Adeno-associated virus compositions for targeted gene therapy

Abstract

Described herein are compositions and kits comprising recombinant adeno-associated viruses (rAAVs) with tropism that show increased specificity and improved viral transduction efficiency in target cell types, such as the brain and lung.The rAAV compositions described herein also have tropism that show decreased specificity and reduced viral transduction efficiency in non-target cell types, such as the liver.The rAAV compositions described herein encapsulate transgenes, such as therapeutic nucleic acids, in capsids.When delivered to a subject systemically, rAAVs can increase the specificity of transgenes to target cell types and increase transduction efficiency compared to parental or reference AAVs.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 736,904, filed September 26, 2018, and U.S. Provisional Patent Application No. 62 / 832,812, filed April 11, 2019, both of which are incorporated herein by reference in their entireties.

[0002] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. NS087949 awarded by the National Institutes of Health and Grant No. W911NF-17-2-0036 awarded by DARPA. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy, created on September 20, 2019, is named 38075-733_601_SL.txt and is 10,656,833 bytes in size. [Background technology]

[0004] background Recombinant adeno-associated viruses (rAAVs) are increasingly being used for gene delivery in basic science research and therapeutic applications due to their ability to transduce both dividing and non-dividing cells, their long-term persistence as episomal DNA within infected cells, and their low immunogenicity. These properties make rAAVs attractive for both basic science applications and clinical applications such as gene therapy. Current gene therapy approaches for many disorders focus on direct delivery to improve transduction efficiency and restrict expression to only affected cell types. However, this administration route has met with limited success across many clinical trials and diseases due to limited applicability of local delivery.

[0005] Intravenous administration addresses these limitations and allows for widespread and efficient use in non-invasive ways in tissues and organs throughout the body. Unfortunately, naturally occurring serotypes are limited to transducing certain cell types and organs, with nonspecific overlapping tropism in other cell types and organs. This poses several complications for gene therapy applications, including, but not limited to, off-target effects due to transduction of unaffected organs and cell types (especially the liver) and the need for larger viral loads to achieve sufficient therapeutic levels in the target tissue or organ. In addition, even with systemic delivery, current rAAV-mediated gene therapies lack the specificity to cross the blood-brain barrier (BBB) ​​or epithelium, resulting in limited therapeutic reach during therapeutic intervention for brain and organ (e.g., lung) diseases and conditions.

[0006] Thus, there is a need for rAAVs that can achieve high specificity for targeting particular cell types or organs in a subject when delivered systemically (e.g., intravenously, intranasally) to a subject, and low specificity for unaffected (non-target) organs and cell types. Summary of the Invention [Means for solving the problem]

[0007] overview Disclosed herein are recombinant AAVs (rAAVs) comprising capsids engineered to have specific capsid structures through repeated rounds of positive and negative selection. This engineering results in rAAV variants with altered specificity for a particular tissue or organ compared to the unmodified parent capsid from which the rAAV variants were derived. In some embodiments, the rAAV variants comprise an AAV capsid protein with a 7-mer peptide substitution at residues corresponding to amino acids 452-458 of the AAV9 capsid protein VP1. In certain other embodiments, the substitution is at amino acids 452-458 of AAV9 VP1. In certain other embodiments, the substitution is at amino acids 452-458 of AAV-PHP.B VP1. In still other embodiments, the substitution is at amino acids 452-458 of AAV-PHP.eB VP1. In some embodiments, the substitution reduces the tropism of the rAAV capsid for cells or tissues, thereby enabling reduced off-target effects of therapeutic viral transduction by the rAAV variant, thereby eliminating the need for higher viral doses. In some embodiments, the substitution increases the tropism of the rAAV capsid for cells or tissues, thereby enabling the rAAV variant to achieve broad transduction of the target environment (e.g., target cell type or tissue) in a subject upon systemic delivery (e.g., intravenous injection). Typically, the 7-mer peptide substitution is not ILGTGTS (SEQ ID NO: 45479), QSSQTPR (SEQ ID NO: 45479), or TLAVPFK (SEQ ID NO: 45477). The rAAVs described herein were identified using an improved version of the Cre recombination-based AAV targeted evolution (CREATE) method. The multiplexed CREATE (M-CREATE) method results in enhanced transduction efficiency and / or specificity through the introduction of diversity into capsid protein sequences, unbiased in vivo selection, and recovery of only variants that translocate into defined cell populations, cross the cell membrane, translocate to the nucleus, unpackage their genetic payload, and express their genetic payload. Variant capsids exhibiting the most desirable tropism (e.g., enhanced efficiency and specificity for a particular in vivo environment) are recovered and identified by deep sequencing. The strategy for unbiased selection and analysis involves determining the enrichment scores of variants (by normalizing the target tissue library to the starting viral library) and determining unbiased propagation (in which each variant is equally represented) between iterative selections through synthetic library construction. Detailed characterization of the resulting libraries from sequencing data is also described herein, providing useful insight into the selection of variants against the target.

[0008] Also disclosed herein are AAV capsid libraries comprising AAV capsid proteins with 7-mer peptide substitutions at residues corresponding to amino acids 452-458 of AAV9 VP1. In some embodiments, the library is a library of capsids comprising 7-mer peptide substitutions at amino acids 452-458 of the AAV9 variants AAV-PHP.eB or AAV-PHP.B. Such libraries are particularly useful for positive selection across different brain cell types (e.g., endothelial cells, neurons, and astrocytes) and negative selection across the liver in vivo, and such libraries have been shown herein to generate large pools of AAV9 variants with enhanced ability to cross the BBB, broadly transduce the central nervous system (CNS), and detarget off-target in vivo environments, such as the liver. In other embodiments, the library is an AAV capsid library comprising AAV capsid proteins with 7-mer peptide substitutions at amino acids 452-458 of AAV9. Such libraries are particularly useful for selection across various organs in vivo, such as spinal cord, brain, liver, stomach, intestine, lung, heart, testes, spleen, adipose (fat) and muscle.

[0009] Also disclosed herein are methods and kits for producing therapeutic recombinant AAV (rAAV) particles, as well as methods and pharmaceutical compositions or formulations comprising the rAAV particles for the treatment of diseases or conditions affecting the CNS, PNS, or target in vivo environment (e.g., the lung), e.g., in a subject in need thereof.

[0010] Aspects disclosed herein provide a recombinant AAV (rAAV) comprising: (a) a variant AAV capsid comprising a variant AAV capsid protein comprising an amino acid substitution of three or more amino acids within a 7-mer peptide sequence at the three-fold symmetry axis of a corresponding parent AAV capsid protein, wherein the 7-mer peptide sequence is at an amino acid position corresponding to amino acid residues 452-458 of AAV9 VP1 (SEQ ID NO: 1), and the amino acid substitution is not ILGTGTS (SEQ ID NO: 45479), QSSQTPR (SEQ ID NO: 45479), or TLAVPFK (SEQ ID NO: 45477); and (b) a heterologous polynucleotide comprising a nucleotide sequence encoding a gene product. In some embodiments, the variant AAV capsid has increased tropism for a target tissue or target cell when measured in a subject, compared to the tropism of the corresponding parent AAV capsid. In some embodiments, the target tissue or target cell comprises a tissue or cell of the central nervous system (CNS) or peripheral nervous system (PNS), or a combination thereof. In some embodiments, the variant AAV capsid has reduced tropism for non-target tissues, including liver tissue, or non-target cells, including liver cells, compared to the tropism of the corresponding parent AAV capsid when measured in a subject. In some embodiments, the three or more amino acids comprise three consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V, at amino acid positions of the parent AAV capsid protein corresponding to amino acid residues 452-454, 453-455, 454-456, 455-457, or 456-458 of AAV9 VP1 (SEQ ID NO: 1). In some embodiments, the three or more amino acids comprise four consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions in the parent AAV capsid protein corresponding to amino acid residues 452-455, 453-456, 454-457, or 455-458 of AAV9 VP1 (SEQ ID NO: 1).In some embodiments, the three or more amino acids comprise at least five consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions in the parent AAV capsid protein corresponding to amino acid residues 452-456, 453-457, or 454-458 of AAV9 VP1 (SEQ ID NO: 1). In some embodiments, the three or more amino acids are (a) a substituted A, D, E, G, H, M, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 452 of AAV9 VP1 (SEQ ID NO: 1); (b) a substituted A, D, E, G, K, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 453 of AAV9 VP1 (SEQ ID NO: 1); (c) a substituted A, D, E, G, K, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 454 of AAV9 VP1 (SEQ ID NO: 1); (d) a substituted A, D, E, G, K, N, P, Q, S, or T at the amino acid position corresponding to amino acid residue 455 of AAV9 VP1 (SEQ ID NO: 1); (e) a substituted A, D, E, G, H, K, N, P, Q, S, or T at the amino acid position corresponding to amino acid residue 456 of AAV9 VP1 (SEQ ID NO: 1); and (g) three or more of a substitution A, D, E, G, K, N, P, S, T, or V at the amino acid position corresponding to amino acid residue 457 of AAV9 VP1 (SEQ ID NO: 1), and a substitution A, E, G, H, K, L, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 458 of AAV9 VP1 (SEQ ID NO: 1).In some embodiments, the three or more amino acids are (a) a substituted A, D, E, G, H, K, L, M, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 452 of AAV9 VP1 (SEQ ID NO: 1); (b) a substituted A, D, G, H, M, N, P, Q, S, T, or V at the amino acid position corresponding to amino acid residue 453 of AAV9 VP1 (SEQ ID NO: 1); (c) a substituted A, D, E, G, K, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 454 of AAV9 VP1 (SEQ ID NO: 1); (d) a substituted A, D, E, G, K, N, P, Q, S, T, or V at the amino acid position corresponding to amino acid residue 455 of AAV9 VP1 (SEQ ID NO: 1); (f) a substitution A, D, G, K, N, P, Q, S, or T at the amino acid position corresponding to amino acid residue 456 of AAV9 VP1 (SEQ ID NO: 1), a substitution A, G, H, L, M, N, P, Q, S, T, or V at the amino acid position corresponding to amino acid residue 457 of AAV9 VP1 (SEQ ID NO: 1), and (g) a substitution A, D, E, G, H, I, K, L, N, Q, R, S, T, or V at the amino acid position corresponding to amino acid residue 458 of AAV9 VP1 (SEQ ID NO: 1). In some embodiments, the amino acid substitutions comprise an amino acid sequence selected from DGAATKN (SEQ ID NO: 3943), and DGQSSKS (SEQ ID NO: 2764).In some embodiments, the three or more amino acids are (a) a substituted A, D, G, L, N, Q, S, or T at the amino acid at a position corresponding to amino acid residue 452 of AAV9 VP1 (SEQ ID NO: 1); (b) a substituted A, G, N, P, Q, R, S, or T at the amino acid at a position corresponding to amino acid residue 453 of AAV9 VP1 (SEQ ID NO: 1); (c) a substituted A, D, G, N, S, or T at the amino acid at a position corresponding to amino acid residue 454 of AAV9 VP1 (SEQ ID NO: 1); (d) a substituted A, D, G, K, N, P, Q, S, or T at the amino acid at a position corresponding to amino acid residue 455 of AAV9 VP1 (SEQ ID NO: 1); (e) a substituted A, G, K, N, P, R, S, or T at the amino acid at a position corresponding to amino acid residue 456 of AAV9 VP (SEQ ID NO: 1); and (g) three or more of an A, G, K, N, P, R, S, T, or V substitution at the amino acid at the position corresponding to amino acid residue 457 of AAV9 VP1 (SEQ ID NO: 1); and (g) an A, G, K, L, R, S, T, or V substitution at the amino acid at the position corresponding to amino acid residue 458 of AAV9 VP1 (SEQ ID NO: 1). In some embodiments, the amino acid substitutions comprise an amino acid sequence selected from the group consisting of LQTSSPG (SEQ ID NO:2933), QQGKQSV (SEQ ID NO:79), SINTKTN (SEQ ID NO:45475), SNGTKQT (SEQ ID NO:442), GSGKTAA (SEQ ID NO:88), MGDKPTR (SEQ ID NO:2466), QPSGGNT (SEQ ID NO:2672), ERGANTK (SEQ ID NO:5192), TTGGHSS (SEQ ID NO:2743), GTTKTSE (SEQ ID NO:3064), GTGTSVL (SEQ ID NO:11958), NQSGTKG (SEQ ID NO:780), KGPGQMG (SEQ ID NO:45476), and GTPSKAG (SEQ ID NO:2741). In some embodiments, the target tissue, or target cells within the tissue, are selected from the group consisting of lung, intestine, kidney, heart, and stomach.In some embodiments, the three or more amino acids include three consecutive amino acids at amino acid positions corresponding to amino acid residues 452-454, 453-455, 454-456, 455-457, or 456-458 of AAV9 VP1, wherein (a) 452 is selected from the group consisting of N, K, R, and T; (b) 453 is selected from the group consisting of L, N, P, and S; (c) 454 is selected from the group consisting of A, D, G, N, S, and T; (d) 455 is selected from the group consisting of L, P, S, and T; (e) 456 is selected from the group consisting of P, R, and S; (f) 457 is selected from the group consisting of G, N, S, and T; and (g) 458 is selected from the group consisting of I, L, and R. In some embodiments, the three or more amino acids include (a) four consecutive amino acids at amino acid positions corresponding to amino acid residues 452-455, 453-456, 454-457, or 455-458 of AAV9 VP1 (SEQ ID NO: 1); (b) five consecutive amino acids at amino acid positions corresponding to amino acid residues 452-456, 453-457, or 453-458 of AAV9 VP1 (SEQ ID NO: 1); (c) six consecutive amino acids at amino acid positions corresponding to amino acid residues 452-457 or 453-458 of AAV9 VP1 (SEQ ID NO: 1); and (d) seven consecutive amino acids at amino acid positions corresponding to amino acid residues 452-458 of AAV9 VP1 (SEQ ID NO: 1). In some embodiments, the target tissue is lung, and the three or more amino acids are provided within an amino acid sequence comprising KDNTPGR (SEQ ID NO: 32538), NNLPRNL (SEQ ID NO: 32867), or any amino acid sequence shown in Figure 13. In some embodiments, the target tissue is intestine, and the three or more amino acids are provided within an amino acid sequence comprising RESSPSL (SEQ ID NO: 26474), KDNTPGR (SEQ ID NO: 26584), or any amino acid sequence shown in Figure 5.In some embodiments, the tissue is kidney, and the three or more amino acids are comprised within an amino acid sequence comprising RVPLSTI (SEQ ID NO:26933), NNLPRNL (SEQ ID NO:27530), KDNTPGR (SEQ ID NO:28509), or any amino acid sequence depicted in FIG. 6. In some embodiments, the tissue is heart, and the three or more amino acids are comprised within an amino acid sequence comprising KDNTPGR (SEQ ID NO:25633), or any amino acid sequence depicted in FIG. 4. In some embodiments, the tissue is stomach, and the three or more amino acids are comprised within an amino acid sequence comprising RESSPSL (SEQ ID NO:31904), or any amino acid sequence depicted in FIG. 12. In some embodiments, the corresponding parent AAV capsid protein is AAV9 VP1 or a variant thereof. In some embodiments, the AAV9 VP1 variant has 90% or higher sequence identity to SEQ ID NO:1. In some embodiments, the AAV9 VP1 variant has 95% or higher sequence identity to SEQ ID NO:1. In some embodiments, the corresponding parent AAV capsid protein further comprises an insertion of an amino acid sequence selected from the group consisting of TLAXPFK (SEQ ID NO: 46424), TLAX (SEQ ID NO: 46425), LAVX (SEQ ID NO: 46426), AVPX (SEQ ID NO: 46427), and VPFX (SEQ ID NO: 46428) at amino acid position corresponding to 588_589 of AAV9 VP1 (SEQ ID NO: 1), where X is any amino acid except V. In some embodiments, the parent AAV capsid protein is from AAV-PHP.B or AAV-PHP.eB. In some embodiments, the rAAV is isolated and purified.

[0011] Aspects disclosed herein provide pharmaceutical formulations comprising the disclosed rAAV and a pharmaceutical excipient. In some embodiments, the pharmaceutical formulation is formulated for intravenous, intraarterial, intranasal, intrathecal, intracisternal, or subcutaneous injection.

[0012] Aspects disclosed herein provide a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an rAAV of the present disclosure or a pharmaceutical formulation of the present disclosure, wherein the gene product is a therapeutic gene product. In some embodiments, the administering step is by intravenous, intraarterial, intranasal, intrathecal, intracisternal, or subcutaneous injection. In some embodiments, the disease or condition is selected from the group consisting of pulmonary fibrosis, surfactant protein disorders, peroxisomal biogenesis disorders, or COPD. In some embodiments, the disease or condition is a central nervous system (CNS) or peripheral nervous system (PNS) disorder.

[0013] A method for producing an rAAV of the present disclosure, comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first nucleic acid sequence encoding a therapeutic gene expression product surrounded by 5' and 3' inverted terminal repeat (ITR) sequences; (ii) a second nucleic acid sequence encoding a viral genome comprising the 5' ITR sequence, a replication (Rep) gene, a capsid (Cap) gene, and the 3' ITR, wherein the Cap gene encodes an rAAV capsid protein; and (iii) a third nucleic acid sequence encoding a first helper virus protein selected from the group consisting of E4orf6, E2a, and VA RNA, and optionally encoding a second helper virus protein comprising E1a or E1b55k; (b) expressing the AAV capsid protein in the cell; (c) constructing an rAAV of the present disclosure; and (d) packaging the first nucleic acid sequence into the rAAV. Aspects disclosed herein provide variant adeno-associated virus (AAV) capsids that comprise (a) at least one of decreased specificity and decreased transduction efficiency, as measured in the liver of the primate subject when delivered intravenously to the primate subject, compared to a corresponding parent AAV capsid having the AAV capsid protein of SEQ ID NO: 1, and (b) at least one of increased specificity and improved transduction efficiency, as measured in the central nervous system (CNS) of the primate subject when delivered intravenously to the primate subject, compared to a corresponding parent AAV capsid. In some embodiments, the variant AAV capsid further comprises a variant AAV capsid protein comprising an amino acid substitution of three or more amino acids within a 7-mer peptide sequence at amino acid positions corresponding to amino acid residues 452-458 of AAV9 VP1 (SEQ ID NO: 1). In some embodiments, the three or more amino acids comprise three consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions in the parent AAV capsid protein corresponding to amino acid residues 452-454, 453-455, 454-456, 455-457, or 456-458 of AAV9 VP1 (SEQ ID NO: 1).In some embodiments, the 7-mer peptide sequence is DGAATKN (SEQ ID NO: 3943). In some embodiments, the variant AAV capsid protein further comprises an amino acid sequence that is at least 96% identical to the amino acid sequence between amino acids 217 and 736 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is not ILGTGTS (SEQ ID NO: 45479), QSSQTPR (SEQ ID NO: 45479), or TLAVPFK (SEQ ID NO: 45477). In some embodiments, the variant AAV capsid protein further comprises an insertion of an amino acid sequence selected from the group consisting of TLAXPFK (SEQ ID NO: 46424), TLAX (SEQ ID NO: 46425), LAVX (SEQ ID NO: 46426), AVPX (SEQ ID NO: 46427), and VPFX (SEQ ID NO: 46428) at an amino acid position corresponding to 588_589 of AAV9 VP1 (SEQ ID NO: 1), wherein X is any amino acid other than V. Embodiments disclosed herein provide variant adeno-associated virus (AAV) capsids comprising: (a) a variant AAV capsid protein comprising an amino acid sequence having amino acid substitutions of three or more amino acids within a 7-mer peptide sequence at amino acid positions corresponding to amino acid residues 452-458 of AAV9 VP1 (SEQ ID NO: 1); and (b) at least one of increased specificity and improved transduction efficiency in a target tissue compared to the corresponding parent AAV capsid protein of SEQ ID NO: 1 or SEQ ID NO: 2, as measured in the target tissue of a subject when delivered intranasally or intravenously to the subject, wherein the target tissue is selected from the group consisting of lung, intestine, kidney, heart, and stomach.In some embodiments, the three or more amino acids include three consecutive amino acids at amino acid positions corresponding to amino acid residues 452-454, 453-455, 454-456, 455-457, or 456-458 of AAV9 VP1, wherein (a) 452 is selected from the group consisting of N, K, R, and T; (b) 453 is selected from the group consisting of L, N, P, and S; (c) 454 is selected from the group consisting of A, D, G, N, S, and T; (d) 455 is selected from the group consisting of L, P, S, and T; (e) 456 is selected from the group consisting of P, R, and S; (f) 457 is selected from the group consisting of G, N, S, and T; and (g) 458 is selected from the group consisting of I, L, and R. In some embodiments, the target tissue is lung, and the three or more amino acids are provided within an amino acid sequence comprising KDNTPGR (SEQ ID NO: 32538), NNLPRNL (SEQ ID NO: 32867), or any amino acid sequence depicted in FIG. 13. In some embodiments, the target tissue is intestine, and the three or more amino acids are provided within an amino acid sequence comprising RESSPSL (SEQ ID NO: 26474), KDNTPGR (SEQ ID NO: 26584), or any amino acid sequence depicted in FIG. 5. In some embodiments, the tissue is kidney, and the three or more amino acids are provided within an amino acid sequence comprising RVPLSTI (SEQ ID NO: 26933), NNLPRNL (SEQ ID NO: 27530), KDNTPGR (SEQ ID NO: 28509), or any amino acid sequence depicted in FIG. 6. In some embodiments, the tissue is heart, and the three or more amino acids are provided within an amino acid sequence comprising KDNTPGR (SEQ ID NO: 25633), or any amino acid sequence depicted in FIG. 4. In some embodiments, the tissue is stomach, and the three or more amino acids are comprised within an amino acid sequence comprising RESSPSL (SEQ ID NO: 31904), or any of the amino acid sequences in Figure 12. In some embodiments, the variant AAV capsid protein further comprises an amino acid sequence that is at least 96% identical to an amino acid sequence between amino acids 217 and 736 of SEQ ID NO: 1. In some embodiments, the variant AAV capsids of the present disclosure are isolated and purified.In some embodiments, the variant AAV capsids of the present disclosure are formulated as pharmaceutical formulations further comprising a pharmaceutically acceptable carrier, hi some embodiments, the pharmaceutical formulations are formulated for intranasal or intravenous administration.

[0014] Disclosed aspects provide a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a disclosed rAAV or a disclosed pharmaceutical formulation. In some embodiments, the disease or condition is selected from the group consisting of pulmonary fibrosis, surfactant protein disorders, peroxisomal biogenesis disorders, chronic obstructive pulmonary disease (COPD), a CNS disease or condition, and a PNS disease or condition.

[0015] Embodiments disclosed herein provide a recombinant adeno-associated virus (rAAV) capsid comprising a variant AAV capsid protein, which has at least one of increased specificity and improved transduction efficiency when measured in target tissues of a primate subject compared to the corresponding parent AAV capsid, and at least one of decreased specificity and reduced transduction efficiency when measured in non-target tissues of a primate subject compared to the corresponding parent AAV capsid. In some cases, the specificity or transduction efficiency is measured after intravenous, intraarterial, intrathecal, or subcutaneous administration of the rAAV capsid to a subject. In some cases, the rAAV capsid is chimeric. In some cases, the rAAV, or the variant AAV protein contained therein, results in increased localization of the rAAV in target tissues compared to the parent AAV capsid or capsid protein.

[0016] In some cases, the variant AAV capsid protein (and rAAV capsid) comprises an amino acid substitution with an amino acid selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at one or more amino acid positions corresponding to one or more of residues 452-458 of the amino acid sequence of the AAV9 VP1 capsid protein set forth in SEQ ID NO: 1. In some cases, the amino acid sequence of the AAV9 capsid protein is set forth in amino acids 217-736 in SEQ ID NO: 1. In some cases, the target tissue is the central nervous system (CNS). In some cases, the CNS comprises a region selected from the neocortex, basal ganglia, hippocampus, thalamus, cerebellum, brainstem, and spinal cord. In some embodiments, the target tissue is the peripheral nervous system (PNS). In some cases, the PNS comprises a ganglion. In some cases, the ganglion comprises a trigeminal ganglion or a dorsal root ganglion. In some embodiments, the target tissue is a non-dividing cell. In some cases, the non-dividing cells are selected from the group consisting of neurons, astrocytes, microglial cells, oligodendrocytes, and Schwann cells. In some cases, the non-target tissue is the liver. In some cases, VP1, VP2, and VP3 of the AAV capsid comprise amino acid substitutions. In some cases, the amino acid substitutions are at the three-fold symmetry axis of the corresponding parent AAV capsid protein. In some cases, the amino acid substitutions comprise at least three consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions corresponding to residues 452-454, 453-455, 454-456, 455-457, or 456-458 of the amino acid sequence of the AAV9 VP1 capsid protein. In some embodiments, the amino acid substitutions comprise at least four consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions corresponding to residues 452-455, 453-456, 454-457, or 455-458 of the amino acid sequence of the AAV9 VP1 capsid protein.In some embodiments, the amino acid substitutions comprise at least five consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions corresponding to residues 452-456, 453-457, or 454-458 of the amino acid sequence of the AAV9 VP1 capsid protein. In some cases, the corresponding parent AAV capsid (or AAV capsid protein) is a native AAV9 capsid. In some cases, the corresponding parent AAV capsid is a variant of AAV9 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6.

[0017] In some cases, the amino acid substitution at the amino acid position corresponding to residue 452 of AAV9 VP1 is selected from the group consisting of N452A, N452D, N452E, N452G, N452H, N452M, N452N, N452Q, N452S, N452T, and N452V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 453 of AAV9 VP1 is selected from the group consisting of G453A, G453D, G453E, G453G, G453K, G453N, G453Q, G453S, G453T, and G453V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 454 of AAV9 VP1 is selected from the group consisting of S454A, S454D, S454E, S454G, S454K, S454N, S454Q, S454S, S454T, and S454V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 455 of AAV9 VP1 is selected from the group consisting of G455A, G455D, G455E, G455G, G455K, G455N, G455P, G455Q, G455S, and G455T. In some cases, the amino acid substitution at the amino acid position corresponding to residue 456 of AAV9 VP1 is selected from the group consisting of Q456A, Q456D, Q456E, Q456G, Q456H, Q456K, Q456N, Q456P, Q456Q, Q456S, and Q456T. In some cases, the amino acid substitution at the amino acid position corresponding to residue 457 of AAV9 VP1 is selected from the group consisting of N457A, N457D, N457E, N457G, N457K, N457N, N457P, N457S, N457T, and N457V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 458 of AAV9 VP1 is selected from the group consisting of Q458A, Q458E, Q458G, Q458H, Q458K, Q458L, Q458N, Q458Q, Q458S, Q458T, and Q458V. In some cases, the amino acid substitution at the amino acid position corresponding to residues 452-458 of AAV9 VP1 is DGAATKN (SEQ ID NO: 3943). In some cases, the amino acid substitution comprises an amino acid sequence selected from Figure 3 or Table 1, or a combination thereof.

[0018] In some cases, the variant AAV capsid protein further comprises an insertion after the amino acid at a position corresponding to residue 588 of the amino acid sequence of AAV9 VP1 of an amino acid sequence selected from the group consisting of TLAXPFK (SEQ ID NO: 46424), TLAX (SEQ ID NO: 46425), LAVX (SEQ ID NO: 46426), AVPX (SEQ ID NO: 46427), and VPFX (SEQ ID NO: 46428), wherein X is any amino acid other than V. In some embodiments, the substitution comprises the amino acid sequence shown in FIG.

[0019] In some cases, the target tissue is the brain, and the rAAV capsid, or variant AAV capsid protein contained therein, has increased brain specificity compared to the parent AAV capsid or capsid protein (e.g., AAV9). In some cases, the non-target tissue is the liver, and the rAAV capsid, or variant AAV capsid protein, has decreased liver specificity compared to the parent AAV capsid or capsid protein (e.g., AAV9). In some cases, the target tissue is the central nervous system (CNS), and the rAAV capsid, or variant AAV capsid protein, has at least a 12-fold increase in viral transduction efficiency into the central nervous system (CNS) compared to the corresponding parent AAV capsid or capsid protein (e.g., AAV9). In some cases, the target tissue is the spinal cord, and the rAAV capsid or variant AVV capsid protein exhibits at least a 20-fold increase in viral transduction efficiency into the spinal cord compared to the corresponding parent AAV capsid or capsid protein. In some cases, the target tissue is a neuron, and the rAAV capsid or variant AVV capsid protein exhibits increased specificity for neurons compared to the corresponding parent AAV capsid or capsid protein. In some cases, the non-target tissue is the liver, and the rAAV capsid or variant AVV capsid protein exhibits at least a 50-fold decrease in viral transduction efficiency into the liver compared to the corresponding parent AAV capsid or capsid protein. In some cases, the non-target tissue is a Purkinje cell, and the rAAV capsid or variant AVV capsid protein exhibits at least a 4-fold decrease in viral transduction efficiency into Purkinje cells compared to the corresponding parent AAV capsid or capsid protein. In some cases, the non-target tissue is the liver, and the rAAV capsid or variant AAV capsid protein has at least 100-fold reduced viral transduction efficiency in the liver compared to the corresponding parent AAV capsid or capsid protein.In some cases, the AAV further comprises a heterologous nucleic acid encoding a therapeutic gene expression product.In some cases, the AAV capsid is isolated and purified.In some cases, the AAV capsid described herein is formulated as a pharmaceutical preparation, further comprising a pharmaceutically acceptable carrier.In some cases, the pharmaceutical preparation further comprises a heterologous nucleic acid encoding a therapeutic gene expression product.In some cases, the pharmaceutical preparation further comprises a pharmaceutical excipient.In some cases, the pharmaceutical preparation is formulated for intravenous, intraarterial, intrathecal or subcutaneous administration to treat CNS or PNS diseases or conditions.

[0020] Embodiments disclosed herein provide rAAV capsids, including variant AAV capsid proteins having an amino acid substitution with an amino acid selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at an amino acid position corresponding to residues 452-458 in the amino acid sequence of AAV9 VP1, wherein the rAAV capsid and variant AAV capsid proteins have at least one of decreased specificity for non-target tissues and decreased transduction efficiency in a subject compared to the corresponding parent AAV capsid or capsid protein, and at least one of increased specificity for target tissues and increased transduction efficiency in a subject compared to the corresponding parent AAV capsid or capsid protein. In some cases, the rAAV capsid is chimeric. In some cases, the amino acid substitution is at the three-fold symmetry axis of the rAAV capsid. In some cases, the rAAV, or the variant AAV proteins contained therein, result in increased localization of the rAAV within target tissues compared to the corresponding parent AAV capsid or capsid proteins.

[0021] In some cases, the target tissue is the central nervous system (CNS) or the peripheral nervous system (PNS). In some cases, the CNS includes a region selected from the neocortex, basal ganglia, hippocampus, thalamus, cerebellum, brainstem, and spinal cord. In some cases, the PNS includes a ganglion. In some cases, the ganglion includes a trigeminal ganglion or a dorsal root ganglion. In some embodiments, the target tissue is a non-dividing cell. In some cases, the CNS includes cells selected from the group consisting of neurons, spinal cord, astrocytes, oligodendrocytes, and Schwann cells. In some cases, the non-target tissue is the liver.

[0022] In some cases, the amino acid substitutions comprise at least three consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions corresponding to residues 452-454, 453-455, 454-456, 455-457, or 456-458 of the amino acid sequence of the AAV9 capsid protein. In some cases, the amino acid substitutions comprise at least four consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions corresponding to residues 452-455, 453-456, 454-457, or 455-458 of the amino acid sequence of the AAV9 capsid protein. In some cases, the amino acid substitutions comprise at least five consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at an amino acid position corresponding to residues 452-456, 453-457, or 454-458 of the amino acid sequence of an AAV9 capsid protein. In some cases, the AAV capsid protein is an AAV9 capsid protein.

[0023] In some cases, the amino acid substitution at the amino acid position corresponding to residue 452 of AAV9 VP1 is selected from the group consisting of N452A, N452D, N452E, N452G, N452H, N452M, N452N, N452Q, N452S, N452T, and N452V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 453 of AAV9 VP1 is selected from the group consisting of G453A, G453D, G453E, G453G, G453K, G453N, G453Q, G453S, G453T, and G453V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 454 of AAV9 VP1 is selected from the group consisting of S454A, S454D, S454E, S454G, S454K, S454N, S454Q, S454S, S454T, and S454V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 455 of AAV9 VP1 is selected from the group consisting of G455A, G455D, G455E, G455G, G455K, G455N, G455P, G455Q, G455S, and G455T. In some cases, the amino acid substitution at the amino acid position corresponding to residue 456 of AAV9 VP1 is selected from the group consisting of Q456A, Q456D, Q456E, Q456H, Q456H, Q456K, Q456N, Q456P, Q456Q, Q456S, and Q456T. In some cases, the amino acid substitution at the amino acid position corresponding to residue 457 of AAV9 VP1 is selected from the group consisting of N457A, N457D, N457E, N457G, N457K, N457N, N457P, N457S, N457T, and N457V. In some cases, the amino acid substitution at the amino acid position corresponding to residue 458 of AAV9 VP1 is selected from the group consisting of Q458A, Q458E, Q458G, Q458H, Q458K, Q458L, Q458N, Q458Q, Q458S, Q458T, and Q458V. In some cases, the amino acid substitution comprises at least three consecutive amino acids in an amino acid sequence selected from the group consisting of DGAATKN (SEQ ID NO: 3943), DGQSSKS (SEQ ID NO: 2764), and an amino acid sequence set forth in Table 1.In some cases, the amino acid sequence of AAV9 VP1 is set forth in SEQ ID NO: 1. In some cases, the amino acid sequence of AAV9 VP1 is set forth in amino acids 217-736 in SEQ ID NO: 1. In some cases, the AAV capsid proteins VP1, VP2, and VP3 comprise amino acid substitutions. In some cases, the corresponding parent AAV capsid or capsid protein is a native AAV9 VP1 capsid. In some cases, the native AAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In some cases, the corresponding parent AAV capsid or capsid protein is a variant of AAV9 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6.

[0024] In some cases, the rAAV capsid, or variant AAV capsid protein, further comprises an insertion of an amino acid sequence selected from the group consisting of TLAXPFK (SEQ ID NO: 46424), TLAX (SEQ ID NO: 46425), LAVX (SEQ ID NO: 46426), AVPX (SEQ ID NO: 46427), and VPFX (SEQ ID NO: 46428) at amino acid positions 588-589 within the amino acid sequence of the AAV capsid protein, wherein X is any amino acid other than V.

[0025] In some cases, the target tissue is the brain, and the rAAV capsid or variant AVV capsid protein has increased brain specificity compared to the corresponding parent AAV capsid or capsid protein. In some cases, the non-target tissue is the liver, and the rAAV capsid or variant AVV capsid protein has decreased liver specificity compared to the corresponding parent AAV capsid or capsid protein. In some cases, the target tissue is the central nervous system (CNS), and the rAAV capsid or variant AVV capsid protein has at least a 12-fold increase in viral transduction efficiency into the CNS compared to the corresponding parent AAV capsid or capsid protein. In some cases, the target tissue is the spinal cord, and the rAAV capsid or variant AVV capsid protein has at least a 20-fold increase in viral transduction efficiency into the spinal cord compared to the corresponding parent AAV capsid or capsid protein. In some cases, the target tissue is a neuron, and the rAAV capsid or variant AVV capsid protein has increased neuronal specificity compared to the corresponding parent AAV capsid or capsid protein. In some cases, the non-target tissue is the liver, and the rAAV capsid or variant AVV capsid protein has at least a 50-fold reduction in viral transduction efficiency in the liver compared to the corresponding parent AAV capsid or capsid protein. In some cases, the non-target tissue is a Purkinje cell, and the rAAV capsid or variant AVV capsid protein has at least a 4-fold reduction in viral transduction efficiency in Purkinje cells compared to the corresponding parent AAV capsid or capsid protein. In some cases, the off-target tissue is the liver, and the rAAV capsid, or variant AVV capsid protein, has at least a 100-fold reduction in viral transduction efficiency in the liver compared to the corresponding parent AAV capsid or capsid protein. In some cases, the rAAV capsid, or variant AVV capsid protein described herein is isolated and purified.In some cases, the rAAV capsid or variant capsid protein described herein is formulated as a pharmaceutical preparation, further comprising a pharmaceutically acceptable carrier. In some cases, the pharmaceutical preparation further comprises a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the pharmaceutical preparation further comprises a pharmaceutical excipient. In some cases, the pharmaceutical preparation is formulated for intravenous, intraarterial, intrathecal or subcutaneous administration to treat CNS or PNS diseases or conditions.

[0026] Embodiments disclosed herein provide a recombinant AAV (rAAV) capsid comprising a variant AAV capsid protein comprising at least four consecutive amino acids at an amino acid position corresponding to residues selected from the group consisting of 452-455, 453-456, 454-457, and 455-458 of the amino acid sequence of an AAV9 capsid protein according to VP1 numbering, which confers at least one of increased specificity for a target tissue and improved transduction efficiency in a subject, wherein (a) the amino acid at the amino acid position corresponding to residue 452 is selected from the group consisting of A, D, G, L, N, Q, S, and T, and (b) the amino acid at the amino acid position corresponding to residue 453 is selected from the group consisting of A, G, N, P, Q, R, S, and T. (c) the amino acid at the amino acid position corresponding to residue 454 is selected from the group consisting of A, D, G, N, S, and T; (d) the amino acid at the amino acid position corresponding to residue 455 is selected from the group consisting of A, D, G, K, N, P, Q, S, and T; (e) the amino acid at the amino acid position corresponding to residue 456 is selected from the group consisting of A, G, K, N, P, R, S, and T; (f) the amino acid at the amino acid position corresponding to residue 457 is selected from the group consisting of A, G, K, N, P, R, S, T, and V; and (g) the amino acid at the amino acid position corresponding to residue 458 is selected from the group consisting of A, G, K, L, R, S, T, and V. In some cases, the AAV capsid protein does not contain the amino acid sequence ILGTGTS (SEQ ID NO: 45479), QSSQTPR (SEQ ID NO: 45479), or TLAVPFK (SEQ ID NO: 45477) at amino acid positions corresponding to residues selected from the group consisting of 452-455, 453-456, 454-457, and 455-458 of AAV9 VP1. In some cases, the amino acid substitution is at a three-fold symmetry axis of the rAAV capsid. In some cases, the specificity or transduction efficiency is measured after systemic administration of the rAAV capsid to a subject. In some cases, systemic administration includes intra-arterial, intravenous, or subcutaneous administration.In some cases, the rAAV capsid, or variant AAV capsid protein therein, is from AAV 9. In some cases, the rAAV, or variant AAV protein contained therein, results in increased localization of the rAAV in target tissues compared to the corresponding parent AAV capsid or capsid protein.

[0027] In some cases, the substitution comprises an amino acid sequence depicted in Figure 3. In some such cases, at least four consecutive amino acids are provided within an amino acid sequence selected from the group consisting of LQTSSPG (SEQ ID NO:2933), SINTKTN (SEQ ID NO:45475), ERGANTK (SEQ ID NO:5192), TTGGHSS (SEQ ID NO:2743), GTTKTSE (SEQ ID NO:3064), GTGTSVL (SEQ ID NO:11958), and GTPSKAG (SEQ ID NO:2741). In some cases, the substitution comprises an amino acid sequence depicted in Figure 2. In some such cases, at least four consecutive amino acids are provided within an amino acid sequence selected from the group consisting of QQGKQSV (SEQ ID NO:79), SNGTKQT (SEQ ID NO:442), GSGKTAA (SEQ ID NO:88), MGDKPTR (SEQ ID NO:2466), QPSGGNT (SEQ ID NO:2672), and NQSGTKG (SEQ ID NO:780). In some embodiments, the at least four contiguous amino acids are comprised within an amino acid sequence selected from the group consisting of LQTSSPG (SEQ ID NO:2933), QQGKQSV (SEQ ID NO:79), SINTKTN (SEQ ID NO:45475), SNGTKQT (SEQ ID NO:442), GSGKTAA (SEQ ID NO:88), MGDKPTR (SEQ ID NO:2466), QPSGGNT (SEQ ID NO:2672), ERGANTK (SEQ ID NO:5192), TTGGHSS (SEQ ID NO:2743), GTTKTSE (SEQ ID NO:3064), GTGTSVL (SEQ ID NO:11958), NQSGTKG (SEQ ID NO:780), and GTPSKAG (SEQ ID NO:2741). In some cases, the at least four contiguous amino acids are comprised within an amino acid sequence set forth in Table 2. In some cases, the target tissue is the central nervous system (CNS). In some cases, the CNS includes a region selected from the neocortex, basal ganglia, hippocampus, thalamus, cerebellum, brainstem, and spinal cord. In some cases, the target tissue is the peripheral nervous system (PNS). In some cases, the PNS includes a ganglion. In some cases, the ganglion is a trigeminal ganglion or a dorsal root ganglion. In some cases, the target tissue is a non-dividing cell. In some cases, the CNS includes a cell selected from the group consisting of a neuron, an astrocyte, a microglial cell, an oligodendrocyte, and a Schwann cell.In some cases, the target tissue is the CNS, and the AAV capsid has at least a 12-fold increase in viral transduction efficiency into the CNS compared to the parent AAV capsid or AAV capsid protein. In some cases, the target tissue is the brain, and the AAV capsid has increased specificity for the brain compared to the parent AAV capsid or AAV capsid protein. In some cases, the target tissue is the spinal cord, and the AAV capsid has at least a 20-fold increase in viral transduction efficiency into the spinal cord compared to the parent AAV capsid or AAV capsid protein. In some cases, the target tissue is a neuron, and the AAV capsid has increased specificity for neurons compared to the parent AAV capsid or AAV capsid protein. In some cases, the rAAV capsid or variant AAV capsid protein described herein is isolated and purified. In some cases, the rAAV capsid or variant AAV protein described herein is formulated as a pharmaceutical preparation, further comprising a pharmaceutically acceptable carrier. In some cases, the pharmaceutical preparation further comprises a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the pharmaceutical preparation further comprises a pharmaceutical excipient. In some cases, the pharmaceutical preparation is formulated for intravenous, intraarterial, intrathecal or subcutaneous administration to treat CNS or PNS diseases or conditions.

[0028] Embodiments disclosed herein provide a rAAV capsid comprising an AAV capsid protein comprising at least four consecutive amino acids from the amino acid sequence shown in Figure 2 or Table 2 at amino acid positions corresponding to residues selected from the group consisting of 452-455, 453-456, 454-457, and 455-458 of AAV9 VP1, wherein the rAAV capsid provides at least one of increased specificity and improved transduction efficiency as measured in a target tissue in a subject compared to the parent AAV capsid or capsid protein. In some cases, the at least four consecutive amino acids are at a three-fold symmetry axis of the rAAV capsid. In some cases, the AAV capsid protein does not contain the amino acid sequence ILGTGTS (SEQ ID NO: 45479), QSSQTPR (SEQ ID NO: 45479), or TLAVPFK (SEQ ID NO: 45477) at amino acid positions corresponding to residues selected from the group consisting of 452-455, 453-456, 454-457, and 455-458 of AAV9 VP1. In some cases, the specificity or transduction efficiency is measured after systemic administration of the AAV capsid to a subject. In some cases, the rAAV capsid is chimeric. In some cases, the rAAV, or a variant AAV protein contained therein, results in increased localization of the rAAV in target tissues compared to the parent AAV capsid or capsid protein.

[0029] In some cases, the at least four consecutive amino acids include at least 4, 5, 6, or 7 consecutive amino acids in an amino acid sequence selected from the group consisting of LQTSSPG (SEQ ID NO: 2933), QQGKQSV (SEQ ID NO: 79), SINTKTN (SEQ ID NO: 45475), SNGTKQT (SEQ ID NO: 442), GSGKTAA (SEQ ID NO: 88), MGDKPTR (SEQ ID NO: 2466), QPSGGNT (SEQ ID NO: 2672), ERGANTK (SEQ ID NO: 5192), TTGGHSS (SEQ ID NO: 2743), GTTKTSE (SEQ ID NO: 3064), GTGTSVL (SEQ ID NO: 11958), NQSGTKG (SEQ ID NO: 780), and GTPSKAG (SEQ ID NO: 2741).

[0030] In some cases, the target tissue is the central nervous system (CNS). In some cases, the CNS includes a region selected from the neocortex, basal ganglia, hippocampus, thalamus, cerebellum, brainstem, and spinal cord. In some embodiments, the target tissue is the peripheral nervous system (PNS). In some cases, the PNS includes a ganglion. In some cases, the ganglion is a trigeminal ganglion or a dorsal root ganglion. In some cases, the target tissue is a non-dividing cell. In some cases, the CNS includes a cell selected from the group consisting of a neuron, an astrocyte, a microglial cell, an oligodendrocyte, and a Schwann cell.

[0031] In some cases, the rAAV capsid, or the variant AAV capsid protein contained therein, further comprises an insertion of an amino acid sequence selected from the group consisting of TLAXPFK (SEQ ID NO: 46424), TLAX (SEQ ID NO: 46425), LAVX (SEQ ID NO: 46426), AVPX (SEQ ID NO: 46427), and VPFX (SEQ ID NO: 46428) at an amino acid position corresponding to residues 588-589 of the amino acid sequence of AAV9 VP1, wherein X is any amino acid other than V. In some cases, the parent AAV capsid or capsid protein is AAV9. In some cases, the amino acid sequence of the AAV9 capsid protein is set forth in SEQ ID NO: 1. In some cases, the amino acid sequence of the AAV9 capsid protein is set forth in amino acids 217-736 of SEQ ID NO: 1.

[0032] In some cases, the target tissue is the brain, and the rAAV capsid or variant AAV capsid protein has increased brain specificity compared to the parent AAV capsid or capsid protein. In some cases, the target tissue is the CNS, and the rAAV capsid or variant AAV capsid protein has at least a 12-fold increase in viral transduction efficiency into the CNS compared to the parent AAV capsid or capsid protein. In some cases, the target tissue is the spinal cord, and the rAAV capsid or variant AAV capsid protein has at least a 20-fold increase in viral transduction efficiency into the spinal cord compared to the parent AAV capsid or capsid protein. In some cases, the target tissue is a neuron, and the rAAV capsid or variant AAV capsid protein has increased neuron specificity compared to the parent AAV capsid or capsid protein. In some cases, the rAAV capsid or variant AAV capsid protein described herein is isolated and purified.In some cases, the rAAV capsid or variant AAV capsid protein described herein is formulated as a pharmaceutical preparation, which further comprises a pharmaceutically acceptable carrier.In some cases, the pharmaceutical preparation further comprises a heterologous nucleic acid encoding a therapeutic gene expression product.In some cases, the pharmaceutical preparation further comprises a pharmaceutical excipient.

[0033] Embodiments provided herein provide rAAV capsids comprising a variant AAV capsid protein and having at least one of increased specificity and improved transduction efficiency when measured in a target tissue of a subject compared to a corresponding parental AAV capsid or capsid protein, wherein the AAV capsid protein comprises three amino acids (X1-X2-X3) at amino acid positions corresponding to residues selected from the group consisting of 452-454, 453-455, 454-456, 455-457, and 456-458 of AAV9 VP1, wherein (a) X1 is selected from the group consisting of N, K, R, L, and T, (b) X2 is selected from the group consisting of N, S, L, E, D, and P, and (c) X3 is selected from the group consisting of N, S, A, D, G, L, and T. In some cases, the target tissue is selected from the group consisting of lung, heart, intestine, kidney, and stomach. In some cases, the target tissue is a non-dividing cell. In some cases, the tissue is lung, and the amino acid sequence is KDNTPGR (SEQ ID NO: 32538), or any amino acid sequence shown in FIG. 13. In some cases, the tissue is lung, and the amino acid sequence is NNLPRNL (SEQ ID NO: 32867), or any amino acid sequence shown in FIG. 13. In some cases, the tissue is intestine, and the amino acid sequence is RESSPSL (SEQ ID NO: 29065), or any amino acid sequence shown in FIG. 5. In some cases, the tissue is kidney, and the amino acid sequence is RVPLSTI (SEQ ID NO: 26933), or any amino acid sequence shown in FIG. 6. In some cases, the AAV capsid protein is an AAV9 capsid protein. In some cases, the AAV9 capsid protein is shown in SEQ ID NO: 1. In some cases, the AAV capsid protein further comprises four amino acids at amino acid positions selected from the group consisting of 452-455, 453-456, 454-457, and 455-458, where X4 is selected from the group consisting of T, S, P, and L. In some cases, the AAV capsid protein further comprises five amino acids at amino acid positions selected from the group consisting of 452-456, 453-457, and 453-458, where X5 is selected from the group consisting of P, R, and S.In some cases, the AAV capsid protein further comprises six amino acids at amino acid positions 452-457 or 453-458, where X6 is selected from the group consisting of N, G, S, and T. In some cases, the AAV capsid protein further comprises seven amino acids at amino acid positions 452-458, where X7 is selected from the group consisting of R, L, and I. In some cases, the AAV capsid proteins VP1, VP2, and VP3 comprise the amino acid sequences. In some cases, the corresponding parent AAV capsid is a native AAV9 capsid. In some cases, the amino acid sequence of the VP1 protein of a native AAV9 capsid is set forth in SEQ ID NO: 1. In some cases, the corresponding parent AAV is a native AAV5 capsid. In some cases, the amino acid sequence of the VP1 protein of a native AAV5 capsid is set forth in SEQ ID NO: 2.

[0034] In some cases, the rAAV or variant AAV capsid protein described herein has at least one of increased specificity for a target tissue and improved transduction efficiency when measured in a subject compared to the corresponding parent AAV capsid or capsid protein. In some cases, the target tissue is a lung cell type that is an alveolar type II epithelial (ATII) cell, and the rAAV capsid or variant AAV capsid has at least 60-fold improved viral transduction efficiency into ATII cells compared to the parent AAV capsid or capsid protein. In some cases, the target tissue is a lung cell type that is an ATII cell, and the rAAV capsid or variant AAV capsid has at least 60-fold improved viral transduction efficiency into ATII cells compared to the parent AAV capsid or capsid protein. In some cases, the rAAV capsid or variant AAV capsid has at least 15-fold improved lung viral transduction efficiency compared to the parent AAV capsid or capsid protein. In some cases, the rAAV capsid or variant AAV capsid has at least 30-fold improved lung viral transduction efficiency compared to the parent AAV capsid or capsid protein. AAV capsids comprising these AAV capsid proteins are also envisioned. In some cases, the rAAV capsid or variant AAV capsid described herein is isolated and purified. In some cases, the rAAV capsid or variant AAV capsid described herein is formulated as a pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some cases, the pharmaceutical formulation further comprises a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the pharmaceutical formulation further comprises a pharmaceutical excipient. In some cases, the pharmaceutical preparation is formulated for intravenous, intraarterial, subcutaneous, or intranasal administration to treat a disease or condition of a target tissue.

[0035] Embodiments disclosed herein provide an rAAV capsid comprising a variant AAV capsid protein comprising three amino acids at amino acid positions corresponding to residues selected from the group consisting of 452-454, 453-455, 454-456, 455-457, and 456-458 of AAV9 VP1, wherein the three amino acids are provided within the amino acid sequence provided in one or more of Figures 4-14. In some cases, the rAAV capsid or variant AAV capsid protein has at least one of increased specificity and improved transduction efficiency when measured in a target tissue in a subject. In some cases, the target tissue is selected from the group consisting of lung, intestine, liver, stomach, heart, muscle, adipose tissue, spleen, kidney, or reproductive organs (e.g., testes, ovaries). In some cases, the target tissue is a non-dividing cell. In some cases, the amino acid sequence is shown in Figure 4. In some cases, the amino acid sequence is shown in Figure 5. In some cases, the amino acid sequence is shown in Figure 6. In some cases, the amino acid sequence is shown in Figure 7. In some cases, the amino acid sequence is shown in Figure 8. In some cases, the amino acid sequence is shown in Figure 9. In some cases, the amino acid sequence is shown in Figure 10. In some cases, the amino acid sequence is shown in Figure 11. In some cases, the amino acid sequence is shown in Figure 12. In some cases, the amino acid sequence is shown in Figure 13. In some cases, the amino acid sequence is shown in Figure 14. In some embodiments, the rAAV, or the variant AAV protein contained therein, results in increased localization of the rAAV in target tissues compared to the parent AAV capsid or capsid protein.

[0036] In some cases, the parent AAV capsid is native AAV9. In some cases, the amino acid sequence of the VP1 protein of the native AAV9 capsid protein is set forth in SEQ ID NO:1.

[0037] In some cases, the three amino acids further comprise four amino acids at amino acid positions corresponding to residues selected from the group consisting of 452-455, 453-456, 454-457, and 455-458 of AAV9 VP1, whose amino acid sequences are shown in one or more of Figures 4-14. In some cases, the three amino acids further comprise five amino acids at amino acid positions corresponding to residues selected from the group consisting of 452-456, 453-457, and 453-458 of AAV9 VP1, whose amino acid sequences are shown in one or more of Figures 4-14. In some cases, the three amino acids further comprise six amino acids at amino acid positions corresponding to residues 452-457 or 453-458, whose amino acid sequences are shown in one or more of Figures 4-14. In some cases, the three amino acids further comprise seven amino acids at amino acid positions corresponding to residues 452-458, the amino acid sequences of which are shown in one or more of Figures 4-14. In some cases, VP1, VP2, and VP3 of the rAAV capsid comprise the amino acid sequence: In some cases, the rAAV capsid, or variant AAV capsid protein described herein, is isolated and purified. In some cases, the AAV capsid, or variant AAV capsid protein described herein, is formulated as a pharmaceutical formulation further comprising a pharmaceutically acceptable carrier. In some cases, the pharmaceutical formulation further comprises a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the pharmaceutical formulation further comprises a pharmaceutical excipient. In some cases, the pharmaceutical formulation is formulated for intravenous, intraarterial, subcutaneous, or intranasal administration to treat a disease or condition of a target tissue.

[0038] Aspects disclosed herein provide a method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a rAAV capsid disclosed herein, wherein the rAAV capsid encapsulates a nucleic acid sequence encoding a therapeutic gene expression product. In some cases, the mammalian subject is a primate. In some cases, the mammalian subject is a non-human primate. In some cases, the mammalian subject is a human. In some cases, the therapeutic gene expression product is a protein. In other cases, the therapeutic gene expression product is RNA, such as a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or a microRNA (miRNA). In some cases, the administering step comprises intravenous or intranasal administration. In some cases, the disease or condition is a disease or condition of the central nervous system or peripheral nervous system. In some cases, the disease or condition is a disease or condition of the CNS or PNS. In some cases, the disease or condition may be adrenoleukodystrophy, Alzheimer's disease, amyotrophic lateral sclerosis, Angelman syndrome, ataxia-telangiectasia, Charcot-Marie-Tooth syndrome, classic radicular chondrodysplasia punctata (RCDP), Cockayne syndrome, hearing loss, Dravet syndrome, Duchenne muscular dystrophy, epilepsy, essential tremor, Fragile X syndrome, Friedreich's ataxia, frontotemporal dementia (FTD), Gaucher disease, glioblastoma, Huntington's disease, infantile Refsum disease ( IRD), Lesch-Nyhan syndrome, maple syrup urine disease, Menkes syndrome, myotonic dystrophy, narcolepsy, neurofibromatosis, Niemann-Pick disease, Parkinson's disease, phenylketonuria, Prader-Willi syndrome, Refsum disease, Rett syndrome, spinal muscular atrophy, spinocerebellar ataxia, Tangier disease, Tay-Sachs disease, tuberous sclerosis complex, von Hippel-Lindau syndrome, Williams syndrome, Wilson's disease, and Zellweger syndrome.In some cases, the therapeutic gene expression product may be sarcoglycan alpha (SGCA), glutamic acid decarboxylase 65 (GAD65), glutamic acid decarboxylase 67 (GAD67), CLN2, nerve growth factor (NGF), survival motor neuron 1, telomere type (SMN1), factor X (FIX), retinoid isomerohydrolase (RPE65), sarcoplasmic / endoplasmic reticulum Ca2+-ATPase (SERCA2a), β-glucocerebrosidase (GCase), frataxin ( The therapeutic gene expression product is effective to modulate the activity or expression of a target gene or gene expression product selected from the group consisting of: FXN, huntingtin (HTN), methyl-CpG-binding protein 2 (MECP2), peroxisome biogenesis factor (PEX), progranulin (GRN), antitubulin, copper-zinc superoxide dismutase (SOD1), glucosylceramidase beta (GBA), NPC intracellular cholesterol transporter 1 (NPC1), and NLRP3 inflammasome. In some cases, the therapeutic gene expression product comprises a gene editing component. In some cases, the gene editing component is selected from the group consisting of artificial site-specific RNA endonucleases (ASREs), zinc finger endonucleases (ZFNs), transcription factor-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) / Cas enzymes, and CRISPR / Cas guide RNAs.

[0039] Embodiments disclosed herein include a plasmid vector comprising a nucleic acid sequence encoding an AAV capsid described herein. In some cases, the plasmid vector is bacterial. In some cases, the plasmid vector is derived from Escherichia coli. In some cases, the nucleic acid sequence comprises, from 5' to 3', (1) a 5' inverted terminal repeat (ITR) sequence, (2) a replication (Rep) gene, (3) a capsid (Cap) gene, and (4) a 3' ITR, wherein the Cap gene encodes an AAV capsid protein described herein. In some cases, the plasmid vector encodes a pseudotyped AAV capsid protein. In some cases, the Cap gene is derived from a deoxyribose nucleic acid (DNA) sequence set forth in any one of SEQ ID NOs: 6-10. In some cases, the nucleic acid sequence comprising the Cap gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the DNA sequences set forth in SEQ ID NOs: 46364-46383. In some cases, the 5'ITR and 3'ITR are derived from the AAV2 serotype. In some cases, the 5'ITR and 3'ITR are derived from the AAV5 serotype. In some cases, the 5'ITR and 3'ITR are derived from the AAV9 serotype.

[0040] Embodiments disclosed herein provide a method of production, comprising: (a) introducing into a cell a nucleic acid comprising: (i) a first nucleic acid sequence encoding a therapeutic gene expression product, the first nucleic acid sequence being flanked at its 5' and 3' ends by inverted terminal repeat (ITR) sequences; (ii) a second nucleic acid sequence encoding a viral genome comprising the 5' ITR sequence, a replication (Rep) gene, a capsid (Cap) gene, and the 3' ITR, wherein the Cap gene encodes an AAV capsid protein described herein; and (iii) a third nucleic acid sequence encoding a first helper virus protein selected from the group consisting of E4orf6, E2a, and VA RNA, and optionally encoding a second helper virus protein comprising E1a or E1b55k; (b) expressing the AAV capsid proteins described herein in the cell; (c) constructing AAV particles comprising the AAV capsid proteins disclosed herein; and (d) packaging the first nucleic acid sequence into the AAV particles. In some cases, the nucleic acid is contained in a plasmid and the cell is a mammalian cell. In some cases, the cell is an immortalized cell. In some cases, the immortalized cell is an embryonic stem cell. In some cases, the embryonic stem cell is a human embryonic stem cell. In certain cases, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. In other cases, the nucleic acid is contained in a virus, such as a baculovirus. In some such cases, the cell is an insect cell. In certain such cases, the cell is an SF9 cell. In some cases, the Cap gene is derived from a deoxyribose nucleic acid (DNA) set forth in any one of SEQ ID NOs: 6-10. In some cases, the nucleic acid sequence comprising the Cap gene is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the DNA sequences set forth in SEQ ID NOs: 46364-46383. In some cases, the 5'ITR and 3'ITR are derived from the AAV2 serotype. In some cases, the 5'ITR and 3'ITR are derived from the AAV5 serotype. In some cases, the 5'ITR and 3'ITR are derived from the AAV9 serotype.In some cases, the first nucleic acid sequence (encoding the therapeutic gene expression product) and the second nucleic acid sequence (encoding the cap gene) are in a trans relationship. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are in a cis relationship. In some cases, the first nucleic acid sequence, the second nucleic acid sequence, and the third nucleic acid sequence (encoding the helper virus) are in a trans relationship.

[0041] Embodiments disclosed herein provide kits including: (a) a first vector including, in a 5' to 3' direction, a first nucleic acid sequence encoding a viral genome, the first nucleic acid sequence including: (i) a 5' inverted terminal repeat (ITR) sequence; (ii) a replication (Rep) gene; (iii) a capsid (Cap) gene encoding an AAV capsid protein described herein; and (iv) a 3' ITR; and (b) optionally, a second vector including a second nucleic acid sequence encoding a helper virus protein, the second nucleic acid sequence including at least one of E4orf6, E2a, VA RNA, E1a, and E1b55k. In some cases, the kit further includes a cell. In some cases, the cell is a mammalian cell. In some cases, the cell is an immortalized cell. In some cases, the immortalized cell is an embryonic stem cell. In some cases, the embryonic stem cell is a human embryonic stem cell. In some cases, the human embryonic stem cell is a human embryonic kidney 293 (HEK-293) cell. In some cases, the kit further comprises a vector comprising a heterologous nucleic acid encoding a therapeutic gene expression product. In some cases, the vector is episomal. In certain embodiments, for example, the following are provided: (Item 1) a) a variant AAV capsid comprising a variant AAV capsid protein comprising an amino acid substitution of three or more amino acids within a 7-mer peptide sequence at the three-fold axis of symmetry of a corresponding parent AAV capsid protein, wherein the 7-mer peptide sequence is at an amino acid position corresponding to amino acid residues 452 to 458 of AAV9 VP1 (SEQ ID NO: 1), and the amino acid substitution is not ILGTGTS (SEQ ID NO: 45479), QSSQTPR (SEQ ID NO: 45479), or TLAVPFK (SEQ ID NO: 45477); and b) a heterologous polynucleotide comprising a nucleotide sequence encoding a gene product; Recombinant AAV (rAAV) containing (Item 2) 2. The rAAV of item 1, wherein the variant AAV capsid has increased tropism for a target tissue or target cell when measured in a subject compared to the tropism of the corresponding parent AAV capsid. (Item 3) The rAAV of any preceding item, wherein the target tissue or target cell comprises a tissue or cell of the central nervous system (CNS) or peripheral nervous system (PNS), or a combination thereof. (Item 4) The rAAV of any preceding item, wherein the variant AAV capsid has reduced tropism for non-target tissues, including liver tissue, or non-target cells, including liver cells, when measured in a subject, compared to the tropism of the corresponding parent AAV capsid. (Item 5) The rAAV of any preceding item, wherein the three or more amino acids comprise three consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V, at amino acid positions in the parent AAV capsid protein corresponding to amino acid residues 452-454, 453-455, 454-456, 455-457, or 456-458 of AAV9 VP1 (SEQ ID NO: 1). (Item 6) The rAAV of any preceding item, wherein the three or more amino acids comprise four consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V, at amino acid positions in the parent AAV capsid protein corresponding to amino acid residues 452-455, 453-456, 454-457, or 455-458 of AAV9 VP1 (SEQ ID NO: 1). (Item 7) The rAAV of any preceding item, wherein the three or more amino acids comprise at least five consecutive amino acids, each independently selected from the group consisting of A, D, E, G, K, H, M, N, P, LQ, S, T, and V at amino acid positions in the parent AAV capsid protein corresponding to amino acid residues 452-456, 453-457, or 454-458 of AAV9 VP1 (SEQ ID NO: 1). (Item 8) the three or more amino acids being a) a substituted A, D, E, G, H, M, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 452 of AAV9 VP1 (SEQ ID NO: 1); b) a substituted A, D, E, G, K, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 453 of AAV9 VP1 (SEQ ID NO: 1); c) a substituted A, D, E, G, K, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 454 of AAV9 VP1 (SEQ ID NO: 1); d) a substituted A, D, E, G, K, N, P, Q, S, or T at the amino acid position corresponding to amino acid residue 455 of AAV9 VP1 (SEQ ID NO: 1); e) a substituted A, D, E, G, H, K, N, P, Q, S, or T at the amino acid position corresponding to amino acid residue 456 of AAV9 VP1 (SEQ ID NO: 1); f) a substituted A, D, E, G, K, N, P, S, T, or V at the amino acid position corresponding to amino acid residue 457 of AAV9 VP1 (SEQ ID NO: 1), and g) a substituted A, E, G, H, K, L, N, Q, S, T, or V at the amino acid position corresponding to amino acid residue 458 of AAV9 VP1 (SEQ ID NO: 1). The rAAV of any preceding item, comprising three or more of: (Item 9) The rAAV of any preceding item, wherein the amino acid substitution comprises an amino acid sequence selected from DGAATKN (SEQ ID NO: 3943), and DGQSSKS (SEQ ID NO: 2764). (Item 10) the three or more amino acids being a) a substituted A, D, G, L, N, Q, S, or T at the amino acid at the position corresponding to amino acid residue 452 of AAV9 VP1 (SEQ ID NO: 1); b) a substituted A, G, N, P, Q, R, S at the amino acid at the position corresponding to amino acid residue 453 of AAV9 VP1 (SEQ ID NO: 1); or c) a substituted A, D, G, N, S, or T at the amino acid at the position corresponding to amino acid residue 454 of AAV9 VP1 (SEQ ID NO: 1); d) a substituted A, D, G, K, N, P, Q, S, or T at the amino acid at the position corresponding to amino acid residue 455 of AAV9 VP1 (SEQ ID NO: 1); e) a substituted A, G, K, N, P, R, S, or T at the amino acid at the position corresponding to amino acid residue 456 of AAV9 VP (SEQ ID NO: 1); f) a substituted A, G, K, N, P, R, S, T, or V at the amino acid at the position corresponding to amino acid residue 457 of AAV9 VP1 (SEQ ID NO: 1); and g) a substituted A, G, K, L, R, S, T, or V at the amino acid at the position corresponding to amino acid residue 458 of AAV9 VP1 (SEQ ID NO: 1); 5. The rAAV of item 4, comprising three or more of: (Item 11) The rAAV of any preceding item, wherein the amino acid substitution comprises an amino acid sequence selected from the group consisting of LQTSSPG (SEQ ID NO: 2933), QQGKQSV (SEQ ID NO: 79), SINTKTN (SEQ ID NO: 45475), SNGTKQT (SEQ ID NO: 442), GSGKTAA (SEQ ID NO: 88), MGDKPTR (SEQ ID NO: 2466), QPSGGNT (SEQ ID NO: 2672), ERGANTK (SEQ ID NO: 5192), TTGGHSS (SEQ ID NO: 2743), GTTKTSE (SEQ ID NO: 3064), GTGTSVL (SEQ ID NO: 11958), NQSGTKG (SEQ ID NO: 780), KGPGQMG (SEQ ID NO: 45476), and GTPSKAG (SEQ ID NO: 2741). (Item 12) 12. The rAAV of any one of items 2 to 11, wherein the target tissue or the target cell within the tissue is selected from the group consisting of lung, intestine, kidney, heart, and stomach. (Item 13) the three or more amino acids include three consecutive amino acids at amino acid positions corresponding to amino acid residues 452 to 454, 453 to 455, 454 to 456, 455 to 457, or 456 to 458 of AAV9 VP1; a) 452 is selected from the group consisting of N, K, R and T; b) 453 is selected from the group consisting of L, N, P and S; c) 454 is selected from the group consisting of A, D, G, N, S and T; d) 455 is selected from the group consisting of L, P, S and T; e) 456 is selected from the group consisting of P, R and S; f) 457 is selected from the group consisting of G, N, S and T, and g) 458 is selected from the group consisting of I, L and R; The rAAV according to any of the preceding items. (Item 14) the three or more amino acids being a) four consecutive amino acids at amino acid positions corresponding to amino acid residues 452 to 455, 453 to 456, 454 to 457, or 455 to 458 of AAV9 VP1 (SEQ ID NO: 1); b) 5 consecutive amino acids at amino acid positions corresponding to amino acid residues 452 to 456, 453 to 457, or 453 to 458 of AAV9 VP1 (SEQ ID NO: 1); c) 6 consecutive amino acids at amino acid positions corresponding to amino acid residues 452 to 457 or 453 to 458 of AAV9 VP1 (SEQ ID NO: 1), and d) 7 consecutive amino acids at amino acid positions corresponding to amino acid residues 452 to 458 of AAV9 VP1 (SEQ ID NO: 1) The rAAV of any preceding item, comprising: (Item 15) 15. The rAAV of any one of paragraphs 12 to 14, wherein the target tissue is lung and the three or more amino acids are provided within an amino acid sequence comprising KDNTPGR (SEQ ID NO: 32538), NNLPRNL (SEQ ID NO: 32867), or any amino acid sequence shown in FIG. 13. (Item 16) 15. The rAAV of any one of paragraphs 12 to 14, wherein the target tissue is the intestine and the three or more amino acids are provided within an amino acid sequence comprising RESSPSL (SEQ ID NO: 26474), KDNTPGR (SEQ ID NO: 26584), or any amino acid sequence shown in FIG. 5. (Item 17) 15. The rAAV of any one of paragraphs 12 to 14, wherein the tissue is kidney and the three or more amino acids are provided within an amino acid sequence comprising RVPLSTI (SEQ ID NO: 26933), NNLPRNL (SEQ ID NO: 27530), KDNTPGR (SEQ ID NO: 28509), or any amino acid sequence shown in FIG. (Item 18) 15. The rAAV of any one of paragraphs 12 to 14, wherein the tissue is heart and the three or more amino acids are provided within an amino acid sequence comprising KDNTPGR (SEQ ID NO: 25633), or any amino acid sequence shown in FIG. 4. (Item 19) 15. The rAAV of any one of paragraphs 12 to 14, wherein the tissue is stomach and the three or more amino acids are provided within an amino acid sequence comprising RESSPSL (SEQ ID NO: 31904), or any amino acid sequence in FIG. 12. (Item 20) The rAAV of any preceding item, wherein the corresponding parent AAV capsid protein is AAV9 VP1 or a variant thereof. (Item 21) 21. The rAAV of item 20, wherein the AAV9 VP1 variant has 90% or more sequence identity to SEQ ID NO:1. (Item 22) 21. The rAAV of item 20, wherein the AAV9 VP1 variant has 95% or more sequence identity to SEQ ID NO:1. (Item 23) 46426), AVPX (SEQ ID NO: 46427), and VPFX (SEQ ID NO: 46428) at an amino acid position corresponding to 588-589 of the AAV9 VP1 (SEQ ID NO: 1), wherein X is any amino acid other than V. (Item 24) The rAAV of any of the preceding items, wherein the parent AAV capsid protein is from AAV-PHP.B or AAV-PHP.eB. (Item 25) The rAAV of any preceding item, which is isolated and purified. (Item 26) A pharmaceutical formulation comprising the rAAV described in any of the preceding items and a pharmaceutical excipient. (Item 27) 27. The pharmaceutical preparation according to item 26, formulated for intravenous, intraarterial, intranasal, intrathecal, intracisternal or subcutaneous injection. (Item 28) A method for treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the rAAV of any one of items 1 to 25 or the pharmaceutical formulation of any one of items 26 to 27, wherein the gene product is a therapeutic gene product. (Item 29) 29. The method of claim 28, wherein the administering step is by intravenous, intraarterial, intranasal, intrathecal, intracisternal or subcutaneous injection. (Item 30) 29. The method of claim 28, wherein the disease or condition is selected from the group consisting of pulmonary fibrosis, surfactant protein disorders, peroxisomal biogenesis disorders, or chronic obstructive pulmonary disease (COPD). (Item 31) 29. The method of claim 28, wherein the disease or condition is a central nervous system (CNS) or peripheral nervous system (PNS) disorder. (Item 32) A method for producing the rAAV according to any one of items 1 to 25, comprising: a) In cells, a) a first nucleic acid sequence encoding a therapeutic gene expression product surrounded by 5' and 3' inverted terminal repeat (ITR) sequences; b) a second nucleic acid sequence encoding a viral genome including a 5' ITR sequence, a replication (Rep) gene, a capsid (Cap) gene, and a 3' ITR, wherein the Cap gene encodes the rAAV capsid protein; and c) a third nucleic acid sequence encoding a first helper virus protein selected from the group consisting of E4orf6, E2a, and VA RNA, and optionally encoding a second helper virus protein comprising E1a or E1b55k; introducing a nucleic acid comprising: b) expressing the AAV capsid proteins in the cells; c) constructing the rAAV according to any one of items 1 to 23; and d) packaging the first nucleic acid sequence into the rAAV. A method comprising: (Item 33) a) at least one of decreased specificity and decreased transduction efficiency as compared to the corresponding parent AAV capsid having the AAV capsid protein of SEQ ID NO: 1, as measured in the liver of a primate subject when delivered intravenously to said primate subject; and b) at least one of increased specificity and improved transduction efficiency as measured in the central nervous system (CNS) of said primate subject when delivered intravenously to said primate subject, compared to said corresponding parental AAV capsid. A variant adeno-associated virus (AAV) capsid comprising: (Item 34) a) a variant AAV capsid protein comprising an amino acid sequence having an amino acid substitution of three or more amino acids within a 7-mer peptide sequence at amino acid positions corresponding to amino acid residues 452 to 458 of AAV9 VP1 (SEQ ID NO: 1); and b) at least one of increased specificity and improved transduction efficiency in a target tissue of a subject as compared to the corresponding parent AAV capsid protein of SEQ ID NO: 1 or SEQ ID NO: 2, as measured in said target tissue when delivered intranasally or intravenously to said subject. wherein the target tissue is selected from the group consisting of lung, intestine, kidney, heart and stomach. Variant adeno-associated virus (AAV) capsid. Incorporation by Reference

[0042] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0043] The embodiments are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. It is emphasized that, according to common practice, the various features of the drawings are not to scale. Rather, dimensions of the various features have been arbitrarily enlarged or reduced for clarity. The drawings include the following figures: [Brief explanation of the drawings]

[0044] [Figure 1A-C]Figures 1A-1D provide an overview of virus engineering according to this embodiment. Figure 1A shows a surface model of three monomers containing AAV9 three-fold symmetry (circled), illustrating the localization of the seven amino acid substitutions (red) introduced at amino acids 452-458 of this library, as well as the two amino acid substitutions and seven amino acid insertions (green) previously introduced at amino acids 588-589 by AAV-PHP.eB. Figure 1B shows the location of two loops within a single AAV9 monomer, showing their proximity and their relationship to the capsid surface (AAV inside is down, outside is up). Figure 1C shows the spike created by the 588 loop (left) and 455 loop (right) of one AAV9 monomer interacting with the 495 loop (center) of a second monomer. Figure 1D shows a schematic diagram of the engineering process ("TATT NNKNNK...NNKNNK CAAC" disclosed as SEQ ID NOs: 45492-45493 and "TATT AACGGT...AATCAA CAAC" disclosed as SEQ ID NOs: 45494-45495). Using PCR, diversity is introduced into the rAAV9 genome, which harbors a Cre-inducible switch encompassing the polyadenylation sequence, in the form of a nine amino acid substitution / insertion (AAV-PHP.eB; green band) and / or substitution (AA452-458; red band). The DNA capsid library is transfected into HEK-293T cells, and a diverse viral capsid library is harvested 60 hours later. The viral library is then systemically injected into a group of Cre-transgenic animals. After 3 weeks of expression, tissues are harvested and DNA is extracted from all organs. Using PCR, sequences are selectively recovered from only capsids transduced into Cre+ cells that invert their polyadenylation sequences. The recovered sequences are then prepared for next-generation sequencing (NGS) by PCR, with a unique double-index barcode appended to each specific Cre-tissue combination. After NGS, data are mined using positive and negative selection for enrichment (increased prevalence in a particular tissue relative to other sequences, normalized to their presence in the injected viral library) and specificity (increased prevalence in a particular tissue or cell type relative to other tissues or other cell types).After one or two rounds of selection, individual variants are assayed based on their enrichment and specificity scores. The italicized sequence represents AAV9, which is highly enriched in the liver (right) but not in the brain (left). [Figure 1D]Figures 1A-1D provide an overview of virus engineering according to this embodiment. Figure 1A shows a surface model of three monomers containing AAV9 three-fold symmetry (circled), illustrating the localization of the seven amino acid substitutions (red) introduced at amino acids 452-458 of this library, as well as the two amino acid substitutions and seven amino acid insertions (green) previously introduced at amino acids 588-589 by AAV-PHP.eB. Figure 1B shows the location of two loops within a single AAV9 monomer, showing their proximity and their relationship to the capsid surface (AAV inside is down, outside is up). Figure 1C shows the spike created by the 588 loop (left) and 455 loop (right) of one AAV9 monomer interacting with the 495 loop (center) of a second monomer. Figure 1D shows a schematic diagram of the engineering process ("TATT NNKNNK...NNKNNK CAAC" disclosed as SEQ ID NOs: 45492-45493 and "TATT AACGGT...AATCAA CAAC" disclosed as SEQ ID NOs: 45494-45495). Using PCR, diversity is introduced into the rAAV9 genome, which harbors a Cre-inducible switch encompassing the polyadenylation sequence, in the form of a nine amino acid substitution / insertion (AAV-PHP.eB; green band) and / or substitution (AA452-458; red band). The DNA capsid library is transfected into HEK-293T cells, and a diverse viral capsid library is harvested 60 hours later. The viral library is then systemically injected into a group of Cre-transgenic animals. After 3 weeks of expression, tissues are harvested and DNA is extracted from all organs. Using PCR, sequences are selectively recovered from only capsids transduced into Cre+ cells that invert their polyadenylation sequences. The recovered sequences are then prepared for next-generation sequencing (NGS) by PCR, with a unique double-index barcode appended to each specific Cre-tissue combination. After NGS, data are mined using positive and negative selection for enrichment (increased prevalence in a particular tissue relative to other sequences, normalized to their presence in the injected viral library) and specificity (increased prevalence in a particular tissue or cell type relative to other tissues or other cell types).After one or two rounds of selection, individual variants are assayed based on their enrichment and specificity scores. The italicized sequence represents AAV9, which is highly enriched in the liver (right) but not in the brain (left).

[0045] [Figure 2-1] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-2] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-3] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-4] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-5] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-6] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-7]FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-8] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-9] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-10] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-11] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-12] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-13] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-14]FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-15] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-16] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-17] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-18] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 2-19] FIG. 2 shows 7-mer variant AAV capsid amino acid sequences that are positively enriched in the central nervous system (CNS) after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0046] [Figure 3-1]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-2] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-3] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-4]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-5] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-6] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-7]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-8] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-9] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-10]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-11] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-12] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-13]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-14] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-15] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-16]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-17] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-18] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-19]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-20] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-21] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-22]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-23] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-24] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-25]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-26] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-27] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-28]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-29] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-30] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-31]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-32] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-33] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-34]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-35] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-36] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-37]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-38] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-39] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-40]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-41] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-42] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-43]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-44] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-45] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-46]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-47] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-48] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-49]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-50] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-51] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-52]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-53] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-54] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-55]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-56] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-57] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-58]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-59] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-60] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-61]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-62] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-63] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-64]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-65] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-66] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-67]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-68] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-69] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-70]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-71] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-72] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-73]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-74] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-75] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-76]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-77] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-78] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-79]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-80] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-81] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-82]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-83] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-84] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-85]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-86] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-87] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-88]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-89] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-90] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-91]3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-92] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue. [Figure 3-93] 3 shows a 7-mer variant AAV capsid amino acid sequence with specific tropism for the CNS and detargeting to the liver after two rounds of in vivo selection, and a DNA sequence encoding the 7-mer variant AAV capsid amino acid sequence. "Enrichment" is the prevalence of a given variant in a tissue compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score higher than 1 indicates positive enrichment, and an enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant could not be detected in the tissue.

[0047] [Figure 4-1] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 4-2] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 4-3] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 4-4] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 4-5] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 4-6] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 4-7] FIG. 4 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the heart after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0048] [Figure 5-1] FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 5-2]FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 5-3] FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 5-4] FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 5-5] FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 5-6] FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 5-7] FIG. 5 shows 7-mer variant AAV capsid amino acid sequences with gut-specific tropism and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection.

[0049] [Figure 6-1] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-2]FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-3] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-4] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-5] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-6] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-7] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-8] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-9] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-10] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-11] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-12] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-13] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-14] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-15] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-16] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-17]FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-18] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-19] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-20] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-21] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-22] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-23] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-24] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-25] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-26] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 6-27] FIG. 6 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the kidney after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0050] [Figure 7-1] FIG. 7 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the liver after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 7-2] FIG. 7 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the liver after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0051] [Figure 8-1] FIG. 8 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for muscle after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 8-2] FIG. 8 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for muscle after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 8-3]FIG. 8 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for muscle after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 8-4] FIG. 8 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for muscle after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 8-5] FIG. 8 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for muscle after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0052] [Figure 9] FIG. 9 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the pancreas and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection.

[0053] [Figure 10-1] FIG. 10 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the spleen after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 10-2] FIG. 10 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the spleen after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 10-3] FIG. 10 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the spleen after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 10-4]FIG. 10 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the spleen after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0054] [Figure 11-1] FIG. 11 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the stomach and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 11-2] FIG. 11 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the stomach and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection.

[0055] [Figure 12-1] FIG. 12 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the testis after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 12-2] FIG. 12 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the testis after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 12-3] FIG. 12 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the testis after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 12-4] FIG. 12 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the testis after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0056] [Figure 13-1] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-2] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-3] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-4] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-5] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-6] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-7] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-8] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-9] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-10] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-11] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences. [Figure 13-12] FIG. 13 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for the lung after two rounds of in vivo selection, and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences.

[0057] [Figure 14-1] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-2] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-3] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-4]FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-5] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-6] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-7] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-8] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection. [Figure 14-9] FIG. 14 shows 7-mer variant AAV capsid amino acid sequences with specific tropism for adipose tissue and DNA sequences encoding the 7-mer variant AAV capsid amino acid sequences after two rounds of in vivo selection.

[0058] [Figure 15A-B]Figures 15A-15B show next-generation sequencing data and in vivo transduction efficiencies of candidate variants. Figure 15A shows the relative abundance (log enrichment) for two candidate variants, AAV.CAP-A4 and AAV.CAP-A14, as well as a randomly selected variant. The NGS data show significant enrichment of AAV.CAP-A4 in the lung and negative enrichment in several peripheral organs. Conversely, AAV.CAP-A14 is enriched in neurons in the stomach and intestine and negatively enriched in other organs, such as the lung. Figure 15B shows that in vivo transduction 2 weeks after systemic administration of 5 x 10 vg of rAAV:CAG-mNeonGreen confirms the NGS data from 2A, with AAV.CAP-A4 strongly transducing the lung compared to AAV9, and AAV.CAP-A14 strongly transducing neurons in the stomach and intestine. For the avoidance of doubt, AAV.CAP-A4 is KDNTPGR, set forth in SEQ ID NO:32538 (lung enrichment), SEQ ID NO:25633 (heart enrichment), SEQ ID NO:26584 (intestine enrichment), and SEQ ID NO:28509 (kidney enrichment). AAV.CAP-A14 is RESSPSL, set forth in SEQ ID NO:26474 (intestine enrichment), SEQ ID NO:29065 (kidney enrichment), and SEQ ID NO:31904 (stomach enrichment).

[0059] [Figure 16A-B]Figures 16A-16D show that AAV.CAP-A4 tropism is strongly enriched in submucosal cells within the lung. ssAAV9:CAG-NLSx2-EGFP, ssAAV5:CAG-NLSx2-EGFP, or ssAAV.CAP-A4:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figure 16A provides a comparison of the total number of cells transduced by AAV.CAP-A4 compared to its parent capsid, AAV9. Figure 16B shows that overall transduction, as assessed by EGFP expression, is increased 15- to 30-fold compared to AAV5 and AAV9. Figure 16C provides cell-type specificity quantification, assessed by colocalization of EGFP signal and antibody staining, demonstrating a 30- to 60-fold increase in AAV.CAP-A4 transduction of ATII cells compared with AAV5 and AAV9 (respectively). A significant number of cells transduced by AAV.CAP-A4 are alveolar type II pneumocytes. Figure 16D shows cell-type specificity quantification, identifying 60% of transduced cells in the lung as ATII cells. Quantification: n = 3 mice per group for AAV9, AAV5, and AAV.CAP-A4, mean ± SE, one-way ANOVA (*P ≤ 0.05; ns, P ≥ 0.05). Scale bar is 50 μm. [Figure 16C-D]Figures 16A-16D show that AAV.CAP-A4 tropism is strongly enriched in submucosal cells within the lung. ssAAV9:CAG-NLSx2-EGFP, ssAAV5:CAG-NLSx2-EGFP, or ssAAV.CAP-A4:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figure 16A provides a comparison of the total number of cells transduced by AAV.CAP-A4 compared to its parent capsid, AAV9. Figure 16B shows that overall transduction, as assessed by EGFP expression, is increased 15- to 30-fold compared to AAV5 and AAV9. Figure 16C provides cell-type specificity quantification, assessed by colocalization of EGFP signal and antibody staining, demonstrating a 30- to 60-fold increase in AAV.CAP-A4 transduction of ATII cells compared with AAV5 and AAV9 (respectively). A significant number of cells transduced by AAV.CAP-A4 are alveolar type II pneumocytes. Figure 16D shows cell-type specificity quantification, identifying 60% of transduced cells in the lung as ATII cells. Quantification: n = 3 mice per group for AAV9, AAV5, and AAV.CAP-A4, mean ± SE, one-way ANOVA (*P ≤ 0.05; ns, P ≥ 0.05). Scale bar is 50 μm.

[0060] [Figure 17A]Figures 17A-17E show the characterization of a seven amino acid (AA) substitution library in AAV-PHP.eB. Figure 17A shows the distribution of seven amino acid substitutions in AAV-PHP.eB, demonstrating a relatively uniform distribution (with a few notable exceptions), low prevalence of stop codons, and no bias toward the wild-type (WT) AAV9 sequence (NGSGQNQ (SEQ ID NO: 545)) after PCR generation of the DNA library and packaging of the viral capsid library. Figure 17B shows a subset of the best-performing variants (SEQ ID NOs: 45496-45513) obtained from two rounds of positive and negative selection, respectively, showing strong differences from the WT AAV9 sequence. TI: threonine and isoleucine residues located at positions 450-451; QT: glutamine and threonine residues located at positions 459-460. Figure 17C shows a heatmap plotting the log-enrichment scores of the best-performing subsets, demonstrating specificity for and enrichment within neuronal populations, targets for which AAV-PHP.eB is already biased. Figure 17D shows that ssAAV9:CAG-mNeonGreen, ssAAV-PHP.eB:CAG-mNeonGreen, ssAAV.CAP-B1:CAG-mNeonGreen (LQTSSPG; SEQ ID NO: 2933), ssAAV.CAP-B2:CAG-mNeonGreen (QQGKQSV; SEQ ID NO: 79), ssAAV.CAP-B4:CAG-mNeonGreen (SINTKTN; SEQ ID NO: 45475), and ssAAV.CAP-B7:CAG-mNeonGreen (SNGTKQT; SEQ ID NO: 442) were injected intravenously into male adult mice at 5 x 10 vg / mouse and mNeonGreen fluorescence was assessed two weeks later.Figure 17E shows the results of ssAAV.CAP-B8: CAG-mNeonGreen (GSGKTAA; SEQ ID NO: 88), ssAAV.CAP-B9: CAG-mNeonGreen (MGDKPTR; SEQ ID NO: 2466), ssAAV.CAP-B10: CAG-mNeonGreen (DGAATKN; SEQ ID NO: 3943), ssAAV.CAP-B11: CAG-mNeonGreen (QPSGGNT; SEQ ID NO: 2672), ssAAV.CAP-B14: CAG-mNeonGreen (ERGANTK; SEQ ID NO: 5192), ssAAV.CAP-B16: CAG-mNeonGreen (TTGGHSS; SEQ ID NO: 2743), ssAAV.CAP-B17: CAG-mNeonGreen ( Figure 17D and Figure 17E show that ssAAV.CAP-B18:CAG-mNeonGreen (GTGTSVL; SEQ ID NO: 11958), ssAAV.CAP-B19:CAG-mNeonGreen (NQSGTKG; SEQ ID NO: 780), ssAAV.CAP-B22:CAG-mNeonGreen (DGQSSKS; SEQ ID NO: 2764), ssAAV.CAP-B23:CAG-mNeonGreen (KGPGQMG; SEQ ID NO: 45476), or ssAAV.CAP-B25:CAG-mNeonGreen (GTPSKAG; SEQ ID NO: 2741) were intravenously injected into male adult mice at 5 x 10 vg / mouse, and mNeonGreen fluorescence was assessed 2 weeks later. In Figures 17D and 17E, a direct comparison of the transduction profiles of the best-performing variants demonstrates a strong correlation between the validated tropism and that predicted by the NGS data. The scale bar is 2 mm. [Figure 17B]Figures 17A-17E show the characterization of a seven amino acid (AA) substitution library in AAV-PHP.eB. Figure 17A shows the distribution of seven amino acid substitutions in AAV-PHP.eB, demonstrating a relatively uniform distribution (with a few notable exceptions), low prevalence of stop codons, and no bias toward the wild-type (WT) AAV9 sequence (NGSGQNQ (SEQ ID NO: 545)) after PCR generation of the DNA library and packaging of the viral capsid library. Figure 17B shows a subset of the best-performing variants (SEQ ID NOs: 45496-45513) obtained from two rounds of positive and negative selection, respectively, showing strong differences from the WT AAV9 sequence. TI: threonine and isoleucine residues located at positions 450-451; QT: glutamine and threonine residues located at positions 459-460. Figure 17C shows a heatmap plotting the log-enrichment scores of the best-performing subsets, demonstrating specificity for and enrichment within neuronal populations, targets for which AAV-PHP.eB is already biased. Figure 17D shows that ssAAV9:CAG-mNeonGreen, ssAAV-PHP.eB:CAG-mNeonGreen, ssAAV.CAP-B1:CAG-mNeonGreen (LQTSSPG; SEQ ID NO: 2933), ssAAV.CAP-B2:CAG-mNeonGreen (QQGKQSV; SEQ ID NO: 79), ssAAV.CAP-B4:CAG-mNeonGreen (SINTKTN; SEQ ID NO: 45475), and ssAAV.CAP-B7:CAG-mNeonGreen (SNGTKQT; SEQ ID NO: 442) were injected intravenously into male adult mice at 5 x 10 vg / mouse and mNeonGreen fluorescence was assessed two weeks later.Figure 17E shows the results of ssAAV.CAP-B8: CAG-mNeonGreen (GSGKTAA; SEQ ID NO: 88), ssAAV.CAP-B9: CAG-mNeonGreen (MGDKPTR; SEQ ID NO: 2466), ssAAV.CAP-B10: CAG-mNeonGreen (DGAATKN; SEQ ID NO: 3943), ssAAV.CAP-B11: CAG-mNeonGreen (QPSGGNT; SEQ ID NO: 2672), ssAAV.CAP-B14: CAG-mNeonGreen (ERGANTK; SEQ ID NO: 5192), ssAAV.CAP-B16: CAG-mNeonGreen (TTGGHSS; SEQ ID NO: 2743), ssAAV.CAP-B17: CAG-mNeonGreen ( Figure 17D and Figure 17E show that ssAAV.CAP-B18:CAG-mNeonGreen (GTGTSVL; SEQ ID NO: 11958), ssAAV.CAP-B19:CAG-mNeonGreen (NQSGTKG; SEQ ID NO: 780), ssAAV.CAP-B22:CAG-mNeonGreen (DGQSSKS; SEQ ID NO: 2764), ssAAV.CAP-B23:CAG-mNeonGreen (KGPGQMG; SEQ ID NO: 45476), or ssAAV.CAP-B25:CAG-mNeonGreen (GTPSKAG; SEQ ID NO: 2741) were intravenously injected into male adult mice at 5 x 10 vg / mouse, and mNeonGreen fluorescence was assessed 2 weeks later. In Figures 17D and 17E, a direct comparison of the transduction profiles of the best-performing variants demonstrates a strong correlation between the validated tropism and that predicted by the NGS data. The scale bar is 2 mm. [Figure 17C]Figures 17A-17E show the characterization of a seven amino acid (AA) substitution library in AAV-PHP.eB. Figure 17A shows the distribution of seven amino acid substitutions in AAV-PHP.eB, demonstrating a relatively uniform distribution (with a few notable exceptions), low prevalence of stop codons, and no bias toward the wild-type (WT) AAV9 sequence (NGSGQNQ (SEQ ID NO: 545)) after PCR generation of the DNA library and packaging of the viral capsid library. Figure 17B shows a subset of the best-performing variants (SEQ ID NOs: 45496-45513) obtained from two rounds of positive and negative selection, respectively, showing strong differences from the WT AAV9 sequence. TI: threonine and isoleucine residues located at positions 450-451; QT: glutamine and threonine residues located at positions 459-460. Figure 17C shows a heatmap plotting the log-enrichment scores of the best-performing subsets, demonstrating specificity for and enrichment within neuronal populations, targets for which AAV-PHP.eB is already biased. Figure 17D shows that ssAAV9:CAG-mNeonGreen, ssAAV-PHP.eB:CAG-mNeonGreen, ssAAV.CAP-B1:CAG-mNeonGreen (LQTSSPG; SEQ ID NO: 2933), ssAAV.CAP-B2:CAG-mNeonGreen (QQGKQSV; SEQ ID NO: 79), ssAAV.CAP-B4:CAG-mNeonGreen (SINTKTN; SEQ ID NO: 45475), and ssAAV.CAP-B7:CAG-mNeonGreen (SNGTKQT; SEQ ID NO: 442) were injected intravenously into male adult mice at 5 x 10 vg / mouse and mNeonGreen fluorescence was assessed two weeks later.Figure 17E shows the results of ssAAV.CAP-B8: CAG-mNeonGreen (GSGKTAA; SEQ ID NO: 88), ssAAV.CAP-B9: CAG-mNeonGreen (MGDKPTR; SEQ ID NO: 2466), ssAAV.CAP-B10: CAG-mNeonGreen (DGAATKN; SEQ ID NO: 3943), ssAAV.CAP-B11: CAG-mNeonGreen (QPSGGNT; SEQ ID NO: 2672), ssAAV.CAP-B14: CAG-mNeonGreen (ERGANTK; SEQ ID NO: 5192), ssAAV.CAP-B16: CAG-mNeonGreen (TTGGHSS; SEQ ID NO: 2743), ssAAV.CAP-B17: CAG-mNeonGreen ( Figure 17D and Figure 17E show that ssAAV.CAP-B18:CAG-mNeonGreen (GTGTSVL; SEQ ID NO: 11958), ssAAV.CAP-B19:CAG-mNeonGreen (NQSGTKG; SEQ ID NO: 780), ssAAV.CAP-B22:CAG-mNeonGreen (DGQSSKS; SEQ ID NO: 2764), ssAAV.CAP-B23:CAG-mNeonGreen (KGPGQMG; SEQ ID NO: 45476), or ssAAV.CAP-B25:CAG-mNeonGreen (GTPSKAG; SEQ ID NO: 2741) were intravenously injected into male adult mice at 5 x 10 vg / mouse, and mNeonGreen fluorescence was assessed 2 weeks later. In Figures 17D and 17E, a direct comparison of the transduction profiles of the best-performing variants demonstrates a strong correlation between the validated tropism and that predicted by the NGS data. The scale bar is 2 mm. [Figure 17D]Figures 17A-17E show the characterization of a seven amino acid (AA) substitution library in AAV-PHP.eB. Figure 17A shows the distribution of seven amino acid substitutions in AAV-PHP.eB, demonstrating a relatively uniform distribution (with a few notable exceptions), low prevalence of stop codons, and no bias toward the wild-type (WT) AAV9 sequence (NGSGQNQ (SEQ ID NO: 545)) after PCR generation of the DNA library and packaging of the viral capsid library. Figure 17B shows a subset of the best-performing variants (SEQ ID NOs: 45496-45513) obtained from two rounds of positive and negative selection, respectively, showing strong differences from the WT AAV9 sequence. TI: threonine and isoleucine residues located at positions 450-451; QT: glutamine and threonine residues located at positions 459-460. Figure 17C shows a heatmap plotting the log-enrichment scores of the best-performing subsets, demonstrating specificity for and enrichment within neuronal populations, targets for which AAV-PHP.eB is already biased. Figure 17D shows that ssAAV9:CAG-mNeonGreen, ssAAV-PHP.eB:CAG-mNeonGreen, ssAAV.CAP-B1:CAG-mNeonGreen (LQTSSPG; SEQ ID NO: 2933), ssAAV.CAP-B2:CAG-mNeonGreen (QQGKQSV; SEQ ID NO: 79), ssAAV.CAP-B4:CAG-mNeonGreen (SINTKTN; SEQ ID NO: 45475), and ssAAV.CAP-B7:CAG-mNeonGreen (SNGTKQT; SEQ ID NO: 442) were injected intravenously into male adult mice at 5 x 10 vg / mouse and mNeonGreen fluorescence was assessed two weeks later.Figure 17E shows the results of ssAAV.CAP-B8: CAG-mNeonGreen (GSGKTAA; SEQ ID NO: 88), ssAAV.CAP-B9: CAG-mNeonGreen (MGDKPTR; SEQ ID NO: 2466), ssAAV.CAP-B10: CAG-mNeonGreen (DGAATKN; SEQ ID NO: 3943), ssAAV.CAP-B11: CAG-mNeonGreen (QPSGGNT; SEQ ID NO: 2672), ssAAV.CAP-B14: CAG-mNeonGreen (ERGANTK; SEQ ID NO: 5192), ssAAV.CAP-B16: CAG-mNeonGreen (TTGGHSS; SEQ ID NO: 2743), ssAAV.CAP-B17: CAG-mNeonGreen ( Figure 17D and Figure 17E show that ssAAV.CAP-B18:CAG-mNeonGreen (GTGTSVL; SEQ ID NO: 11958), ssAAV.CAP-B19:CAG-mNeonGreen (NQSGTKG; SEQ ID NO: 780), ssAAV.CAP-B22:CAG-mNeonGreen (DGQSSKS; SEQ ID NO: 2764), ssAAV.CAP-B23:CAG-mNeonGreen (KGPGQMG; SEQ ID NO: 45476), or ssAAV.CAP-B25:CAG-mNeonGreen (GTPSKAG; SEQ ID NO: 2741) were intravenously injected into male adult mice at 5 x 10 vg / mouse, and mNeonGreen fluorescence was assessed 2 weeks later. In Figures 17D and 17E, a direct comparison of the transduction profiles of the best-performing variants demonstrates a strong correlation between the validated tropism and that predicted by the NGS data. The scale bar is 2 mm. [Figure 17E]Figures 17A-17E show the characterization of a seven amino acid (AA) substitution library in AAV-PHP.eB. Figure 17A shows the distribution of seven amino acid substitutions in AAV-PHP.eB, demonstrating a relatively uniform distribution (with a few notable exceptions), low prevalence of stop codons, and no bias toward the wild-type (WT) AAV9 sequence (NGSGQNQ (SEQ ID NO: 545)) after PCR generation of the DNA library and packaging of the viral capsid library. Figure 17B shows a subset of the best-performing variants (SEQ ID NOs: 45496-45513) obtained from two rounds of positive and negative selection, respectively, showing strong differences from the WT AAV9 sequence. TI: threonine and isoleucine residues located at positions 450-451; QT: glutamine and threonine residues located at positions 459-460. Figure 17C shows a heatmap plotting the log-enrichment scores of the best-performing subsets, demonstrating specificity for and enrichment within neuronal populations, targets for which AAV-PHP.eB is already biased. Figure 17D shows that ssAAV9:CAG-mNeonGreen, ssAAV-PHP.eB:CAG-mNeonGreen, ssAAV.CAP-B1:CAG-mNeonGreen (LQTSSPG; SEQ ID NO: 2933), ssAAV.CAP-B2:CAG-mNeonGreen (QQGKQSV; SEQ ID NO: 79), ssAAV.CAP-B4:CAG-mNeonGreen (SINTKTN; SEQ ID NO: 45475), and ssAAV.CAP-B7:CAG-mNeonGreen (SNGTKQT; SEQ ID NO: 442) were injected intravenously into male adult mice at 5 x 10 vg / mouse and mNeonGreen fluorescence was assessed two weeks later.Figure 17E shows the results of ssAAV.CAP-B8: CAG-mNeonGreen (GSGKTAA; SEQ ID NO: 88), ssAAV.CAP-B9: CAG-mNeonGreen (MGDKPTR; SEQ ID NO: 2466), ssAAV.CAP-B10: CAG-mNeonGreen (DGAATKN; SEQ ID NO: 3943), ssAAV.CAP-B11: CAG-mNeonGreen (QPSGGNT; SEQ ID NO: 2672), ssAAV.CAP-B14: CAG-mNeonGreen (ERGANTK; SEQ ID NO: 5192), ssAAV.CAP-B16: CAG-mNeonGreen (TTGGHSS; SEQ ID NO: 2743), ssAAV.CAP-B17: CAG-mNeonGreen ( Figure 17D and Figure 17E show that ssAAV.CAP-B18:CAG-mNeonGreen (GTGTSVL; SEQ ID NO: 11958), ssAAV.CAP-B19:CAG-mNeonGreen (NQSGTKG; SEQ ID NO: 780), ssAAV.CAP-B22:CAG-mNeonGreen (DGQSSKS; SEQ ID NO: 2764), ssAAV.CAP-B23:CAG-mNeonGreen (KGPGQMG; SEQ ID NO: 45476), or ssAAV.CAP-B25:CAG-mNeonGreen (GTPSKAG; SEQ ID NO: 2741) were intravenously injected into male adult mice at 5 x 10 vg / mouse, and mNeonGreen fluorescence was assessed 2 weeks later. In Figures 17D and 17E, a direct comparison of the transduction profiles of the best-performing variants demonstrates a strong correlation between the validated tropism and that predicted by the NGS data. The scale bar is 2 mm.

[0061] [Figure 18A-B]Figures 18A-18D show that AAV.CAP-B10 tropism is strongly brain-biased, with significant liver detargeting. ssAAV9:CAG-NLSx2-EGFP, ssAAV-PHP.eB:CAG-NLSx2-EGFP, or ssAAV.CAP-B10:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figure 18A shows a comparison of BBB crossing and brain transduction for AAV9, AAV-PHP.eB, and AAV.CAP-B10, demonstrating a progressive improvement in brain transduction efficiency after repeated manipulation of WT capsids. Figure 18B shows the same comparison in the liver, demonstrating a progressive decrease in transduction efficiency. Figure 18C provides quantification of the total number of transduced cells in the brain, showing that there was no significant increase in total transduction for AAV.CAP-B10 compared to AAV-PHP.eB, and that both were significantly increased compared to AAV9. Comparison of the mean brightness per cell shows a significant increase for AAV.CAP-B10 compared to AAV9, but not for AAV-PHP.eB. Figure 18D provides quantification of the total number of transduced cells in the liver, showing a significant decrease for AAV.CAP-B10 compared to both AAV9 and AAV-PHP.eB. Brightness per cell was also significantly decreased comparing AAV.CAP-B10 to AAV9, with no significant difference observed with AAV-PHP.eB. For quantification: n = 6 mice per group, mean ± SE, Brown-Forsythe and Welch ANOVA test for transduction and Kruskal-Wallis test for brightness (*P ≤ 0.05; ns, P ≥ 0.05). The scale bar is 1 mm. [Figure 18C-D]Figures 18A-18D show that AAV.CAP-B10 tropism is strongly brain-biased, with significant liver detargeting. ssAAV9:CAG-NLSx2-EGFP, ssAAV-PHP.eB:CAG-NLSx2-EGFP, or ssAAV.CAP-B10:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figure 18A shows a comparison of BBB crossing and brain transduction for AAV9, AAV-PHP.eB, and AAV.CAP-B10, demonstrating a progressive improvement in brain transduction efficiency after repeated manipulation of WT capsids. Figure 18B shows the same comparison in the liver, demonstrating a progressive decrease in transduction efficiency. Figure 18C provides quantification of the total number of transduced cells in the brain, showing that there was no significant increase in total transduction for AAV.CAP-B10 compared to AAV-PHP.eB, and that both were significantly increased compared to AAV9. Comparison of the mean brightness per cell shows a significant increase for AAV.CAP-B10 compared to AAV9, but not for AAV-PHP.eB. Figure 18D provides quantification of the total number of transduced cells in the liver, showing a significant decrease for AAV.CAP-B10 compared to both AAV9 and AAV-PHP.eB. Brightness per cell was also significantly decreased comparing AAV.CAP-B10 to AAV9, with no significant difference observed with AAV-PHP.eB. For quantification: n = 6 mice per group, mean ± SE, Brown-Forsythe and Welch ANOVA test for transduction and Kruskal-Wallis test for brightness (*P ≤ 0.05; ns, P ≥ 0.05). The scale bar is 1 mm.

[0062] [Figure 19A-B]Figures 19A-19F show that AAV.CAP-B10 is strongly biased toward neurons in the brain. ssAAV-PHP.eB:CAG-NLSx2-EGFP or ssAAV.CAP-BB10:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figures 19A-19B show that AAV.CAP-B10 demonstrated no significant increase in the total number of transduced neurons compared to AAV-PHP.eB across multiple brain regions. Figures 19C-19D show that AAV.CAP-B10 demonstrates significantly reduced transduction of astrocytes compared to AAV-PHP.eB across all brain regions quantified. Figures 19E-19F show that AAV.CAP-B10 demonstrates significantly reduced transduction of oligodendrocytes compared to AAV-PHP.eB across all brain regions quantified. Quantification: n = 6 mice per group, mean ± SE, Mann-Whitney test (*P ≤ 0.05; ns, P ≥ 0.05). Scale bar is 200 μm. [Figure 19C-D] Figures 19A-19F show that AAV.CAP-B10 is strongly biased toward neurons in the brain. ssAAV-PHP.eB:CAG-NLSx2-EGFP or ssAAV.CAP-BB10:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figures 19A-19B show that AAV.CAP-B10 demonstrated no significant increase in the total number of transduced neurons compared to AAV-PHP.eB across multiple brain regions. Figures 19C-19D show that AAV.CAP-B10 demonstrates significantly reduced transduction of astrocytes compared to AAV-PHP.eB across all brain regions quantified. Figures 19E-19F show that AAV.CAP-B10 demonstrates significantly reduced transduction of oligodendrocytes compared to AAV-PHP.eB across all brain regions quantified. Quantification: n = 6 mice per group, mean ± SE, Mann-Whitney test (*P ≤ 0.05; ns, P ≥ 0.05). Scale bar is 200 μm. [Figure 19E-F]Figures 19A-19F show that AAV.CAP-B10 is strongly biased toward neurons in the brain. ssAAV-PHP.eB:CAG-NLSx2-EGFP or ssAAV.CAP-BB10:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figures 19A-19B show that AAV.CAP-B10 demonstrated no significant increase in the total number of transduced neurons compared to AAV-PHP.eB across multiple brain regions. Figures 19C-19D show that AAV.CAP-B10 demonstrates significantly reduced transduction of astrocytes compared to AAV-PHP.eB across all brain regions quantified. Figures 19E-19F show that AAV.CAP-B10 demonstrates significantly reduced transduction of oligodendrocytes compared to AAV-PHP.eB across all brain regions quantified. Quantification: n = 6 mice per group, mean ± SE, Mann-Whitney test (*P ≤ 0.05; ns, P ≥ 0.05). Scale bar is 200 μm.

[0063] [Figure 20A] Figures 20A-20C show the amino acid contribution across 7-mer substitutions to variants enriched in the brain and detargeted from the liver. Figure 20A shows the amino acid contribution across 7-mer substitutions to variants enriched in the brain. Figure 20B shows the amino acid contribution across 7-mer substitutions to variants negatively enriched (detargeted) from the liver. Figure 20C shows the amino acid contribution across 7-mer substitutions to variants enriched in the brain and negatively enriched (detargeted) from the liver. We analyzed 1000 variants with the highest enrichment in the brain of hSyn-Cre animals, 1000 variants with the lowest enrichment in the liver of Tek-Cre animals, and all variants with positive enrichment in the brain and negative enrichment in the liver. The z-scores at each position for all amino acids are plotted. [Figure 20B]Figures 20A-20C show the amino acid contribution across 7-mer substitutions to variants enriched in the brain and detargeted from the liver. Figure 20A shows the amino acid contribution across 7-mer substitutions to variants enriched in the brain. Figure 20B shows the amino acid contribution across 7-mer substitutions to variants negatively enriched (detargeted) from the liver. Figure 20C shows the amino acid contribution across 7-mer substitutions to variants enriched in the brain and negatively enriched (detargeted) from the liver. We analyzed 1000 variants with the highest enrichment in the brain of hSyn-Cre animals, 1000 variants with the lowest enrichment in the liver of Tek-Cre animals, and all variants with positive enrichment in the brain and negative enrichment in the liver. The z-scores at each position for all amino acids are plotted. [Figure 20C] Figures 20A-20C show the amino acid contribution across 7-mer substitutions to variants enriched in the brain and detargeted from the liver. Figure 20A shows the amino acid contribution across 7-mer substitutions to variants enriched in the brain. Figure 20B shows the amino acid contribution across 7-mer substitutions to variants negatively enriched (detargeted) from the liver. Figure 20C shows the amino acid contribution across 7-mer substitutions to variants enriched in the brain and negatively enriched (detargeted) from the liver. We analyzed 1000 variants with the highest enrichment in the brain of hSyn-Cre animals, 1000 variants with the lowest enrichment in the liver of Tek-Cre animals, and all variants with positive enrichment in the brain and negative enrichment in the liver. The z-scores at each position for all amino acids are plotted.

[0064] [Figure 21]Figure 21 shows that AAV.CAP-B10 is detargeted from peripheral organs. ssAAV9:CAG-NLSx2-EGFP, ssAAV-PHP.eB:CAG-NLSx2-EGFP, or ssAAV.CAP-B10:CAG-NLSx2-EGFP was intravenously injected into male adult mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Transduction efficiency in peripheral tissues shows a significant increase and decrease in spinal cord transduction for AAV.CAP-B10 compared with AAV9 or AAV-PHP.eB, respectively. In the DRG, AAV.CAP-B10 is significantly reduced compared with AAV9 but not compared with AAV-PHP.eB. In the myenteric and submucosal plexuses of the intestine, AAV.CAP-B10 is significantly reduced compared with AAV9 but not compared with AAV-PHP.eB. In the lung, AAV.CAP-B10 was significantly reduced compared to AAV.PHP.eB, but not compared to AAV9. In the kidney, spleen, pancreas, and testis, AAV.CAP-B10 was significantly reduced compared to AAV9, but not compared to AAV-PHP.eB. Quantification: n = 6 mice per group, except for the spinal cord and DRG (in these cases, n = 3 mice for AAV9 and AAV-PHP.eB, and n = 5 mice for AAV.CAP-B10). Mean ± SE. ANOVA for spinal cord, Brown-Forsythe and Welch ANOVA test for myenteric plexus and pancreas, Kruskal-Wallis test for DRG, submucosal plexus, lung, kidney, spleen, and testis (*P ≤ 0.05; ns, P ≥ 0.05).

[0065] [Figure 22A-B]Figures 22A-22B show that AAV.CAP-B10 significantly detargets Purkinje cells in the cerebellum. ssAAV-PHP.eB:CAG-NLSx2-EGFP or ssAAVCAP-B10:CAG-NLSx2-EGFP were intravenously injected into adult male mice at 1 x 10 vg / mouse. GFP fluorescence was assessed after 3 weeks of expression. Figure 22A shows immunofluorescence in cerebellar sections. Figure 22B shows quantification of Purkinje cell transduction in the cerebellum, demonstrating significantly fewer Purkinje cells transduced by AAV.CAP-B10 compared with AAV-PHP.eB. Quantification: n = 6 mice per group, mean ± SE, Mann-Whitney test (*P ≤ 0.05; ns, P ≥ 0.05). Scale bar is 200 µm.

[0066] [Figure 23A-D]Figures 23A-23F show the characterization of pooled expression in non-human primates. Figure 23A shows the experimental paradigm for pooled injection of novel variants AAV.CAP-B1, AAV.CAP-B2, AAV.CAP-B8, AAV.CAP-B10, AAV.CAP-B18, and AAV.CAP-B22, as well as controls AAV9 and AAV-PHP.eB. Human FXN fused to an HA tag is packaged into each variant under the control of a ubiquitous CAG promoter, and each variant is distinguished by a unique 12-bp RNA barcode in the 5'UTR. Two marmosets were injected with a dose of 1.2 x 10 vg / kg, of which each variant in the pool contributed 1 / 8. Figure 23B shows six sections sectioned through the anterior-posterior axis of the marmoset brain and the cerebellum. Counterstaining for the HA tag on the FXN transgene demonstrates robust expression distributed throughout the brain after IV administration of the pool of variants. Figure 23C shows NGS quantification of RNA barcode expression for each of the eight variants in the pool from two marmosets, demonstrating dramatic increases for several variants, including a >12-fold increase in RNA levels for AAV.CAP-B22 and a >5-fold increase for AAV.CAP-B10 compared to AAV9. Figure 23D shows zoomed-in frames from various cortical and subcortical regions demonstrating significant and widespread transduction across most brain regions, including the cortex, hippocampus, and cerebellum. Figure 23E shows counterstaining for the HA tag in the liver, demonstrating minimal overall transduction from the viral pool. NGS quantification demonstrates relative detargeting from the liver, with AAV.CAP-B22 contributing similar RNA levels to AAV9 and AAV.CAP-B10 contributing less than fivefold. Figure 23F shows that cardiac transduction was increased from AAV9 for several of the variants, with AAV.CAP-B22 contributing nearly five-fold more RNA in comparison. Adrenal cortex transduction by the variants varied compared to AAV9. RNA was collected from two animals for analysis of liver transduction and from one animal for heart and adrenal cortex transduction. [Figure 23E-F] Figures 23A-23F show the characterization of pooled expression in non-human primates. Figure 23A shows the experimental paradigm for pooled injection of novel variants AAV.CAP-B1, AAV.CAP-B2, AAV.CAP-B8, AAV.CAP-B10, AAV.CAP-B18, and AAV.CAP-B22, as well as controls AAV9 and AAV-PHP.eB. Human FXN fused to an HA tag is packaged into each variant under the control of a ubiquitous CAG promoter, and each variant is distinguished by a unique 12-bp RNA barcode in the 5'UTR. Two marmosets were injected with a dose of 1.2 x 10 vg / kg, of which each variant in the pool contributed 1 / 8. Figure 23B shows six sections sectioned through the anterior-posterior axis of the marmoset brain and the cerebellum. Counterstaining for the HA tag on the FXN transgene demonstrates robust expression distributed throughout the brain after IV administration of the pool of variants. Figure 23C shows NGS quantification of RNA barcode expression for each of the eight variants in the pool from two marmosets, demonstrating dramatic increases for several variants, including a >12-fold increase in RNA levels for AAV.CAP-B22 and a >5-fold increase for AAV.CAP-B10 compared to AAV9. Figure 23D shows zoomed-in frames from various cortical and subcortical regions demonstrating significant and widespread transduction across most brain regions, including the cortex, hippocampus, and cerebellum. Figure 23E shows counterstaining for the HA tag in the liver, demonstrating minimal overall transduction from the viral pool. NGS quantification demonstrates relative detargeting from the liver, with AAV.CAP-B22 contributing similar RNA levels to AAV9 and AAV.CAP-B10 contributing less than fivefold. Figure 23F shows that cardiac transduction was increased from AAV9 for several of the variants, with AAV.CAP-B22 contributing nearly five-fold more RNA in comparison. Adrenal cortex transduction by the variants varied compared to AAV9. RNA was collected from two animals for analysis of liver transduction and from one animal for heart and adrenal cortex transduction. DETAILED DESCRIPTION OF THE INVENTION

[0067] Detailed Description of Disclosure Provided herein are modified adeno-associated (AAV) virus capsid protein compositions that, when administered to a subject, are useful for the integration of a transgene into a target cell or environment (e.g., a cell type or tissue) in the subject. The modified AAV capsid proteins of the present disclosure contain at least one insertion or substitution of an amino acid in a corresponding parent AAV capsid protein, which insertion or substitution confers a desired targeting property, such as increased or decreased specificity compared to a reference AAV capsid protein, e.g., the corresponding unmodified parent capsid protein, or increased or decreased transgene transduction efficiency compared to a reference AAV capsid protein, e.g., the corresponding unmodified parent capsid protein.

[0068] The most commonly engineered loop in AAV is the 588 loop, because it is the heparan sulfate binding site of AAV2 and is suitable for peptide presentation. The only known receptor for AAV9 is N-linked terminal galactose, but evidence suggests that others exist. Modifications of the AAV9 588 loop have been shown to result in increased specificity and improved transduction efficiency for the target in vivo environment compared to the corresponding parent AAV capsid protein in rodent models, but these desired targeting properties are not as pronounced in non-human primate models, resulting in limited utility for treating human disease. Non-limiting examples of modified AAV capsid proteins include AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, and AAV-PHP.

[0069] Disclosed herein are modified AAV capsid proteins that exhibit desirable tropism in both rodent and non-human primate models of human disease, comprising at least one amino acid insertion or substitution compared to the corresponding parent AAV capsid protein, wherein the insertion or substitution is within a loop corresponding to the amino acid 455 loop (AA455 loop) of AAV9. The AA455 loop protrudes furthest from the surface of the capsid and is associated with neutralizing antibody binding. The AA455 loop is believed to play a significant role in cell surface receptor binding, either naturally or through interaction with the 588 loop. In some cases, the parent AAV capsid protein is AAV5, AAV9, or a modified AAV5 or AAV9 (e.g., AAV-PHP.eB, AAV-PHP.B, etc.).

[0070] The most common method of AAV-mediated transgene delivery is by direct injection into the target in vivo environment, which is disadvantageous for many reasons, including the risk of injury or death, pain, and higher costs compared to less invasive methods. Previous AAV-mediated delivery via intravenous administration, while eliminating the need for direct injection, suffers from reduced specificity and transduction efficiency for the target in vivo environment, resulting in off-target transduction events and requiring a higher viral load to reach sufficient therapeutic levels in the target in vivo environment. This is particularly evident when AAV must cross the blood-brain barrier (BBB) ​​or epithelial lining to reach the target in vivo environment, such as to treat diseases or conditions of the central nervous system (CNS) and lungs.

[0071] Disclosed herein is a method comprising systemically administering a modified AAV capsid of the present disclosure, which encapsidates a viral vector containing a transgene (e.g., a therapeutic nucleic acid), and which exhibits increased specificity and improved transduction efficiency compared to a reference AAV capsid protein, e.g., a corresponding parent capsid protein. The modified AAV capsid protein of the present disclosure is capable of crossing the BBB and transducing a transgene into specific target cell types (e.g., neurons) in both rodent and non-human primate models. In addition, the modified AAV capsid protein of the present disclosure can target lung cell types (e.g., type II epithelial (ATII) cells) with, in some cases, a 60-fold increase in transgene transduction efficiency compared to a reference AAV capsid protein, e.g., a corresponding parent capsid protein. The modified AAV capsid proteins of the present disclosure can detarget non-target in vivo environments such as the liver, in some cases by exhibiting a 100-fold reduction in transgene transduction efficiency compared to a reference AAV capsid protein, such as the corresponding parent capsid protein. Thus, the modified AAV capsid proteins of the present disclosure are suitable for transgene therapy to treat human diseases.

[0072] The present specification discloses a transgene contained in a recombinant AAV (rAAV) vector and a transgene that is encapsulated by the modified AAV capsid protein of the present disclosure.The transgene disclosed herein can be delivered to a subject for various purposes, for example, to treat a disease or condition in the subject.The transgene can be a gene editing component that modulates the activity or expression of a target gene or gene expression product.Alternatively, the transgene can be a gene that encodes a therapeutic gene expression product that is effective in modulating the activity or expression of its own or another target gene or gene expression product.

[0073] Also provided is a method for producing recombinant AAV (rAAV) virions, i.e., rAAV particles, comprising an AAV capsid comprising a modified AAV capsid protein and a heterologous polynucleotide encoding a therapeutic nucleic acid. The modified capsid protein is produced by introducing a heterologous polynucleotide encoding a transgene (e.g., containing a therapeutic nucleic acid), a first plasmid vector encoding an AAV genome having the modified AAV capsid protein, and a second plasmid vector encoding a helper virus protein required for the assembly of the modified capsid structure and the packaging of the transgene into the modified capsid into cells (e.g., immortalized stem cells). The assembled rAAV particles can be isolated and purified from cells using suitable methods known in the art.

[0074] Also provided herein are vectors comprising nucleic acid sequences encoding the modified AAV capsid proteins of the present disclosure. For example, the vectors of the present disclosure comprise nucleic acid sequences encoding two AAV viral genes, Rep (replication) and Cap (capsid), and the Cap gene encoding the viral capsid proteins VP1, VP2, and VP3 is modified to produce the modified AAV capsid proteins of the present disclosure. The vectors can comprise viral genomes from one or more AAV serotypes (e.g., AAV5, AAV9), or variant AAV serotypes (e.g., AAV-PHP.eB, AAV-PHP.B, etc.). Compendium

[0075] Recombinant AAV (rAAV)-mediated gene delivery utilizes the AAV viral transduction mechanism for the nuclear expression of episomal heterologous nucleic acids (e.g., transgenes, therapeutic nucleic acids). Upon delivery to the host in vivo environment, rAAV (1) binds or attaches to cell surface receptors on target cells, (2) undergoes endocytosis, (3) is transported to the nucleus, (4) the viral envelope is removed to release the encapsidated heterologous nucleic acid, (5) converts the heterologous nucleic acid from single-stranded to double-stranded DNA as a template for transcription within the nucleus, and (6) transcribes ("transduces") the episomal heterologous nucleic acid within the nucleus of the host cell. rAAV engineered to have increased specificity (binding to cell surface receptors on target cells) and improved transduction efficiency (transcription of episomal heterologous nucleic acids within host cells) are desirable for gene therapy applications.

[0076] The AAV capsid is composed of three capsid protein monomers: VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in a 1:1:10 ratio to form the viral capsid. VP1 encompasses the entire VP2 protein, plus an approximately 137-amino acid N-terminal region (VP1u), while VP2 encompasses the entire VP3 protein, plus an approximately 65-amino acid N-terminal region (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence with VP3, in some cases a region beginning at amino acid 217 (e.g., AA217-736).

[0077] The AAV VP3 structure contains a highly conserved region common to all serotypes, a core eight-stranded β-barrel motif (βB-βI), and a small α-helix (αA). The loop region inserted between the β strands consists of the characteristic HI loop between β strands H and I, the DE loop between β strands D and E, and nine variable regions (VRs) that form the apex of the loops. These VRs are found on the capsid surface and may be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigen specificity. For example, the AA455 loop, which protrudes furthest from the capsid surface, has been implicated in neutralizing antibody binding and is thought to play a significant role in cell surface receptor binding, either naturally or through interaction with the 588 loop.

[0078] Recombinant adeno-associated viruses (rAAVs) are disclosed herein having an AAV capsid containing a modified AAV capsid protein in the AA455 loop that provides desired tropism, characterized by higher efficiency and specificity for transduction into specific cell types, such as brain cell types. In particular, the modified AAV capsids disclosed herein enable rAAV-mediated transduction of heterologous genes (e.g., transgenes), while preventing off-target transduction events in peripheral organs such as the liver by detargeting others. Also disclosed herein are rAAVs having AAV capsids containing modified AAV capsid proteins that provide tropism, characterized by higher efficiency and specificity for transduction into specific organs or environments, such as the lungs, intestines, stomach, heart, muscle, adipose tissue, spleen, kidney, or reproductive organs (e.g., testes or ovaries). The rAAVs described herein are useful for a wide range of applications, including, but not limited to, disease treatment. composition

[0079] rAAV capsid and variant AAV capsid proteins

[0080] Disclosed herein are recombinant AAVs (rAAVs) having variant capsid proteins (e.g., rAAV capsid proteins) engineered with modified capsid proteins (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using a method of the present disclosure (e.g., M-CREATE). In some embodiments, the AAV capsid proteins are used in methods of delivering therapeutic nucleic acids (e.g., transgenes) to a subject. In some cases, the rAAV capsid proteins have desired AAV tropism, making them particularly suitable for certain therapeutic applications, for example, for treating diseases or disorders in a subject, such as those disclosed herein.

[0081] rAAV capsid proteins, such as those listed in Tables 3-4, are engineered for optimized entry into and through the blood-brain barrier (BBB) ​​of a subject upon systemic administration of the rAAV to the subject. Previous AAV-mediated delivery methods for therapeutic transgenes to the brain required intracranial injection, an invasive procedure that is uncomfortable and, in some cases, painful for the subject. For example, intracranial injection can cause cerebral hemorrhage. In addition, intracranial delivery is poorly diffused and highly heterogeneous. The rAAV capsid proteins listed in Tables 3-4 have been engineered to have tropism that obviates the need for intracranial injection while also achieving widespread, efficient transduction of the capsid-encapsulated transgene. In particular, the tropism includes at least one of increased specificity and improved efficiency (e.g., viral transduction efficiency) for the subject's central nervous system (CNS) and / or the subject's peripheral nervous system (PNS) compared to the corresponding parent or reference AAV. The rAAVs disclosed herein, such as those shown in Table 1, can also avoid off-target viral transduction by detargeting certain peripheral organs (e.g., the liver).

[0082] Also disclosed herein are rAAVs with engineered capsid proteins that are optimized for targeting to specific organs or tissues within a subject. In some embodiments, the organ is the heart. In some embodiments, the organ is the lung. In some embodiments, the organ is the liver. In some embodiments, the organ is the intestine. In some embodiments, the organ is the stomach. In some embodiments, the organ is the spleen. In some embodiments, the organ is the kidney. In some embodiments, the tissue is fat (adipose). In some embodiments, the tissue is muscle. The muscle can be cardiac muscle. The muscle can be skeletal muscle. In some embodiments, the organ is the pancreas. In some embodiments, the organ is a reproductive organ, e.g., the testes or ovaries. In further embodiments, the rAAVs disclosed herein can also reduce or avoid off-target viral transduction by detargeting certain peripheral organs. In a non-limiting example, the rAAV of this embodiment, Table 1, has increased specificity and transduction for the brain (e.g., the target environment) compared to a reference AAV (e.g., AAV9), but has very low transduction efficiency and no specificity for the liver (e.g., the non-target environment).

[0083] The engineered AAV capsid proteins described herein may, in some cases, have an amino acid insertion or substitution that is heterologous to the parent AAV capsid protein at the amino acid position of the insertion or substitution. In some embodiments, the amino acid is not endogenous to the parent AAV capsid protein at the amino acid position of the insertion or substitution. The amino acid may also be an amino acid that naturally occurs at the same or equivalent amino acid position in a different AAV capsid protein as the insertion or substitution.

[0084] Typically, the insertion or substitution involves a 5-, 6-, or 7-amino acid polymer (represented as a 5-mer, 6-mer, or 7-mer, respectively) inserted or substituted in the 455 loop of the parent AAV capsid protein. The 7-mers described herein, in which each of the seven amino acids is encoded by the deoxyribose nucleic acid (DNA) sequence NNK, were advantageously generated using polymerase chain reaction (PCR) with degenerate primers. "N" is any of the four DNA nucleotides, and K is guanine (G) or thymine (T). This method of generating random 7-mer amino acid sequences allows for 1.28 billion possible combinations at the protein level. Because the resulting 7-mers are random, some amino acids in the 7-mer may naturally occur at the amino acid position of the AAC capsid protein, while other amino acids may be different.

[0085] Recombinant AAVs (rAAVs) were generated, each containing a unique 7-mer in the 455 loop, each encapsulating a reporter gene that allows for selective amplification and recovery of sequences that effectively transduce the reporter gene into the target in vivo environment of the transgenic animal when administered systemically to multiple transgenic animals. Provided herein are 7-mers that are found to be positively enriched in the target in vivo environment (e.g., central nervous system, lungs) and negatively enriched in the non-target in vivo environment (e.g., liver). "Enrichment" refers to the prevalence of a given 7-mer in tissues of the in vivo environment compared to its prevalence in the viral library administered to the transgenic animal. An enrichment score greater than 0 indicates positive enrichment. An enrichment score less than 0 indicates negative enrichment. An enrichment score of 0 indicates that the variant is absent in the tissue. A subset of rAAVs with the desired enrichment profile were individually tested in vivo to determine precise systemic expression (e.g., specificity and transduction efficiency). rAAVs from this subset exhibiting the desired tropism, including increased specificity and, in some cases, improved transduction efficiency, are believed to be uniquely suited for targeted rAAV-mediated transgene delivery, useful for a wide variety of purposes (e.g., therapeutics, diagnostics, scientific discovery).

[0086] rAAV particles with the 7-mer insertions or substitutions described herein have improved transduction efficiency into the target in vivo environment (e.g., tissue or cell type). In some cases, the improved transduction efficiency comprises a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 75-fold, or 100-fold or more improvement compared to a reference AAV, e.g., a corresponding parental capsid protein. In some cases, the increase in transduction efficiency is at least 30-fold. In some cases, the increase in transduction efficiency is at least 40-fold. In some cases, the increase in transduction efficiency is at least 50-fold. In some cases, the increase in transduction efficiency is at least 60-fold. In some cases, the increase in transduction efficiency is at least 80-fold. In some cases, the increase in transduction efficiency is at least 90-fold. In some cases, the increase in transduction efficiency is at least 100-fold.

[0087] rAAV particles with 7-mer insertions or substitutions described herein have reduced transduction efficiency into off-target in vivo environments (e.g., tissues or cell types). In some cases, off-target gene transfer is 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, 35 fold, 36 fold, 37 fold, 38 fold, 39 fold, 40 fold, 41 fold, 42 fold, 43 fold, 44 fold, 45 fold, 46 fold, 47 fold, 48 fold, 49 fold, 50 fold, 51 fold, 52 fold, 53 fold, 54 fold, 55 fold, 56 fold, 57 fold, 58 fold, 59 fold, 60 fold, 61 fold, 62 fold, 63 fold, 64 fold, 65 fold, 66 fold, 67 fold, 68 fold, 69 fold, 70 fold, 71 fold, 72 fold, 73 fold, 74 fold, 75 fold, 76 fold, 77 fold, 78 fold, 79 fold, 80 fold, 81 fold, 82 fold, 83 fold, 84 fold, 85 fold, 86 fold, 87 fold, 88 fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, 75-fold, or 100-fold or less reduction in off-target gene introgression. In some cases, off-target gene introgression is reduced by at least 20-fold. In some cases, off-target gene introgression is reduced by at least 30-fold. In some cases, off-target gene introgression is reduced by at least 40-fold. In some cases, off-target gene introgression is reduced by 50-fold or less. In some cases, off-target gene transfer is reduced by at least 60-fold. In some cases, off-target gene transfer is reduced by at least 80-fold. In some cases, off-target gene transfer is reduced by at least 90-fold. In some cases, off-target gene transfer is reduced by at least 100-fold.

[0088] rAAV particles with the 7-mer insertions or substitutions described herein have increased or decreased specificity for the target in vivo environment (e.g., tissue or cell type) compared to a reference AAV, such as a corresponding parent capsid protein. Detecting whether an rAAV has greater or less specificity for the target in vivo environment than a reference AAV involves measuring the level of a gene expression product (e.g., RNA or protein) expressed from a heterologous nucleic acid encapsidated by the rAAV in a tissue sample obtained from the target in vivo environment in a subject, and comparing the measured level with a reference level (e.g., a gene expression product expressed from a heterologous nucleic acid encapsidated by a reference AAV (e.g., AAV9)). Suitable methods for measuring the expression of gene expression products include luciferase reporter assays and quantitative polymerase chain reaction (qPCR).

[0089] Increased specificity correlates with increased enrichment in the target in vivo environment, which enrichment is represented in some cases by the enrichment scores shown in Figures 2-14 herein. As a non-limiting example, AAV.CAP-B10 (SEQ ID NO: 3943; DGAATKN), which is shown herein to be positively enriched in the brain (enrichment score of approximately 0.950) and not enriched in the liver (enrichment score of about 0) relative to the reference AAV9, also exhibited increased reporter gene expression (e.g., as measured by luciferase reporter assay) in the brain but not in the liver relative to the reference AAV9. Without being bound by any particular theory, the inventors of the present disclosure would expect this correlation to be observed for all rAAVs disclosed herein, and further would expect that a more significant enrichment score (whether negative or positive) would correlate with more significant specificity for the in vivo environment(s) as indicated by the measured levels of gene expression product in the in vivo environment(s).

[0090] As disclosed herein, transduction efficiency can be measured by at least one of (1) the number of cells in the target in vivo or non-target in vivo environment that express a heterologous nucleic acid encapsidated by the modified AAV capsid protein disclosed herein, and (2) the amount of expression of the heterologous nucleic acid in a single cell. If an increased presence or level of rAAV-mediated transduction into the target in vivo environment is observed compared to a reference AAV, specificity for the target in vivo environment can be inferred. If an absence or reduced level of rAAV-mediated transduction into the non-target in vivo environment is observed compared to a reference AAV, a lack or reduced specificity for the non-target in vivo environment can be inferred.

[0091] Natural AAV serotypes have been shown to exhibit distinct tropism for various tissues and organs. The rAAV particles of the present disclosure contain a modified (e.g., chimeric) AAV9 capsid. AAV9 has been shown to exhibit stronger tropism for the central nervous system (CNS), lung, heart, liver, muscle, and testis compared to AAV serotypes 1-8. Direct comparisons of AAV serotypes 1-9 administered intravenously via tail vein injection in mice showed that AAV9 exhibited increased specificity for viral transduction for the liver, lung, muscle, brain, testis, and heart compared to AAV serotypes 1-8. Additionally, AAV9 exhibited higher transduction efficiency levels for the liver, muscle, brain, and heart compared to AAV serotypes 1-8. However, the 7-mers disclosed herein can be incorporated into capsid proteins from AAV serotypes other than AAV9, such as AAV2 or AAV8.

[0092] AAV capsid proteins from natural AAV serotypes, such as AAV9, which have specific tropism for the liver, activate the innate immune response, which in some cases causes a severe inflammatory response in subjects that can lead to multiple organ failure. By improving the transduction efficiency of natural AAV serotypes for target in vivo tissues (e.g., the brain) and reducing the specificity of AAV capsid proteins for the liver, the rAAV particles of the present disclosure reduce the immunogenic properties of AAV-mediated transgene delivery and prevent activation of the innate immune response.

[0093] rAAV may contain chimeric AAV capsids. A "chimeric" AAV capsid refers to a capsid having an exogenous amino acid or amino acid sequence (e.g., a 7-mer substitution). rAAV may contain mosaic AAV capsids. A "mosaic" AAV capsid refers to a capsid composed of two or more capsid proteins or polypeptides, each derived from a different AAV serotype. rAAV may also be the result of capsid conversion, which in some cases refers to the packaging of inverted terminal repeats (ITRs) from a first serotype into a capsid of a second serotype, where the first and second serotypes are not the same. In some cases, the capsid gene of a parent AAV serotype is pseudotyped, meaning that the ITRs from a first AAV serotype (e.g., AAV2) are used within a capsid from a second AAV serotype (e.g., AAV9), where the first and second AAV serotypes are not the same. As a non-limiting example, a pseudotyped AAV serotype containing AAV2 ITRs and AAV9 capsid proteins may be designated AAV2 / 9. rAAV may additionally or alternatively contain a capsid engineered to express an exogenous ligand-binding moiety (e.g., a receptor) or a modified native receptor. For example, the 7-mer substitutions described herein can alter the ligand-binding function of the parent AAV to provide increased specificity for a particular cell surface antigen.

[0094] In some cases, the reference AAV disclosed herein is AAV9 because it provides the best comparison of natural AAVs with strong tropism for the CNS, lungs, heart, liver, muscle, and testes. However, the reference AAV may be of any serotype, for example, a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof. For example, the reference AAV may have a serotype selected from the group consisting of AAV-PHP.B, AAV-PHP.eB, and AAV-PHP.S. In many cases, the reference AAV is a parent AAV, for example, a corresponding unmodified AAV from which the variant AAV was engineered.

[0095] The rAAV capsid proteins of the present disclosure contain one or more amino acid substitutions in the amino acid sequence of an AAV capsid protein. The AAV capsid protein from which the engineered AAV capsid proteins of the present disclosure are produced is referred to as the "parent" AAV capsid protein or the "corresponding unmodified capsid protein." In some cases, the parent AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. The complete genome of AAV-1 is set forth in GenBank accession number NC_002077, the complete genome of AAV-2 is set forth in GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is set forth in GenBank accession number NC_1829, the complete genome of AAV-4 is set forth in GenBank accession number NC_001829, the AAV-5 genome is set forth in GenBank accession number AF085716, the complete genome of AAV-6 is set forth in GenBank accession number NC_001862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively, and the AAV-9 genome is set forth in Gao et al., J. Virol., 78: 6381-6388 (2004), the AAV-10 genome is set forth in Mol. Ther., 13(1): 67-76 (2006), the AAV-11 genome is set forth in Virology, 330(2): 375-383 (2004), a portion of the AAV-12 genome is set forth in Genbank accession number DQ813647, and a portion of the AAV-13 genome is set forth in Genbank accession number EU285562.

[0096] In some cases, the parent AAV is derived from an AAV having a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV 12. In other words, the parent AAV is a variant of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. An AAV variant refers to an AAV that has 70% or more sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, for example, 80%, 85%, or 90% or more, 91%, 92%, 93%, 94%, 95% or more, and in some cases 96%, 97%, 98%, or 99% sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. A variant can include, for example, an AAV that contains a heterologous amino acid or heterologous amino acid sequence within the amino acid sequence of an AAV capsid protein. The heterologous amino acid may also naturally occur in a different AAV capsid protein. In some cases, the parent AAV capsid is described in US2019 / 0055578, US2018 / 0230489, US2017 / 0067908, US2019 / 0048041, US9585971, or US2017 / 0166926, all of which are incorporated herein in their entireties.

[0097] In some cases, the parent AAV is AAV9. In some cases, the amino acid sequence of the AAV9 capsid protein comprises SEQ ID NO: 1. In some cases, the parent AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 1, or to a portion of SEQ ID NO: 1. In some cases, the parent AAV capsid protein comprises the entire VP1 region (e.g., amino acids 1-736) set forth in SEQ ID NO: 1. In some cases, the parent AAV capsid protein comprises amino acids 217-736 of SEQ ID NO: 1, which is a common region found in VP1, VP2, and VP3 AAV9 capsid proteins. In some cases, the parent AAV capsid protein comprises amino acids 64-736 of SEQ ID NO: 1, which is a common region found in VP1 and VP2. The parent AAV capsid protein sequences are 1-736, 10-736, 20-736, 30-736, 40-736, 50-736, 60-736, 70-736, 80-736, 90-736, 100-736, 110-736, 120-736, 130-736, 140-736, 150-736, 160-736, 170-736, 180-736, 190-736, 200-736, 210-736, 220-736, 230-736, 240-736, 250-736, 260-736, 270-736, 280-736, 290-736, 300-736, 310-736, 320-736, 330-736, 340-736, 350-736, 360-736, 370-736, 380-736, 390-736, 410-736, 420-736, 430-736, 440-736, 450-736, 460-736, 470-736, 480-736, 490-736, 500-736, 510-736, 520-736, 530-736, 540-736, 550-736, 560-736, 570-736 36, 240 to 736, 250 to 736, 260 to 736, 270 to 736, 280 to 736, 290 to 736, 300 to 736, 310 to 736, 320 to 736, 330 to 736, 340 to 736, 350 to 736, 360 to 736, 370 to 736, 380 to 736, 390 to 736, 400 to 736, 410 to 736, 420 to 736, 430 to 736, 440 to 736, and 450 to 736.

[0098] In some cases, the parent AAV is AAV5. In some cases, the amino acid sequence of the AAV5 capsid protein comprises SEQ ID NO: 2. In some cases, the parent AAV capsid protein sequence is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 2, or to a portion of SEQ ID NO: 2. The parent AAV capsid protein sequence is selected from the group consisting of 1-724, 10-724, 20-724, 30-724, 40-724, 50-724, 60-724, 70-724, 80-724, 90-724, 100-724, 110-724, 120-724, 130-724, 140-724, 150-724, 160-724, 170-724, 180-724, 190-724, 200-724, 210-724, 220-724, 230-724, 240-724, 250-724, 260-724, 270-724, 280-724, 290-724, 300-724, 310-724, 320-724, 330-724, 340-724, 350-724, 360-724, 370-724, 380-724, 390-724, 400-724, 410-724, 420-724, 430-724, 440-724, 450-724, 460-724, 470-724, 480-724, 490-724, 500-724, 510-724, 520-724, 530-724, 540-724, 550-724, 5 4, 240 to 724, 250 to 724, 260 to 724, 270 to 724, 280 to 724, 290 to 724, 300 to 724, 310 to 724, 320 to 724, 330 to 724, 340 to 724, 350 to 724, 360 to 724, 370 to 724, 380 to 724, 390 to 724, 400 to 724, 410 to 724, 420 to 724, 430 to 724, 440 to 724, and 450 to 724.

[0099] In some cases, the parent AAV is AAV-PHP.B. In some cases, the amino acid sequence of the AAV-PHP.B capsid protein comprises SEQ ID NO: 3. In some cases, the parent AAV capsid protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 3, or to a portion of SEQ ID NO: 3. The parent AAV capsid protein sequences are 1-743, 10-743, 20-743, 30-743, 40-743, 50-743, 60-743, 70-743, 80-743, 90-743, 100-743, 110-743, 120-743, 130-743, 140-743, 150-743, 160-743, 170-743, 180-743, 190-743, 200-743, 210-743, 220-743, 230-743, 240-743, 250-743, 260-743, 270-743, 280-743, 290-743, 300-743, 310-743, 320-743, 330-743, 340-743, 350-743, 360-743, 370-743, 380-743, 390-743, 400-743, 410-743, 420-743, 430-743, 440-743, 450-743, 460-743, 470-743, 480-743, 490-743, 500-743, 510-743, 520-743, 530-743, 540-743, 550-743, 560-743 3, 240 to 743, 250 to 743, 260 to 743, 270 to 743, 280 to 743, 290 to 743, 300 to 743, 310 to 743, 320 to 743, 330 to 743, 340 to 743, 350 to 743, 360 to 743, 370 to 743, 380 to 743, 390 to 743, 400 to 743, 410 to 743, 420 to 743, 430 to 743, 440 to 743, and 450 to 743.

[0100] In some cases, the parent AAV is AAV.PHP.S. In some cases, the amino acid sequence of the AAV-PHP.S capsid protein comprises SEQ ID NO: 4. In some cases, the parent capsid protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 4, or to a portion of SEQ ID NO: 4. The parent AAV capsid protein sequence is selected from the group consisting of 1-743, 10-743, 20-743, 30-743, 40-743, 50-743, 60-743, 70-743, 80-743, 90-743, 100-743, 110-743, 120-743, 130-743, 140-743, 150-743, 160-743, 170-743, 180-743, 190-743, 200-743, 210-743, 220-743, 230-743, 240-743, 250-743, 260-743, 270-743, 280-743, 290-743, 300-743, 310-743, 320-743, 330-743, 340-743, 350-743, 360-743, 370-743, 380-743, 390-743, 400-743, 410-743, 420-743, 430-743, 440-743, 450-743, 460-743, 470-743, 480-743, 490-743, 500-743, 510-743, 520-743, 530-743, 540-743, 550-743, 5 3, 240 to 743, 250 to 743, 260 to 743, 270 to 743, 280 to 743, 290 to 743, 300 to 743, 310 to 743, 320 to 743, 330 to 743, 340 to 743, 350 to 743, 360 to 743, 370 to 743, 380 to 743, 390 to 743, 400 to 743, 410 to 743, 420 to 743, 430 to 743, 440 to 743, and 450 to 743.

[0101] In some cases, the parent AAV is AAV-PHP.eB. In some cases, the amino acid sequence of the AAV-PHP.eB capsid protein comprises SEQ ID NO: 5. In some cases, the AAV parent capsid protein is 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous to SEQ ID NO: 5, or to a portion of SEQ ID NO: 5. The parent AAV capsid protein sequence is selected from the group consisting of 1-743, 10-743, 20-743, 30-743, 40-743, 50-743, 60-743, 70-743, 80-743, 90-743, 100-743, 110-743, 120-743, 130-743, 140-743, 150-743, 160-743, 170-743, 180-743, 190-743, 200-743, 210-743, 220-743, 230-743, 240-743, 250-743, 260-743, 270-743, 280-743, 290-743, 300-743, 310-743, 320-743, 330-743, 340-743, 350-743, 360-743, 370-743, 380-743, 390-743, 400-743, 410-743, 420-743, 430-743, 440-743, 450-743, 460-743, 470-743, 480-743, 490-743, 500-743, 510-743, 520-743, 530-743, 540-743, 550-743, 5 3, 240 to 743, 250 to 743, 260 to 743, 270 to 743, 280 to 743, 290 to 743, 300 to 743, 310 to 743, 320 to 743, 330 to 743, 340 to 743, 350 to 743, 360 to 743, 370 to 743, 380 to 743, 390 to 743, 400 to 743, 410 to 743, 420 to 743, 430 to 743, 440 to 743, and 450 to 743.

[0102] In some cases, the insertion is introduced after any one amino acid position selected from 452 to 458 (VP1 numbering) in AAV9, or the equivalent amino acid position in a variant thereof. The amino acid sequence of AAV9 (VP1) is set forth in SEQ ID NO: 1. In some cases, the substitution is an amino acid substitution at any one amino acid position selected from 452 to 458 (VP1 numbering) in AAV9, or the equivalent amino acid position in a variant thereof. In some cases, the substitution is an amino acid substitution at amino acid position 452. In some cases, the substitution is an amino acid substitution at amino acid position 453. In some cases, the substitution is an amino acid substitution at amino acid position 454. In some cases, the substitution is an amino acid substitution at amino acid position 455. In some cases, the substitution is an amino acid substitution at amino acid position 456. In some cases, the substitution is an amino acid substitution at amino acid position 457. In some cases, the substitution is an amino acid substitution at amino acid position 458. In some cases, the substitution comprises the substitution of one amino acid. In some cases, the substitution comprises the substitution of two amino acids. In some cases, the substitution comprises the substitution of three amino acids. In some cases, the substitution comprises the substitution of four amino acids. In some cases, the substitution comprises the substitution of five amino acids. In some cases, the substitution comprises the substitution of six amino acids. In some cases, the substitution comprises the substitution of seven amino acids. The substitutions or insertions of two or more amino acids may be contiguous. The substitutions or insertions of two or more amino acids may be non-contiguous.

[0103] The amino acids at positions 452-458 according to VP1 numbering in SEQ ID NO:1 (AAV9), SEQ ID NO:3 (AAV-PHP.B), SEQ ID NO:4 (AAV-PHP.S), and SEQ ID NO:5 (AAV-PHP.eB) are designated as "NGSGQNQ" (SEQ ID NO:545). In some cases, the amino acids at positions 452-458 in SEQ ID NO:2 (AAV5) are designated as "NLAGRYA" (SEQ ID NO:45478). In some cases, the substitution is at amino acid position N452, G453, S454, G455, Q456, N457, or Q458 of an AAV9 capsid protein or variant thereof, or a combination thereof. In some cases, the substitution is at amino acid position N452, L453, A454, G455, G455, R456, Y457, or A458 of an AAV5 capsid protein or variant thereof, or a combination thereof. In some cases, the substituted amino acid(s) are located at amino acids 452-453, and may include substituted amino acids at positions 452 and 453. In some cases, two amino acids are substituted at amino acid positions selected from the group consisting of 452-453, 453-455, 455-456, 456-457, and 457-458. In some cases, three amino acids are substituted at amino acid positions selected from the group consisting of 452-454, 453-455, 454-456, 455-457, and 456-458. In some cases, four amino acids are substituted at amino acid positions selected from the group consisting of 452-455, 452-456, 453-457, and 454-458. In some cases, five amino acids are substituted at amino acid positions selected from the group consisting of 452-456, 453-457, and 454-458. In some cases, six amino acids are substituted at amino acid positions 452-457 or 453-458. In some cases, seven amino acids are substituted at amino acid positions 452-458.

[0104] The rAAV capsid protein described herein can be isolated and purified.AAV can be isolated and purified by standard methods in the art, for example, by column chromatography or cesium chloride gradient.Methods for purifying AAV from helper viruses are known in the art, and can include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69: 427-443 (2002); U.S. Patent No. 6,566,118 and WO98 / 09657.

[0105] The rAAV capsid protein can be conjugated to a nanoparticle, a second molecule, or a viral capsid protein. In some cases, the nanoparticle or viral capsid protein encapsulates a therapeutic nucleic acid as described herein. In some cases, the second molecule is a therapeutic agent, such as a small molecule, an antibody, an antigen-binding fragment, a peptide, or a protein, such as those described herein. In some cases, the second molecule is a detectable moiety. For example, the modified AAV capsid protein conjugated to a detectable moiety can be used for in vitro, ex vivo, or in vivo biomedical research applications, and the detectable moiety can be used to visualize the modified capsid protein. The modified AAV capsid protein conjugated to a detectable moiety can also be used for diagnostic purposes.

[0106] Central nervous system-targeting rAAV capsid protein

[0107] Disclosed herein are recombinant AAVs (rAAVs) having variant capsid proteins with desired tropism characterized by substitution or insertion of at least one amino acid at the above-mentioned amino acid positions in the corresponding parent AAV capsid protein. In some cases, the rAAV capsid protein has desired tropism, including increased specificity for a target in vivo environment in a subject. In some cases, the target in vivo environment includes the central nervous system (CNS) or peripheral nervous system (PNS). One of the many advantages of the tropism of the rAAV capsid proteins described herein is their ability to target the CNS and penetrate the blood-brain barrier (BBB). In some cases, the desired tropism further includes decreased specificity for a non-target in vivo environment compared to the tropism of the corresponding parent AAV capsid protein. In some cases, the non-target in vivo environment includes a peripheral organ such as the liver. Another advantage of the rAAV capsid proteins described herein is their ability to avoid expression of heterologous nucleic acids in the liver, thereby reducing liver toxicity and the viral dose required for therapeutic efficacy.

[0108] The in vivo environment can be a cell. The cell can be a cell type selected from the group consisting of central nervous system (CNS) cells and peripheral nervous system (PNS) cells. Non-limiting examples of CNS cells include neurons and glial cells. Glial cells can be selected from the group consisting of oligodendrocytes, ependymal cells, and astrocytes. Non-limiting examples of PNS cells include neurons and glial cells. Glial cells can be selected from the group consisting of Schwann cells, satellite cells, and enteric glial cells.

[0109] The in vivo environment can be a tissue, for example, a tissue from an organ or organ system. The organ can be the brain or spinal cord. The tissue can be a region of an organ, such as the cerebrum, cerebellum, brainstem, cortex, striatum, thalamus, lateral ventricle, putamen, hypothalamus, medulla, pons, hippocampus, amygdala, motor cortex, or a combination thereof.

[0110] Disclosed herein is an rAAV capsid protein comprising an amino acid sequence that provides increased specificity and / or improved transduction efficiency for the CNS or PNS of a subject compared to a reference AAV. In some cases, the amino acid sequence also provides decreased specificity or reduced transduction efficiency for peripheral organs (e.g., the liver) compared to the reference AAV. In some cases, the amino acid sequence includes at least one amino acid substitution at an amino acid position selected from the group consisting of 452, 453, 454, 455, 456, 457, and 458 of the amino acid sequence of the AAV capsid protein (e.g., parent AAV). In some embodiments, the parent AAV is AAV9 or a variant thereof.

[0111] Provided herein is an rAAV capsid protein having increased specificity for the CNS or PNS and / or improved transduction efficiency compared to a reference AAV, the rAAV capsid protein comprising an amino acid (X1), where X1 is A, D, G, L, N, Q, S, or T. The rAAV capsid protein can comprise at least two amino acids, where X1 is A, D, G, L, N, Q, S, or T, and X2 is A, G, N, P, Q, R, S, or T. The rAAV can comprise at least three amino acids, where X1 is A, D, G, L, N, Q, S, or T, where X2 is A, G, N, P, Q, R, S, or T, and where X3 is A, D, G, N, S, or T. The rAAV can comprise at least four amino acids, where X1 is A, D, G, L, N, Q, S, or T, X2 is A, G, N, P, Q, R, S, or T, X3 is A, D, G, N, S, or T, and X4 is A, D, G, K, N, P, Q, S, or T. The rAAV can comprise at least five amino acids, where X1 is A, D, G, L, N, Q, S, or T, X2 is A, G, N, P, Q, R, S, or T, X3 is A, D, G, N, S, or T, X4 is A, D, G, K, N, P, Q, S, or T, and X5 is A, G, K, N, P, R, S, or T. The rAAV can comprise at least six amino acids, wherein X1 is A, D, G, L, N, Q, S, or T; X2 is A, G, N, P, Q, R, S, or T; X3 is A, D, G, N, S, or T; X4 is A, D, G, K, N, P, Q, S, or T; X5 is A, G, K, N, P, R, S, or T; and X6 is A, G, K, N, P, R, S, T, or V. The rAAV can comprise at least seven amino acids, where X1 is A, D, G, L, N, Q, S, or T, X2 is A, G, N, P, Q, R, S, or T, X3 is A, D, G, N, S, or T, X4 is A, D, G, K, N, P, Q, S, or T, X5 is A, G, K, N, P, R, S, or T, X6 is A, G, K, N, P, R, S, T, or V, and X7 is A, G, K, L, R, S, T, or V.

[0112] Provided herein are rAAV capsid proteins that have increased specificity and / or improved transduction efficiency for the CNS or PNS compared to a reference AAV, and decreased specificity and / or reduced transduction efficiency for peripheral organs (e.g., the liver) compared to a reference AAV. The rAAV, in some cases, comprises an amino acid (X1), where X1 is G, A, V, N, S, H, L, E, or Q. The rAAV capsid protein can comprise at least two amino acids, where X1 is G, A, V, N, S, H, L, E, or Q, and X2 is A, I, T, P, N, R, T, G, S, K, H, or Q. An rAAV can comprise at least three amino acids, where X1 is G, A, V, N, S, H, L, E, or Q, X2 is A, I, T, P, N, R, T, G, S, K, H, or Q, and X3 is S, N, D, A, T, H, K, or Q. An rAAV can comprise at least four amino acids. In some embodiments, X1 is G, A, V, N, S, H, L, E, or Q, X2 is A, I, T, P, N, R, T, G, S, K, H, or Q, X3 is N, D, A, T, H, K, Q, and X4 is N, G, T, S, D, P, R, T, or Q. The rAAV can comprise at least five amino acids, wherein X1 is G, A, V, N, S, H, L, E, or Q; X2 is A, I, T, P, N, R, T, G, S, K, H, or Q; X3 is N, D, A, T, H, K, Q; X4 is N, G, T, S, D, P, R, T, or Q; and X5 is S, K, P, T, G, D, P, N, or V. The rAAV can comprise at least six amino acids, wherein X1 is G, A, V, N, S, H, L, E, or Q; X2 is A, I, T, P, N, R, T, G, S, K, H, or Q; X3 is N, D, A, T, H, K, Q; X4 is N, G, T, S, D, P, R, T, or Q; X5 is S, K, P, T, G, D, P, N, or V; and X6 is T, A, R, S, N, G, D, P, or V.The rAAV can comprise at least seven amino acids, wherein X1 is G, A, V, N, S, H, L, E, or Q; X2 is A, I, T, P, N, R, T, G, S, K, H, or Q; X3 is N, D, A, T, H, K, Q; X4 is N, G, T, S, D, P, R, T, or Q; X5 is S, K, P, T, G, D, P, N, or V; X6 is T, A, R, S, N, G, D, P, or V; and X7 is G, N, S, L, A, E, K, or Q.

[0113] In some cases, the rAAV comprises an amino acid (X1), where X1 is A, D, E, G, H, M, N, Q, S, T, or V. The rAAV can comprise at least two amino acids, where X1 is A, D, E, G, H, M, N, Q, S, T, or V, and X2 is A, D, E, G, K, N, Q, S, T, or V. The rAAV can comprise at least three amino acids, where X1 is A, D, E, G, H, M, N, Q, S, T, or V, where X2 is A, D, E, G, K, N, Q, S, T, or V, and X3 is A, D, E, G, K, N, Q, S, T, or V. The rAAV can comprise at least four amino acids, where X1 is A, D, E, G, H, M, N, Q, S, T, or V; X2 is A, D, E, G, K, N, Q, S, T, or V; X3 is A, D, E, G, K, N, Q, S, T, or V; and X4 is A, D, E, G, K, N, P, Q, S, or T. The rAAV can comprise at least five amino acids, wherein X1 is A, D, E, G, H, M, N, Q, S, T, or V; X2 is A, D, E, G, K, N, Q, S, T, or V; X3 is A, D, E, G, K, N, Q, S, T, or V; X4 is A, D, E, G, K, N, P, Q, S, or T; and X5 is A, D, E, G, H, K, N, P, Q, S, or T. The rAAV can comprise at least six amino acids, wherein X1 is A, D, E, G, H, M, N, Q, S, T, or V; X2 is A, D, E, G, K, N, Q, S, T, or V; X3 is A, D, E, G, K, N, Q, S, T, or V; X4 is A, D, E, G, K, N, P, Q, S, or T; X5 is A, D, E, G, H, K, N, P, Q, S, or T; and X6 is A, D, E, G, K, N, P, S, T, or V.The rAAV can comprise at least seven amino acids, wherein X1 is A, D, E, G, H, M, N, Q, S, T, or V; X2 is A, D, E, G, K, N, Q, S, T, or V; X3 is A, D, E, G, K, N, Q, S, T, or V; X4 is A, D, E, G, K, N, P, Q, S, or T; X5 is A, D, E, G, H, K, N, P, Q, S, or T; X6 is A, D, E, G, K, N, P, S, T, or V; and X7 is A, E, G, H, K, L, N, Q, S, T, or V.

[0114] In some cases, X1, X2, X3, X4, X5, X6, and X7 are consecutive (X1-X2-X3-X4-X5-X6-X7). Alternatively, X1, X2, X3, X4, X5, X6, and X7 are not consecutive. In some embodiments, any two of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any three of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any four of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any five of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any six of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any seven of X1, X2, X3, X4, X5, X6, and X7 are contiguous. In some embodiments, X1 is at amino acid 452 in the AAV9 capsid protein or its variant. In some embodiments, X2 is at amino acid 453 in the AAV9 capsid protein or its variant. In some embodiments, X3 is at amino acid 454 in the AAV9 capsid protein or its variant. In some embodiments, X4 is at amino acid 455 in the AAV9 capsid protein or its variant. In some embodiments, X5 is at amino acid 456 in the AAV9 capsid protein or its variant. In some embodiments, X6 is at amino acid 457 in the AAV9 capsid protein or its variant. In some embodiments, X7 is at amino acid 458 in the AAV9 capsid protein or its variant. In some embodiments, the amino acid sequence of the rAAV is such that amino acids 452 to 458 of the parent AAV9 capsid protein or a variant thereof do not consist of the amino acid sequence ILGTGTS (SEQ ID NO: 45479) or QSSQTPR (SEQ ID NO: 45479).

[0115] Disclosed herein are rAAV capsid proteins comprising one, two, three, four, five, six, or seven amino acid substitutions in the amino acid sequence set forth in any one of SEQ ID NOs: 11-12739. In some embodiments, the rAAV capsid protein comprises two amino acid substitutions in the amino acid sequence set forth in any one of SEQ ID NOs: 11-12739. In some embodiments, the rAAV capsid protein comprises three amino acid substitutions in the amino acid sequence set forth in any one of SEQ ID NOs: 11-12739. In some embodiments, the rAAV capsid protein comprises four amino acid substitutions in the amino acid sequence set forth in any one of SEQ ID NOs: 11-12739. In some embodiments, the rAAV capsid protein comprises five amino acid substitutions in the amino acid sequence set forth in any one of SEQ ID NOs: 11-12739. In some embodiments, the rAAV capsid protein comprises six amino acid substitutions in the amino acid sequence set forth in any one of SEQ ID NOs: 11-12739.

[0116] Disclosed herein are rAAV capsid proteins with increased specificity for the central nervous system (CNS) or peripheral nervous system (PNS) and / or improved transduction efficiency. Exemplary substitutions include N452D, N452A, N452G, N452L, N452Q, N452S, N452T, G453I, G453N, G453S, G453P, G453R, G453T, S454A, S454Q, S454D, S454G, S454N, S454T, G455A, G455S, G455D, and G455K in the amino acid sequence of an AAV9 capsid protein or a variant thereof. , G455N, G455P, G455Q, G455T, Q456T, Q456S, Q456A, Q456G, Q456K, Q456N, Q456R, Q456P, N457K, N457A, N457G, N457P, N457R, N457S, N457T, N457V, Q458N, Q458A, Q458G, Q458K, Q458L, Q458R, Q458S, Q458T, or Q458V. In some embodiments, the substitution is at least or about 1, 2, 3, 4, 5, 6, or 7 amino acids of the amino acid sequence LQTSSPG (SEQ ID NO: 2933). In some embodiments, the amino acid sequence is QQGKQSV (SEQ ID NO: 79). In some embodiments, the amino acid sequence is SINTKTN (SEQ ID NO:45475). In some embodiments, the amino acid sequence is SNGTKQT (SEQ ID NO:442). In some embodiments, the amino acid sequence is GSGKTAA (SEQ ID NO:88). In some embodiments, the amino acid sequence is MGDKPTR (SEQ ID NO:2466). In some embodiments, the amino acid sequence is DGAATKN (SEQ ID NO:3943). In some embodiments, the amino acid sequence is QPSGGNT (SEQ ID NO:2672). In some embodiments, the amino acid sequence is ERGANTK (SEQ ID NO:5192). In some embodiments, the amino acid sequence is TTGGHSS (SEQ ID NO:2743). In some embodiments, the amino acid sequence is GTTKTSE (SEQ ID NO:3064). In some embodiments, the amino acid sequence is GTGTSVL (SEQ ID NO:11958). In some embodiments, the amino acid sequence is NQSGTKG (SEQ ID NO:780).In some embodiments, the amino acid sequence is DGQSSKS (SEQ ID NO: 2764). In some embodiments, the amino acid sequence is KGPGQMG (SEQ ID NO: 45476). In some embodiments, the amino acid sequence is GTPSKAG (SEQ ID NO: 2741).

[0117] Also provided herein are rAAV capsid proteins with increased specificity and / or improved transduction efficiency for the CNS and PNS, and decreased specificity and / or reduced transduction efficiency for non-target in vivo environments, such as the liver. Exemplary substitutions include N452D, N452G, N452A, N452V, N452S, N452H, N452L, N452E, N452Q, G452A, G452I, G452T, G452P, G452R, G452T, G452S, G452K, G452H, S454A, S454N, S454D, S454G, S454T, S454H, S454K, S454Q, G454A, S454N, S454D, S454G, S454T, S454H, S454K, S454Q, G454B, G454C, G454D, G454G, G454H, S454K, S454Q, G454D, G454G, G454H, G454K, G454Q, G454C, G454D, G454G, G454H, G454K, G ...C, G454D, G454G, G454H, G454K, G454Q, G454C, G454D, G454G, G454H, G454K, G45 55A, G455N, G455T, G455S, G455D, G455P, G455R, G455Q, Q456T, Q456S, Q456K, Q456P, Q456G, Q456D, Q456V, Q456A, Q456N, N457K, N457T, N457A, N457R, N457S, N457G, N457D, N457P, N457V, Q458N, Q458G, Q458S, Q458L, Q458A, Q458E and Q458K. Additional exemplary substitutions include N452A, N452D, N452E, N452G, N452H, N452M, N452N, N452Q, N452S, N452T, N452V, G453A, G453D, G453E, G453G, G453K, G453N, G453Q, G453S, G453T, G453V, S454A, S454D, S454E, S454G, S454K, S454N, S454Q, S454S, S454T, S454V, G455A, G455D, G455E, G455G, G455K, These include G455N, G455P, G455Q, G455S, G455T, Q456A, Q456D, Q456E, Q456H, Q456H, Q456K, Q456N, Q456P, Q456Q, Q456S, Q456T, N457A, N457D, N457E, N457G, N457K, N457N, N457P, N457S, N457T, N457V, Q458A, Q458E, Q458G, Q458H, Q458K, Q458L, Q458N, Q458Q, Q458S, Q458T and Q458V.Additional exemplary substitutions include N452A, N452D, N452E, N452G, N452H, N452K, N452L, N452M, N452N, N452Q, N452S, N452T, N452V, G453A, G453D, G453G, G453H, G453M, G453N, G453P, G453Q, G453S, G453T, G453V, S454A, S454D, S454E, S454G, S454K, S454N, S454Q, S454S, S454T, S454V, G455A, G455D, G455E, G455G, G455 Examples of amino acids that may be used include 5K, G455N, G455P, G455Q, G455S, G455T, Q456A, Q456D, Q456G, Q456K, Q456N, Q456P, Q456Q, Q456S, Q456T, N457A, N457G, N457H, N457L, N457M, N457N, N457P, N457Q, N457S, N457T, N457V, Q458A, Q458D, Q458E, Q458G, Q458H, Q458I, Q458K, Q458L, Q458N, Q458Q, Q458R, Q458S, Q458T and Q458V. In some embodiments, the substitutions are at least or about 1, 2, 3, 4, 5, 6, or 7 amino acids of the amino acid sequence DGAATKN (SEQ ID NO: 3943). In some embodiments, the substitutions are at least or about 1, 2, 3, 4, 5, 6, or 7 amino acids of the amino acid sequence DGQSSKS (SEQ ID NO: 2764).

[0118] In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence of an AAV9 or variant thereof. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence set forth in SEQ ID NO: 1. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence of an AAV5 or variant thereof. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence set forth in SEQ ID NO: 2. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the AAVV-PHP.B capsid protein sequence (SEQ ID NO: 3). In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the AAV-PHP.S capsid protein sequence (SEQ ID NO: 4). In some instances, the amino acid substitution is at an amino acid position selected from 452 to 458 of the AAV-PHP.eB capsid protein sequence (SEQ ID NO: 5). In some embodiments, an rAAV of the disclosure comprises an amino acid substitution at an amino acid position selected from 452 to 458 of the parent AAV capsid protein and an insertion of an amino acid or amino acid sequence at amino acid positions 588 to 589 of the parent AAV capsid protein.

[0119] The rAAV capsid proteins of the present disclosure may also have an amino acid sequence insertion at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises X1LAVPFK (SEQ ID NO: 45481) at amino acid positions 588-589 of the parent AAV9 capsid protein, where X1 is any amino acid other than T, S, or N. In some embodiments, the amino acid sequence insertion comprises X1X2AVPFK (SEQ ID NO: 45482) at amino acid positions 588-589 of the parent AAV9 capsid protein, where X2 is any amino acid other than L or V. In some embodiments, the amino acid sequence insertion comprises X1X2X3VPFK (SEQ ID NO: 45483) at amino acid positions 588-589 of the parent AAV9 capsid protein, where X3 is any amino acid other than A, S, Q, P, and T. In some embodiments, the amino acid sequence insertion comprises X1X2X3X4PFK (SEQ ID NO: 45484) at amino acid positions 588-589 of the parent AAV9 capsid protein, where X4 is any amino acid other than V, T, Q, N, L, and M. In some embodiments, the amino acid sequence insertion comprises TLAX4PFK (SEQ ID NO: 45485) at amino acid positions 588-589 of the parent AAV9 capsid protein, where X is any amino acid other than V, T, Q, N, L, and M. In some embodiments, the rAAV further comprises an amino acid substitution at amino acid position 587 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the substitution is A587D. In some embodiments, the substitution is with an amino acid other than D. In some embodiments, the rAAV further comprises an amino acid substitution at amino acid position 588 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the substitution is Q588G. In some embodiments, the substitution is with an amino acid other than G.

[0120] In some embodiments, the rAAV capsid has a variant AAV capsid protein (e.g., a rAAV capsid protein) that includes at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence LQTSSPG (SEQ ID NO: 2933) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein includes at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence LQTSSPG (SEQ ID NO: 2933) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein includes at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence LQTSSPG (SEQ ID NO: 2933) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46386.

[0121] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence QQGKQSV (SEQ ID NO:79) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO:1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence QQGKQSV (SEQ ID NO:79) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence QQGKQSV (SEQ ID NO:79) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46387.

[0122] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence SINTKTN (SEQ ID NO: 45475) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence SINTKTN (SEQ ID NO: 45475) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence SINTKTN (SEQ ID NO: 45475) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46368.

[0123] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence SNGTKQT (SEQ ID NO: 442) at amino acid positions 452-458 of the parent AAV9 capsid protein represented by SEQ ID NO: 1. In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence SNGTKQT (SEQ ID NO: 442) at amino acid positions 452-458 of the parent AAV9 variant capsid protein represented by SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acids 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46389.

[0124] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GSGKTAA (SEQ ID NO: 88) at amino acid positions 452-458 of the parent AAV9 capsid protein represented by SEQ ID NO: 1. In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GSGKTAA (SEQ ID NO: 88) at amino acid positions 452-458 of the parent AAV9 variant capsid protein represented by SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acids 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46390.

[0125] In some embodiments, the rAAV capsid protein comprises a substitution of at least or about 3, 4, 5, 6, or 7 amino acids for the amino acid sequence MGDKPTR (SEQ ID NO: 2466) at amino acid positions 452-458 of the parent AAV9 capsid protein represented by SEQ ID NO: 1. In some embodiments, the rAAV capsid protein comprises a substitution of at least or about 3, 4, 5, 6, or 7 amino acids for the amino acid sequence MGDKPTR (SEQ ID NO: 2466) at amino acid positions 452-458 of the parent AAV9 variant capsid protein represented by SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acids 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46391.

[0126] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence DGAATKN (SEQ ID NO: 3943) at amino acid positions 452-458 of the parent AAV9 capsid protein represented by SEQ ID NO: 1. In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence DGAATKN (SEQ ID NO: 3943) at amino acid positions 452-458 of the parent AAV9 variant capsid protein represented by SEQ ID NO: 3 or SEQ ID NO: 5. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acids 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46384.

[0127] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence QPSGGNT (SEQ ID NO:2672) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO:1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence QPSGGNT (SEQ ID NO:2672) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence QPSGGNT (SEQ ID NO:2672) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46392.

[0128] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence ERGANTK (SEQ ID NO:5192) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO:1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence ERGANTK (SEQ ID NO:5192) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence ERGANTK (SEQ ID NO:5192) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46393.

[0129] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence TTGGHSS (SEQ ID NO: 2743) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence TTGGHSS (SEQ ID NO: 2743) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence TTGGHSS (SEQ ID NO: 2743) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46394.

[0130] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTTKTSE (SEQ ID NO: 3064) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTTKTSE (SEQ ID NO: 3064) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTTKTSE (SEQ ID NO: 3064) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46395.

[0131] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTGTSVL (SEQ ID NO: 11958) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTGTSVL (SEQ ID NO: 11958) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTGTSVL (SEQ ID NO: 11958) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46396.

[0132] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NQSGTKG (SEQ ID NO: 780) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NQSGTKG (SEQ ID NO: 780) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NQSGTKG (SEQ ID NO: 780) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the insertion of an amino acid sequence comprising at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. In some embodiments, the insertion of the amino acid sequence comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46397.

[0133] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence DGQSSKS (SEQ ID NO:2764) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO:1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence DGQSSKS (SEQ ID NO:2764) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence DGQSSKS (SEQ ID NO:2764) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46385.

[0134] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KGPGQMG (SEQ ID NO: 45476) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KGPGQMG (SEQ ID NO: 45476) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KGPGQMG (SEQ ID NO: 45476) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46398.

[0135] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTPSKAG (SEQ ID NO:2741) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO:1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTPSKAG (SEQ ID NO:2741) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence GTPSKAG (SEQ ID NO:2741) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO:5). In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the insertion of an amino acid sequence comprising at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids. In some embodiments, the insertion of the amino acid sequence comprises TLAVPFK (SEQ ID NO: 45477) at amino acid positions 588-589 of the parent AAV9 capsid protein. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46399.

[0136] Table 1 provides the amino acid sequences of the best-performing rAAV capsid proteins, ranked based on positive enrichment in the brain and negative enrichment in the liver after two rounds of in vivo selection, as well as their encoding DNA sequences. Table 2 provides the amino acid sequences of the best-performing rAAV capsid proteins, ranked based solely on enrichment in the brain after two rounds of in vivo selection, as well as their corresponding DNA sequences. Details on how enrichment scores were calculated are provided in Example 3. An AAV variant was determined to detarget a tissue if its enrichment score in that tissue was less than or equal to 0. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0137] Modified AAV capsid proteins for organ or tissue targeting

[0138] Disclosed herein are recombinant AAV (rAAV) capsid proteins with desired tropism characterized by substitution or insertion of at least one amino acid at the above-mentioned amino acid positions of the parent AAV capsid protein (e.g., AAV5 or AAV9). The AAV capsid proteins of the present disclosure are engineered to have desired tropism, including increased specificity for a target in vivo environment in a subject, such as an organ or organ system. One of the many advantages of the tropism of the rAAV capsid proteins described herein is their ability to selectively and efficiently express heterologous nucleic acids in organs or organ systems while avoiding expression of heterologous nucleic acids in non-target organs, thereby reducing toxicity and the viral dosage required for therapeutic efficacy.

[0139] The in vivo environment can be a tissue or a cell. The in vivo environment can be a tissue, e.g., a tissue from an organ or organ system. Non-limiting examples of organs or organ systems include the liver, intestine, heart, lungs, reproductive organs, muscle, fat, pancreas, brain, and spleen. Non-limiting examples of reproductive organs include the ovaries and testes. The in vivo environment can be a cell. The cell can be a cell type selected from the group consisting of central nervous system (CNS) cells, peripheral nervous system (PNS) cells, liver cells, intestinal cells, lung cells, cardiac cells, adipocytes, muscle cells, kidney cells, muscle cells, pancreatic cells, spleen cells, reproductive organ cells, and stomach cells. The CNS cells can be neurons or glial cells. The glial cells can be selected from the group consisting of oligodendrocytes, ependymal cells, and astrocytes. The PNS cells can be neurons or glial cells. The glial cells can be selected from the group consisting of Schwann cells, satellite cells, and enteric glial cells. The liver cells can be hepatocytes. The intestinal cells can be selected from the group consisting of enterocytes, goblet cells, enteroendocrine cells, cup cells, tuft cells, and Paneth cells. The lung cells can be selected from the group consisting of alveolar type I epithelial cells, alveolar type II epithelial cells, alveolar type I pneumocytes, alveolar type II pneumocytes, capillary endothelial cells, and alveolar macrophages. The cardiac cells can be cardiomyocytes. The gastric cells can be selected from the group consisting of mucous cells, parietal cells, chief cells, and G cells. The muscle cells can be myocytes.

[0140] rAAVs of the present disclosure that are optimized for targeting to specific organs or tissues within a subject have an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 25469-35471. In some embodiments, rAAVs optimized for targeting to the heart have an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 25469-26205. In some embodiments, rAAVs optimized for targeting to the lung have an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 32537-34198. In some embodiments, rAAVs optimized for targeting to the liver have an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 30720-30923. In some embodiments, rAAVs optimized for targeting to the intestine have an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 26206-26873. In some embodiments, rAAVs optimized for targeting to the stomach have an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 31873-32060. In some embodiments, an rAAV optimized for targeting to the spleen has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 31468-31872. In some embodiments, an rAAV optimized for targeting to the kidney has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 26874-30719. In some embodiments, an rAAV optimized for targeting to fat (adipose) has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 34199-35471. In some embodiments, an rAAV optimized for targeting to muscle has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 30924-31451. The muscle may be cardiac muscle. The muscle may be skeletal muscle. In some embodiments, an rAAV optimized for targeting to the pancreas has an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NOs: 31452-31467. In some embodiments, an rAAV optimized for targeting to a reproductive organ, such as the testis, comprises an amino acid sequence comprising an amino acid sequence set forth in SEQ ID NOs: 32061-32536. In some instances, the reproductive organ is an ovary.

[0141] Disclosed herein are rAAV capsid proteins having desired targeting, characterized by at least one amino acid substitution at an amino acid position selected from 452 to 458 of the amino acid sequence of a parent AAV9 capsid protein or a variant thereof. The rAAV capsid protein may contain at least one amino acid substitution in the parent AAV capsid protein. In some cases, X1 is K, R, N, or T. The rAAV capsid protein may contain at least two amino acid substitutions or insertions in the parent AAV capsid protein, where X1 is K, R, N, or T and X2 is D, E, N, or V. The rAAV capsid protein may contain at least three amino acid substitutions or insertions in the parent AAV capsid protein, where X1 is K, R, N, or T, X2 is D, E, N, or V, and X3 is N, S, L, or P. The rAAV capsid protein may comprise at least four amino acid substitutions or insertions in the parent AAV capsid protein, where X1 is K, R, N, or T, X2 is D, E, N, or V, X3 is N, S, L, or P, and X4 is T, S, P, or L. The rAAV capsid protein may comprise at least five amino acid substitutions or insertions in the parent AAV capsid protein, where X1 is K, R, N, or T, X2 is D, E, N, or V, X3 is N, S, L, or P, X4 is T, S, P, or L, and X5 is P, R, or S. The rAAV capsid protein may contain at least six amino acid substitutions or insertions in the parent AAV capsid protein, where X1 is K, R, N, or T; X2 is D, E, N, or V; X3 is N, S, L, or P; X4 is T, S, P, or L; X5 is P, R, or S; and X6 is G, S, N, or T.The rAAV capsid protein may contain at least seven amino acid substitutions or insertions in the parent AAV capsid protein, where X1 is K, R, N, or T; X2 is D, E, N, or V; X3 is N, S, L, or P; IX4 is T, S, P, or L; X5 is P, R, or S; X6 is G, S, N, or T; and X7 is R, L, or I.

[0142] In some embodiments, X1, X2, X3, X4, X5, X6, and X7 are consecutive (X1-X2-X3-X4-X5-X6-X7). In some embodiments, any two of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any three of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any four of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any five of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any six of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, any seven of X1, X2, X3, X4, X5, X6, and X7 are consecutive. In some embodiments, X1, X2, X3, X4, X5, X6, and X7 are not contiguous. In some embodiments, X1 is at amino acid 452 in the AAV9 capsid protein or its variant. In some embodiments, X2 is at amino acid 453 in the AAV9 capsid protein or its variant. In some embodiments, X3 is at amino acid 454 in the AAV9 capsid protein or its variant. In some embodiments, X4 is at amino acid 455 in the AAV9 capsid protein or its variant. In some embodiments, X5 is at amino acid 456 in the AAV9 capsid protein or its variant. In some embodiments, X6 is at amino acid 457 in the AAV9 capsid protein or its variant. In some embodiments, X7 is at amino acid 458 in the AAV9 capsid protein or its variant.

[0143] Disclosed herein are rAAV capsid proteins comprising substitutions of at least one, two, three, four, five, six, or seven amino acids set forth in any one of SEQ ID NOs: 25469-35471. In some embodiments, at least one, two, three, four, five, six, or seven amino acids are provided within an amino acid sequence selected from SEQ ID NOs: 25469-26205. In some embodiments, at least one, two, three, four, five, six, or seven amino acids are provided within an amino acid sequence selected from SEQ ID NOs: 26206-26873. In some embodiments, at least one, two, three, four, five, six, or seven amino acids are provided within an amino acid sequence selected from SEQ ID NOs: 26874-30719. In some embodiments, at least one, two, three, four, five, six, or seven amino acids are provided within an amino acid sequence selected from SEQ ID NOs: 30720-30923. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 30924-31451. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 31452-31467. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 31468-31872. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 31873-32060. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 32061-32536. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 32537-34198. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 amino acids are provided in an amino acid sequence selected from SEQ ID NOs: 34199-35471.In some embodiments, the substitutions include N452K, N452R, N452T, G453D, G453L, G453E, G453V, G453N, G453S, G453L, G453P, S454L, S454P, S454A, S454D, S454G, S454T, G455T, G455S, G455P, G455L, Q456P, Q456R, Q456S, N457G, N457S, N457T, Q458R, Q458L or Q458I, or a combination thereof.

[0144] Disclosed herein are rAAV capsid proteins comprising at least or about 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions of the amino acid sequences provided herein. In some embodiments, the amino acid sequence is KDNTPGR (SEQ ID NO: 32538). In some embodiments, the amino acid sequence is NNLPRNL (SEQ ID NO: 32867). In some embodiments, the amino acid sequence is RESSPSL (SEQ ID NO: 29065). In some embodiments, the amino acid sequence is RVPLSTI (SEQ ID NO: 26933).

[0145] In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence of an AAV9 or variant thereof. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence set forth in SEQ ID NO: 1. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence of an AAV5 or variant thereof. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the capsid amino acid sequence set forth in SEQ ID NO: 2. In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the AAVV-PHP.B capsid protein sequence (SEQ ID NO: 3). In some cases, the amino acid substitution is at an amino acid position selected from 452-458 of the AAV-PHP.S capsid protein sequence (SEQ ID NO: 4). In some instances, the amino acid substitution is at an amino acid position selected from 452 to 458 of the AAV-PHP.eB capsid protein sequence (SEQ ID NO: 5). In some embodiments, an rAAV of the disclosure comprises an amino acid substitution at an amino acid position selected from 452 to 458 of the parent AAV capsid protein and an insertion of an amino acid or amino acid sequence at amino acid positions 588 to 589 of the parent AAV capsid protein.

[0146] The rAAV capsid proteins of the present disclosure may also have an amino acid sequence insertion at amino acid position 588 of the parental AAV9 or AAV5 capsid protein or variant thereof. In some embodiments, the amino acid sequence insertion comprises X1LAVPFK (SEQ ID NO: 45481) at amino acids 588-589 of the parental AAV9 AAV5 capsid protein, where X1 is any amino acid other than T, S, or N. In some embodiments, the amino acid sequence insertion comprises X1X2AVPFK (SEQ ID NO: 45482) at amino acids 588-589 of the parental AAV9 or AAV5 capsid protein, where X2 is any amino acid other than L or V. In some embodiments, the amino acid sequence insertion comprises X1X2X3VPFK (SEQ ID NO: 45483) at amino acids 588-589 of the parental AAV9 or AAV5 capsid protein, where X3 is any amino acid other than A, S, Q, P, and T. In some embodiments, the amino acid sequence insertion comprises X1X2X3X4PFK (SEQ ID NO: 45484) at amino acid positions 588-589 of the parent AAV9 or AAV5 capsid protein, where X4 is any amino acid other than V, T, Q, N, L, and M. In some embodiments, the amino acid sequence insertion comprises TLAX4PFK (SEQ ID NO: 45485) at amino acid positions 588-589 of the parent AAV9 or AAV5 capsid protein, where X is any amino acid other than V, T, Q, N, L, and M. In some embodiments, the rAAV further comprises an amino acid substitution at amino acid position 587 of the parent AAV9 or AAV5 capsid protein or variant thereof. In some embodiments, the substitution is A587D. In some embodiments, the substitution is with an amino acid other than D. In some embodiments, the rAAV further comprises an amino acid substitution at amino acid position 588 of the parent AAV9 or AAV5 capsid protein or variant thereof. In some embodiments, the substitution is Q588G. In some embodiments, the substitution is with an amino acid other than G.

[0147] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KDNTPGR (SEQ ID NO: 32538) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KDNTPGR (SEQ ID NO: 32538) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KDNTPGR (SEQ ID NO: 32538) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KDNTPGR (SEQ ID NO: 32538) at amino acid positions 452-458 of the parent AAV5 capsid protein (SEQ ID NO: 2). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence KDNTPGR (SEQ ID NO: 32538) at amino acid positions 452-458 of the parent AAV5 variant capsid protein. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46400.

[0148] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NNLPRNL (SEQ ID NO: 32867) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NNLPRNL (SEQ ID NO: 32867) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NNLPRNL (SEQ ID NO: 32867) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NNLPRNL (SEQ ID NO: 32867) at amino acid positions 452-458 of the parent AAV5 capsid protein (SEQ ID NO: 2). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence NNLPRNL (SEQ ID NO: 32867) at amino acid positions 452-458 of the parent AAV5 variant capsid protein. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46401.

[0149] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RESSPSL (SEQ ID NO: 29065) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RESSPSL (SEQ ID NO: 29065) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RESSPSL (SEQ ID NO: 29065) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RESSPSL (SEQ ID NO: 29065) at amino acid positions 452-458 of the parent AAV5 capsid protein (SEQ ID NO: 2). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RESSPSL (SEQ ID NO: 29065) at amino acid positions 452-458 of the parent AAV5 variant capsid protein. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46402.

[0150] In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RVPLSTI (SEQ ID NO: 26933) at amino acid positions 452-458 of the parent AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RVPLSTI (SEQ ID NO: 26933) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 3). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RVPLSTI (SEQ ID NO: 26933) at amino acid positions 452-458 of the parent AAV9 variant capsid protein (SEQ ID NO: 5). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RVPLSTI (SEQ ID NO: 26933) at amino acid positions 452-458 of the parent AAV5 capsid protein (SEQ ID NO: 2). In some embodiments, the rAAV capsid protein comprises at least or about 3, 4, 5, 6, or 7 amino acid substitutions for the amino acid sequence RVPLSTI (SEQ ID NO: 26933) at amino acid positions 452-458 of the parent AAV5 variant capsid protein. In some embodiments, the rAAV capsid protein comprises an insertion of at least or about 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids at amino acid positions 588-589 of the parent AAV9 capsid protein or variant thereof. In some embodiments, the rAAV capsid protein comprises the amino acid sequence set forth in SEQ ID NO: 46403.

[0151] heterologous nucleic acid

[0152] Disclosed herein are heterologous nucleic acids, including therapeutic nucleic acids useful for treating or preventing the diseases or conditions disclosed herein, or symptoms of the diseases or conditions. In some embodiments, the therapeutic nucleic acid encodes a therapeutic gene expression product. Non-limiting examples of gene expression products include proteins, polypeptides, peptides, enzymes, antibodies, antigen-binding fragments, nucleic acids (RNA, DNA, antisense oligonucleotides, siRNA, etc.), and gene editing components for use in treating, preventing, and / or ameliorating diseases or disorders, or symptoms of diseases or disorders. In some cases, the therapeutic nucleic acid is introduced into the subject's organism, cell, tissue, or organ by rAAV, such as those disclosed herein.

[0153] Disclosed herein are rAAVs, each comprising a viral genome (e.g., a single-stranded DNA molecule (ssDNA)). In some cases, the viral vector comprises two inverted terminal repeat (ITR) sequences, each approximately 145 bases long, flanking a heterologous nucleic acid or transgene. In some embodiments, the transgene comprises a therapeutic nucleic acid, and in some cases, a promoter in cis with the therapeutic nucleic acid within an open reading frame (ORF). The promoter can initiate transcription of the therapeutic nucleic acid in the nucleus of a target cell. The ITR sequences may be fr...

Claims

1. a) a variant AAV capsid comprising a variant AAV capsid protein that contains an amino acid substitution only within a 7-mer peptide sequence at the three-fold symmetry axis of a corresponding parent AAV capsid protein, wherein the 7-mer peptide sequence is selected from the group consisting of AAV9 a variant AAV capsid at amino acid positions corresponding to amino acid residues 452-458 of VP1 (SEQ ID NO:1), wherein said amino acid substitutions are selected from the group consisting of LQTSSPG (SEQ ID NO:2933), QQGKQSV (SEQ ID NO:79), SINTKTN (SEQ ID NO:45475), SNGTKQT (SEQ ID NO:442), GSGKTAA (SEQ ID NO:88), MGDKPTR (SEQ ID NO:2466), QPSGGNT (SEQ ID NO:2672), ERGANTK (SEQ ID NO:5192), TTGGHSS (SEQ ID NO:2743), GTTKTSE (SEQ ID NO:3064), GTGTSVL (SEQ ID NO:11958), NQSGTKG (SEQ ID NO:780), and GTPSKAG (SEQ ID NO:2741); b) a heterologous polynucleotide comprising a nucleotide sequence encoding a gene product; 1. A recombinant AAV (rAAV) comprising: A rAAV, wherein the variant AAV capsid has increased tropism for a target tissue or target cell when measured in a subject compared to the tropism of the corresponding parent AAV capsid, and the corresponding parent AAV capsid protein is an AAV9 VP1 variant, and the AAV9 VP1 variant has 95% or greater sequence identity to SEQ ID NO:

1.

2. The rAAV of claim 1, wherein the target tissue or target cell comprises a tissue or cell of the central nervous system (CNS) or peripheral nervous system (PNS) or a combination thereof.

3. 2. The rAAV of claim 1, wherein the corresponding parent AAV capsid protein is an AAV9 VP1 variant, and the AAV9 VP1 variant has 96% or greater sequence identity to SEQ ID NO:

1.

4. 2. The rAAV of claim 1, wherein the corresponding parent AAV capsid protein further comprises an insertion of an amino acid sequence selected from the group consisting of TLAXPFK (SEQ ID NO:46424), TLAX (SEQ ID NO:46425), LAVX (SEQ ID NO:46426), AVPX (SEQ ID NO:46427), and VPFX (SEQ ID NO:46428) at an amino acid position corresponding to 588_589 of the AAV9 VP1 (SEQ ID NO:1), where X is any amino acid other than V.

5. The rAAV of any one of claims 1 to 4, wherein the parent AAV capsid protein is from AAV-PHP.B or AAV-PHP.eB.

6. The rAAV of any one of claims 1 to 5, which is isolated and purified.

7. A pharmaceutical formulation comprising the rAAV of any one of claims 1 to 6 and a pharmaceutical excipient.

8. 8. The pharmaceutical preparation of claim 7, formulated for intravenous, intraarterial, intranasal, intrathecal, intracisternal or subcutaneous injection.

9. A composition comprising the rAAV of any one of claims 1 to 6 or the pharmaceutical formulation of claim 7 or 8 for treating a disease or condition in a subject, wherein the gene product is a therapeutic gene product.

10. 10. The composition or pharmaceutical preparation according to claim 9, characterized in that the composition or pharmaceutical preparation is administered by intravenous, intraarterial, intranasal, intrathecal, intracisternal or subcutaneous injection.

11. 10. The composition or pharmaceutical formulation of claim 9, wherein the disease or condition is selected from the group consisting of pulmonary fibrosis, surfactant protein disorders, peroxisomal biogenesis disorders, or chronic obstructive pulmonary disease (COPD).

12. 10. The composition or pharmaceutical formulation of claim 9, wherein the disease or condition is a central nervous system (CNS) or peripheral nervous system (PNS) disorder.

13. a) at least one of reduced specificity and reduced transduction efficiency as compared to a corresponding parent AAV capsid having the AAV capsid protein of SEQ ID NO:1, as measured in the liver of a primate subject when delivered intravenously to said primate subject; and b) at least one of increased specificity and improved transduction efficiency measured in the central nervous system (CNS) of said primate subject when delivered intravenously to said primate subject, as compared to the corresponding parental AAV capsid. A variant adeno-associated virus (AAV) capsid comprising: The variant AAV capsid comprises a variant AAV capsid protein comprising an amino acid substitution only within a 7-mer peptide sequence at the three-fold axis of symmetry of the corresponding parent AAV capsid protein, wherein the 7-mer peptide sequence is at an amino acid position corresponding to amino acid residues 452-458 of AAV9 VP1 (SEQ ID NO: 1), and wherein the amino acid substitution is selected from the group consisting of LQTSSPG (SEQ ID NO: 2933), ERGANTK (SEQ ID NO: 5192), TTGGHSS (SEQ ID NO: 2743), and GTPSKAG (SEQ ID NO: 2741).

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