AAV vector of Embodiment 22, wherein X10 is the amino acid T. Embodiment 24 is the AAV vector of Embodiment 15, wherein X11 is an amino acid selected from the group consisting of H, Q, R, and Y. Embodiment 25 is the AAV vector of Embodiment 24, wherein X11 is the amino acid H. Embodiment 26 is the AAV vector of Embodiment 15, wherein X12 is an amino acid selected from the group consisting of G, N, R, and S. Embodiment 27 is the AAV vector of Embodiment 26, wherein X12 is the amino acid G or S. Embodiment 28 is the AAV vector of Embodiment 15, wherein X13 is an amino acid selected from the group consisting of A, G, S, and T. Embodiment 29 is the AAV vector of Embodiment 28, wherein X13 is the amino acid T. Embodiment 30 is the AAV vector of Embodiment 1, wherein the capsid protein comprises an amino acid sequence selected from Table l a. Embodiment 31 is the AAV vector of Embodiment 30, wherein the amino acid sequence selected from Table la is in hypervariable region IV (HVR IV) relative to wild-ty pe AAV9.

[0152] Embodiment 32 is the AAV vector of Embodiment 31, wherein the capsid protein comprises substitutions at amino acids 451-455 relative to a wild-type AAV9 vector capsid.

[0153] Embodiment 33 is the AAV vector of Embodiment 32, wherein the substitutions at amino acids 451-455 relative to a wild-ty pe AAV9 vector capsid are substituted with an amino acid sequence selected from column 1 of Table lb.

[0154] Embodiment 34 is the AAV vector of Embodiment 33, wherein the capsid protein comprises a 7-mer insert selected from column 2 of Table lb.

[0155] Embodiment 35 is the AAV vector of Embodiment 30, wherein the 7-mer insert is inserted after amino acid 455 relative to a wild-type AAV9 vector.

[0156] Embodiment 36 is the AAV vector of Embodiment 30, wherein the capsid protein comprises an amino acid sequence selected from Table 2a. Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)

[0157] Embodiment 37 is the AAV vector of Embodiment 36, wherein the amino acid sequence selected from Table 2a is in hypervariable region VIII (HVR VIII) relative to wildtype AAV9.

[0158] Embodiment 38 is the AAV vector of Embodiment 37, wherein the capsid protein comprises substitutions at amino acids 584-588 relative to a wild-ty pe AAV9 vector capsid.

[0159] Embodiment 39 is the AAV vector of Embodiment 38, wherein the substitutions at amino acids 584-588 relative to a wild-type AAV9 vector capsid are substituted with an amino acid sequence selected from column 1 of Table 2b.

[0160] Embodiment 40 is the AAV vector of Embodiment 39, wherein the capsid protein comprises a 7-mer insert selected from column 2 of Table 2b.

[0161] Embodiment 41 is the AAV vector of Embodiment 40, wherein the 7-mer insert is inserted after amino acid 588 relative to a wild-type AAV9 vector.

[0162] Embodiment 42 is the AAV vector of Embodiment 1 , wherein the AAV vector exhibits increased muscle and heart tropism as compared to a wild-type AAV vector.

[0163] Embodiment 43 is the AAV vector of Embodiment 1, wherein the capsid protein further comprises a deletion of G267 in reference to an AAV9 capsid or equivalent position in another AAV capsid.

[0164] Embodiment 44 is the AAV vector of Embodiment 43, wherein the vector exhibits reduced liver tropism as compared to a wild-type AAV vector.

[0165] EXAMPLES

[0166] In order that the invention described may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods and compositions provided herein and are not to be construed in any way as limiting their scope.

[0167] Engineered capsid proteins comprising a deletion at position 267 (G267) relative to a wild-type AAV9 vector and further comprising a first amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) in the hypervanable region IV (HVR IV) and a second ammo acid sequence comprising RGD in the hypervariable region VIII (HVR VIII) were generated, thereby generating ‘'dual motif’ or 7thgeneration capsids variants. 7thgeneration capsid variants were benchmarked against wild-type AAV9 and AAVrh74.

[0168] Amino acid sequences in the HVR IV and HVR VIII of 7thgeneration capsid variants are shown in Table 4. For the HVR IV sequence, the first five amino acids include one or Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) more substitutions at amino acids 451-455 relative to the wild-type AAV9 capsid protein and the remaining seven amino acids comprising the RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) motif are inserted after amino acid 455 relative to the wild-type AAV9 capsid protein. For the HVR VIII sequence, the first five amino acids include one or more substitutions at amino acids 584-588 relative to the wild-ty pe AAV9 capsid protein and the remaining seven amino acids comprising the RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) motif are inserted after amino acid 588 relative to the wild-type AAV9 capsid protein.

[0169] Table 4: 7thgeneration capsid sequences

[0170] As shown in FIG. 1-FIG. 4, top 7thgeneration MyoAAV-LD capsid variants transduced human primary myotubes (FIG. 1), NHP skeletal muscle (FIG. 2) and heart (FIG. 3) with higher efficiency compared to naturally occurring capsids AAV9 and AAVrh74. Top 7thgeneration MyoAAV-LD capsid variants were also more efficiently de-targeted from the liver compared to AAV9 and AAVrh74 (FIG. 4).

[0171] INCORPORATION BY REFERENCE

[0172] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32) disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0173] EQUIVALENTS Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subj ect matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)CLAIMSWhat Is Claimed Is:

1. An engineered adeno-associated virus (AAV) capsid protein comprising: a first amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287); and a second amino acid sequence comprising RGD.

2. The engineered AAV capsid protein of claim 1, wherein the first amino acid sequence is in a hypervariable region IV (HVR IV) relative to a wild type AAV9 capsid protein.

3. The engineered AAV capsid protein of claim 1, wherein the first amino acid sequence comprising RGDR (SEQ ID NO: 4286) or RGDY (SEQ ID NO: 4287) is inserted after amino acid 455 in reference to a wild ty pe AAV9 capsid protein.

4. The engineered AAV capsid protein of claim 1, wherein the second amino acid sequence is in a hypervariable region VIII (HVR VIII) relative to a wild type AAV9 capsid protein.

5. The engineered AAV capsid protein of claim 1, wherein the second amino acid sequence comprising RGD is inserted after amino acid 588 in reference to a wild type AAV9 capsid.

6. The engineered AAV capsid protein of claim 1, wherein the first amino acid sequence comprises the amino acid sequence X1NX2X3X4RGDRX5X6L (SEQ ID NO: 4289) or the amino acid sequence X1NX2X3X4RGDYX5X6L (SEQ ID NO: 4290), and wherein each of Xi-Xe is any amino acid.

7. The engineered AAV capsid protein of claim 6, wherein, relative to a wild type AAV9 capsid protein, Xi is located at amino acid 451, X2 is located at amino acid 453, X3 is located at amino acid 454, X4 is located amino acid 455, and RGDRXsXeL (SEQ ID NO: 4292) or RGDYX XsL (SEQ ID NO: 4293) is inserted after amino acid 455.Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)8. The engineered AAV capsid protein of claim 6, wherein:Xi is A, L, M, S, T, or V;X2is A, G, S, T, or Y;X3is A, D, G, N, S, or T;X4is A, G, H, I, M, S, T, or V;Xs is G, Q, or S; andX6is A, G. L, S, T. or Y.

9. The engineered AAV capsid protein of claim 1, wherein the second amino acid sequence comprises the amino acid sequence X7QX8X9X10RGDX11X12X13L (SEQ ID NO: 4294), and wherein each of X7-X13 is any amino acid.

10. The engineered AAV capsid protein of claim 9, wherein, relative to a wild type AAV9 capsid protein, X7 is located at amino acid 584, Xs is located at amino acid 586, X9 is located at amino acid 587, X10 is located at amino acid 588. and RGDX11X12X13L (SEQ ID NO: 4295) is inserted after amino acid 588.

11. The engineered AAV capsid protein of claim 9, wherein:X7 is I, L, N, R, or S;X8is A, E, G, N, S, or T:X9is A, D, G, N, S, or T;X10 is A, G, N, S, or T;Xn is H, Q, R, or Y;X12 is G, N, R, or S; andX13 is A, G, S, or T.

12. The engineered AAV capsid protein of claim 1, wherein the first amino acid sequence comprises any one of SEQ ID NOs: 2-715.

13. The engineered AAV capsid protein of claim 1, wherein the second amino acid sequence comprises any one of SEQ ID NOs: 716-1429.Attorney Docket No.: PAT059914-PCT-SEC01 (KATE-32)14. The engineered AAV capsid protein of claim 1, wherein the first amino acid sequence and the second amino acid sequence each correspond to the HVR IV sequence and the HVR VIII sequence, respectively, set forth in Table 3 for a single capsid protein variant.

15. The engineered AAV capsid protein of claim 1, wherein the engineered AAV capsid protein further comprises a deletion of amino acid G267 relative to a wild type AAV9 vector.

16. The engineered AAV capsid protein of claim 1, wherein the engineered AAV capsid protein exhibits increased muscle and heart tropism compared to a wild t pe AAV capsid protein.

17. The engineered AAV capsid protein of claim 1, wherein the engineered capsid protein exhibits reduced liver tropism as compared to a wild type AAV capsid protein.

18. An adeno-associated virus (AAV) particle comprising the engineered capsid protein of claim 1.

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