Recombinant adeno-associated viruses and uses thereof

Abstract

The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins modified to contain amino acid sequences that confer and / or enhance desired properties. In particular, the present invention provides modified capsid proteins containing peptide inserts from heterologous proteins inserted into or near the variable region IV (VR-IV) of the viral capsid such that the inserts are surface-exposed on the AAV particle. The present invention also provides capsid proteins that target rAAVs to target tissues, particularly capsid proteins containing peptides inserted into the surface-exposed variable region using a method that replaces a stop codon in the capsid gene with such peptide inserts. The present invention also provides methods for generating rAAV vectors into vector libraries and selecting rAAV vectors containing peptide inserts that target rAAVs to specific tissues of interest and thus deliver therapeutic agents for treating disorders.

Description

[Technical Field]

[0001] 1.Technical Field Sequence Listing The Sequence Listing submitted herewith as an XML file named 38013_0030P1_SL.xml, created on August 24, 2023, and having a size of 530,415 bytes, is hereby incorporated by reference in accordance with 37 C.FR §§ 1.831-1.835.

[0002] The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins modified to contain amino acid sequences that confer and / or enhance desired properties when incorporated into the rAAV capsid. In particular, the present invention provides modified capsid proteins containing peptide inserts inserted within or near the variable region IV (VR-IV) of the viral capsid such that the inserts are surface-exposed on the AAV particle. The present invention also provides capsid proteins that target rAAVs to target tissues, particularly capsid proteins derived from rAAV libraries, constructed to reduce parental vector production and thus reduce overexpression of parental capsids in the libraries. Such libraries contain random peptides inserted into the surface-exposed variable region to target rAAVs to tissues of interest, including muscle tissue, and / or improve their transduction and delivery of therapeutic agents to treat muscle disorders. [Background technology]

[0003] 2.Background technology The use of recombinant adeno-associated viruses (AAVs) as gene delivery vectors is a promising avenue for the treatment of many patients with unmet needs and / or rare diseases. Dozens of naturally occurring AAV capsids have been reported, and surveys of the natural diversity of AAV sequences in primate tissues have identified over 100 variants distributed across multiple clades. AAVs belong to the Parvoviridae family and are single-stranded DNA viruses with relatively small genomes and simple genetic components. Our current understanding of these capsids, their utility, and function has enabled efforts to further refine the efficiency and effectiveness of carrying therapeutic genomic DNA, including directing tissue tropism to deliver such DNA to target cells while reducing tropism to tissues where vector transduction and / or transgene expression are undesirable, in order to safely ameliorate severe diseases.

[0004] Due to the promise of low pathogenicity and long-term targeted gene expression, recombinant AAV (rAAV) has been used as a gene transfer vector with a therapeutic sequence packaged in the capsid. Such vectors have been used to deliver various therapeutic genes, and therefore, many gene therapy products are currently under clinical development. Recombinant AAVs, such as AAV9, have demonstrated desirable muscle and neurotropic properties, and clinical trials using recombinant AAV9 for the treatment of muscle diseases such as dystrophinopathy are underway. However, attempts to identify rAAV capsids with desired properties in human subjects are limited by the methods used to select them.

[0005] For example, there remains a need for rAAV vectors with improved targeting to specific tissues and properties for use in high transduction of muscle for delivery therapy in the treatment of disorders such as dystrophinopathy. There is also a need for improved methods for identifying such rAAV vectors with improved tissue-specific targeting and / or improved tissue-specific transduction for delivery of therapeutic agents using lower dosages than are currently available. Summary of the Invention

[0006] 3. Summary of the Invention Recombinant adeno-associated viruses (rAAVs) are provided having capsid proteins modified to include amino acid sequences that confer and / or enhance desired properties, such as tissue targeting, transduction, or expression of the rAAV genome. Specifically, modified capsid proteins are provided that include a peptide insert from a peptide library inserted into or near variable region IV (VR-IV) or, in certain embodiments, variable region VIII (VR-VIII) of the viral capsid, such that the peptide insert is surface-exposed on the AAV particle when the modified capsid protein is incorporated into the rAAV particle. In embodiments, the peptide is 4 to 7 amino acids of one of the peptides having the amino acid sequence of SEQ ID NOs: 1-138 (Tables 4, 5, 14, 15, or 16). In embodiments, the insertion is immediately after the amino acid residue corresponding to one of amino acids 451-461 of the AAV9 capsid protein (SEQ ID NO: 151 and, e.g., as numbered in FIG. 1), including after amino acid 454 (i.e., between amino acids 454 and 455) of the AAV9 capsid protein, or in the AAV9.AAA (e.g., SEQ ID NO: 158) capsid protein, or in the capsid protein of a different AAV type after the residue corresponding to amino acid 454 of AAV9 (see alignment in FIG. 1, or, for AAV types not included in FIG. 1, a similar amino acid sequence alignment of the AAV9 capsid protein sequence, such as 451-461 of AAV9 (SEQ ID NO: 151), is the same as the AAVhu.32 capsid protein sequence (SEQ ID NO: 148) aligned in FIG. 1, and AAV capsid proteins are well known in the art).The capsid protein may be an AAV9 capsid protein, but may also be any of AAV type 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype hu.31 (AAVhu.31), serotype hu.32 (AAVhu.32), serotype rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype The capsid protein may be any AAV capsid protein, such as hu.37 (AVVhu.37), serotype rh39 (AAVrh39), and serotype rh74 (AAVrh74), or the variant AAV9 capsid protein AAV9.AAA (496NNN / AAA498 amino acid substitution) (see, e.g., the alignment depicted in Figure 1), or may be a capsid protein having 90%, 95%, or 99% amino acid sequence identity to one of the foregoing capsid proteins. Thus, provided are modified capsid proteins that include a peptide insert from a heterologous protein (i.e., that is not an AAV capsid protein) inserted immediately after or near the amino acid corresponding to amino acid residue 454 of AAV9, as numbered in Figure 1.

[0007] Also provided are modified capsid proteins that direct rAAV to target tissues, particularly capsid proteins containing peptides (derived from peptide libraries) or peptides that promote tissue targeting and / or cellular uptake and / or expression of the rAAV genome, inserted into the surface-exposed variable region, and targeting rAAV to muscle tissue (skeletal muscle and / or heart), including neurons in the central nervous system and CNS, and retinal tissue, and delivering therapeutic agents for treating neurological and ocular diseases. These peptides, comprising 4, 5, 6, or all 7 consecutive amino acids of one of the peptides in Tables 4, 5, 14, 15, or 16 (SEQ ID NOS: 1-138), are advantageously inserted into the amino acid sequence of the capsid protein such that the inserted peptide is surface-exposed when the capsid protein is incorporated into an AAV particle. These peptides are inserted immediately after or after one of the amino acid residues corresponding to amino acid residues 585-593 of the AAV9 capsid (SEQ ID NO:151) or AAV9.AAA capsid (SEQ ID NO:158; see, e.g., Figure 1 for an alignment), or immediately after or after one of the amino acid residues corresponding to amino acids 451-461 of the AAV9 capsid or AAV9.AAA capsid and the corresponding amino acids at any one of positions 451-461 of the AAV9 capsid (SEQ ID NO:151; see, e.g., Figure 1 for an alignment) or AAV9.AAA capsid. Exemplary modified capsid sequences, including capsid proteins having the amino acid sequence of one of SEQ ID NOs:159-266 (AAV9.AAA parent capsid) and 268-375 (AAVhu.32.AAA parent capsid), are provided in Table 17. In other embodiments, a 4, 5, 6, or 7 amino acid portion of a peptide having the amino acid sequence of SEQ ID NO: 113, 114, or 115 is inserted into AAV5 (SEQ ID NO: 143) immediately after position 577 or 578. In embodiments, capsid proteins and capsid incorporating capsids having the amino acid sequence of SEQ ID NO: 376, 377, or 378 (AAV5 parent capsid) are provided.

[0008] Modified capsid proteins containing peptides that target specific tissues are provided, including those inserted into a surface-exposed variable region of the capsid protein when incorporated into an rAAV vector as a capsid to promote or increase cellular uptake and / or integration of the rAAV genome and / or expression of a transgene within the rAAV genome. In certain embodiments, the peptide targets capsids containing, for example, at least four consecutive amino acids, or at least five, six, or seven consecutive amino acids, of any of the peptides in Tables 4, 5, 14, 15, or 16, and capsids containing one of these peptides immediately following, for example, one of positions 451-461, including after position 454, of AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA, and / or promote transduction or genome integration in cells of that muscle, using capsids to preferentially target rAAV to muscle tissue and, in embodiments, detarget the liver. In embodiments, capsids with peptide inserts target and transduce neurons within the CNS, including for transduction of astrocytes in the CNS. In other embodiments, the inserted peptide is at least four consecutive amino acids, and in addition, the capsid is modified to have one or more amino acid substitutions that may improve targeting, transduction, or reduce immunoneutralizing activity. Such amino acid modifications include the substitution of NNN (asparagine) at positions 496-498 with AAA (alanine) in the AAV9 capsid. Other parent capsids with peptide inserts may also have the substitution of NNN (asparagine) at positions 498-500 with AAA (alanine) in the AAV8 capsid, or the substitution of NNN (asparagine) at positions 497-499 with AAA (alanine) in the AAVhu.32 capsid, or corresponding substitutions in other AAV-type capsids.Additionally, other parent capsids with peptide insertions may also have S263F / S269T / A273T substitutions in AAV9 and corresponding substitutions in other AAV-type capsids, W530R or Q474A in AAV9 and corresponding substitutions in other AAV-type capsids, and / or A269S in AAV8 and corresponding substitutions in other AAV-type capsids.

[0009] Also provided are modified capsid proteins that facilitate transduction of rAAV in one or more tissues, including one or more cell types, upon systemic, intravenous, intraperitoneal, or intramuscular administration, wherein the capsid protein comprises a peptide of Table 4, 5, 14, 15, or 16 (SEQ ID NOS: 1-138) inserted within a surface-exposed variable region (VR) of the capsid, e.g., VR-I, VR-IV, or VR-VIII, or after the first amino acid of VP2, e.g., immediately after residue 138 of the AAV9 capsid (amino acid sequence of SEQ ID NO: 151) or immediately after the corresponding residue in another AAV capsid, or alternatively, modified with one or more of the amino acid substitutions described herein; and transduction of the AAV having the modified capsid in at least one tissue (e.g., muscle (skeletal and / or heart, retina, or CNS including CNS neurons) is facilitated by the use of AAV9, AAV9.AAA, AAVhu.32, or AAVhu.3. 2. Increased (e.g., 1-fold, 2-fold, 5-fold, 10-fold, or 20-fold greater) transduction upon administration compared to transduction of an AAV having a corresponding unmodified capsid (parent capsid) or reference capsid, such as AAA. Such capsids may also exhibit AAV transduction in tissues such as muscle, retina, or CNS, or reduced transduction of one or more tissues, including liver, heart, or astrocytes, upon administration compared to transduction of an AAV having a corresponding unmodified capsid (parent capsid) or reference capsid, such as AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA (e.g., 1-fold, 2-fold, 5-fold, 10-fold, or 20-fold greater). In certain embodiments, transduction is measured by detection of the transgene, such as DNA, RNA transcripts, or expressed protein, in cells; for example, a reporter transgene may be utilized, such as GFP fluorescence.

[0010] In certain embodiments, rAAV incorporating the modified capsids described herein are provided, including rAAVs having genomes containing transgenes of therapeutic interest. Plasmids and cells for producing pools (stocks) of plasmids for the production of rAAVs are described herein. Packaging cells and methods for producing rAAVs containing the modified capsids are also provided herein. Methods of treatment by delivery of the modified rAAVs described herein, and pharmaceutical compositions comprising the modified rAAVs described herein are provided. Methods of producing rAAVs having the modified capsids described herein are also provided.

[0011] Also provided is a method for generating a capsid library with peptide inserts. In embodiments, the method includes providing a starting plasmid containing a gene expression cassette encoding a capsid gene, wherein a stop codon is placed at a target insertion site within the capsid gene, and randomizing a repertoire of nucleic acids encoding randomized peptides to generate a peptide library, 1) each having a nucleic acid encoding a random peptide from the peptide library that is inserted into the target insertion site of the capsid gene, thereby replacing the stop codon, and 2) each encoding a barcode for identification of capsid genes having an insert placed before the 5' end or after the 3' end of the capsid gene. The method includes creating individual plasmids based on a starting plasmid that encodes a peptide insert, collecting the individual plasmids to form a population or collection of plasmids encoding capsids with the peptide insert, and transfecting the collection of plasmids with plasmids encoding recombinant rAAV genomes containing a transgene, including a detectable one, and required genes to generate a collection of rAAV vectors that encapsidate the rAAV genomes containing the transgene, wherein the rAAV vectors have capsids with encoded capsid proteins containing the library peptide insert. In embodiments, the parent AAV is, for example, AAV9, AAV9.AAA, AAVhu.32, AAVhu.32.AAA, or AAV5, as in Table 3, or any other suitable AAV serotype. The insertion site may be within VR-IV, including immediately after or corresponding to one of amino acids 451-461 of AAV9, or within VR-VIII, including immediately after amino acid 588 of AAV9 or corresponding to that position in different AAV capsid types (see Figure 1 for alignment).

[0012] A library of modified capsids is harvested from these cells. In embodiments, the generated rAAV library population has high levels of capsids with peptide inserts, including 85%, 90%, 95%, or 98%, 99%, or even 100%.

[0013] The invention is illustrated by the following examples which describe the construction of modified rAAV9 capsids with peptide inserts designed from an rAAV library that enable detection of desired properties, such as tissue targeting.

[0014] 3.1. Implementation 1. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insert of at least 4 and up to 7 consecutive amino acids, said peptide insert immediately following the amino acid residue corresponding to one of amino acids 451-461 of the AAV9 capsid protein of FIG. 1, and said peptide insert having the amino acid sequence of one of SEQ ID NOs: 1-138; the capsid protein is a wild-type capsid protein or a variant capsid protein having up to three amino acid substitutions; The recombinant adeno-associated virus (rAAV) capsid protein, wherein an rAAV vector prepared from the capsid protein containing the peptide insert has improved target tissue tropism compared to an rAAV vector prepared from a capsid protein without the peptide insert or a reference capsid protein.

[0015] 2. The capsid protein is selected from AAV type 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype hu.31 (AAVhu.31), serotype hu.32 (AAVhu.32), serotype rh10 (AAVrh10), serotype rh20 (AAV rh20), serotype hu.37 (AVVhu.37), serotype rh39 (AAVrh39), and serotype rh74 (AAVrh74), or the variant capsid protein is AAV9.AAA or AAVhu.32.AAA, or a capsid protein having 90%, 95%, or 99% identity thereto.

[0016] 3. The peptide insert is shown in FIG. (a) 450 to 459 of the AAV1 capsid amino acid sequence (SEQ ID NO: 139); (b) 449 to 458 of the AAV2 capsid amino acid sequence (SEQ ID NO: 140); (c) 449 to 459 of the AAV3 capsid amino acid sequence (SEQ ID NO: 141); (d) 443 to 453 of the AAV4 capsid amino acid sequence (SEQ ID NO: 142); (e) 442 to 445 of the AAV5 capsid amino acid sequence (SEQ ID NO: 143); (f) 450 to 459 of the AAV6 capsid amino acid sequence (SEQ ID NO: 144); (g) 451 to 461 of the AAV7 capsid amino acid sequence (SEQ ID NO: 145); (h) 451 to 461 of the AAV8 capsid amino acid sequence (SEQ ID NO: 146); (i) 451 to 461 of the AAV9 capsid amino acid sequence (SEQ ID NO: 151); (j) 451 to 461 of the AAVhu.32 capsid amino acid sequence (SEQ ID NO: 148); (k) 452 to 461 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 152); (l) 452 to 461 of the AAVrh20 capsid amino acid sequence (SEQ ID NO: 155); (m) 452 to 461 of the AAVhu.37 capsid amino acid sequence (SEQ ID NO: 153); (n) 452 to 461 of the AAVrh39 capsid amino acid sequence (SEQ ID NO: 150); (o) 452 to 461 of the AAVrh74 capsid amino acid sequence (SEQ ID NO: 156 or SEQ ID NO: 157), or (p) 452 to 461 of the AAV9.AAA capsid amino acid sequence (SEQ ID NO: 158) 3. The rAAV capsid protein of embodiment 1 or 2, wherein the rAAV capsid protein occurs immediately after one of the amino acid residues in

[0017] 4. The rAAV capsid protein of embodiment 3, wherein the peptide insertion occurs after an amino acid residue corresponding to one of amino acids 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid or variant AAV9.AAA capsid.

[0018] 5. The rAAV capsid protein of embodiment 4, wherein the peptide insertion occurs after an amino acid residue corresponding to amino acid S454 of the AAV9 capsid protein, the AAV.32 capsid protein, a variant AAV9.AAA capsid protein, or a variant AAVhu.32.AAA capsid protein.

[0019] 6. The rAAV capsid protein of any one of embodiments 1 to 5, wherein the peptide is 7 amino acids.

[0020] 7. The rAAV capsid protein of any one of embodiments 1 to 6, wherein the reference capsid protein is AAV9, AAV9.AAA, AAVhu32, or AAVhu32.AAA.

[0021] 8. The peptide insert comprises an amino acid sequence of at least 4 and up to 7 consecutive amino acids of SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 138, wherein SEQ ID NO: 135 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S, G, V, or A; or SEQ ID NO: 136 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X 8. The rAAV capsid protein of any one of embodiments 1-7, wherein SEQ ID NO: 135 is S or A, or SEQ ID NO: 137 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S, G, V, or A, or SEQ ID NO: 138 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S or A.

[0022] 9. The rAAV capsid protein of embodiment 8, wherein the peptide insert has the amino acid sequence of VQVGRAA (SEQ ID NO:5), VQVGRTS (SEQ ID NO:8), AQVGRAS (SEQ ID NO:24), VQVGRVS (SEQ ID NO:36), VQVGRSS (SEQ ID NO:44), VQVGRYS (SEQ ID NO:130), VQVGRAS (SEQ ID NO:131), VQVGRPS (SEQ ID NO:132), VQVVRPS (SEQ ID NO:133), or VQVGHAS (SEQ ID NO:134).

[0023] 10. The rAAV capsid protein of embodiment 8 or 9, having the amino acid sequence of SEQ ID NO: 263, 264, 265, or 266.

[0024] 11. The rAAV capsid protein of any one of embodiments 1-7, wherein the peptide insert comprises an amino acid sequence of at least 4 and up to 7 contiguous amino acids of SGTVIRS (SEQ ID NO: 1), TRQYVPG (SEQ ID NO: 4), VQVGRTS (SEQ ID NO: 8), RGAVQKV (SEQ ID NO: 13), GQVHQAR (SEQ ID NO: 32), TSGGQIR (SEQ ID NO: 38), HMGHSGK (SEQ ID NO: 88), MRAVSQL (SEQ ID NO: 109), PRQYVPG (SEQ ID NO: 110), RSSSGR (SEQ ID NO: 111), VVKSTKS (SEQ ID NO: 112), RHVSASD (SEQ ID NO: 113), VRSDRDQ (SEQ ID NO: 114), or TVVTSIN (SEQ ID NO: 115).

[0025] 12. The rAAV capsid protein of embodiment 11, wherein the peptide insert is SGTVIRS (SEQ ID NO: 1), TRQYVPG (SEQ ID NO: 4), VQVGRTS (SEQ ID NO: 8), RGAVQKV (SEQ ID NO: 13), GQVHQAR (SEQ ID NO: 32), TSGGQIR (SEQ ID NO: 38), HMGHSGK (SEQ ID NO: 88), MRAVSQL (SEQ ID NO: 109), PRQYVPG (SEQ ID NO: 110), RSSSGR (SEQ ID NO: 111), VVKSTKS (SEQ ID NO: 112), RHVSASD (SEQ ID NO: 113), VRSDRDQ (SEQ ID NO: 114), or TVVTSIN (SEQ ID NO: 115).

[0026] 13. The rAAV capsid protein of any one of embodiments 1-7, wherein the peptide insert comprises an amino acid sequence of at least 4 and up to 7 contiguous amino acids of AQVGRAS (SEQ ID NO:24), SVTSVRV (SEQ ID NO:37), SIAKNSA (SEQ ID NO:76), DGRRIGV (SEQ ID NO:116), TSGERRG (SEQ ID NO:117), PSSVQHR (SEQ ID NO:118), SSSVQHR (SEQ ID NO:119), SGMQERR (SEQ ID NO:120), LERGNLE (SEQ ID NO:121), YRDVRQT (SEQ ID NO:122), PSAVQHR (SEQ ID NO:123), YVGGRAV (SEQ ID NO:124), IGSRGVA (SEQ ID NO:125), SDVSRPR (SEQ ID NO:126), GSVRQAA (SEQ ID NO:127), QSPHTSQ (SEQ ID NO:128), or ASQAYHG (SEQ ID NO:128).

[0027] 14. The rAAV capsid protein of embodiment 13, wherein the peptide insert is AQVGRAS (SEQ ID NO:24), SVTSVRV (SEQ ID NO:37), SIAKNSA (SEQ ID NO:76), DGRRIGV (SEQ ID NO:116), TSGERRG (SEQ ID NO:117), PSSVQHR (SEQ ID NO:118), SSSVQHR (SEQ ID NO:119), SGMQERR (SEQ ID NO:120), LERGNLE (SEQ ID NO:121), YRDVRQT (SEQ ID NO:122), PSAVQHR (SEQ ID NO:123), YVGGRAV (SEQ ID NO:124), IGSRGVA (SEQ ID NO:125), SDVSRPR (SEQ ID NO:126), GSVRQAA (SEQ ID NO:127), QSPHTSQ (SEQ ID NO:128), or ASQAYHG (SEQ ID NO:128).

[0028] 15. The rAAV capsid protein of embodiment 14, wherein the peptide insert is AQVGRAS (SEQ ID NO: 24), PSSVQHR (SEQ ID NO: 118), YVGGRAV (SEQ ID NO: 124), IGSRGVA (SEQ ID NO: 125), or GSVRQAA (SEQ ID NO: 127).

[0029] 16. The rAAV capsid protein of embodiment 14, wherein the peptide insert is DGRRIGV (SEQ ID NO: 116), SSSVQHR (SEQ ID NO: 119), YRDVRQT (SEQ ID NO: 122), or PSAVQHR (SEQ ID NO: 123).

[0030] 17. The rAAV capsid protein of embodiment 1, having any one of the amino acid sequences of SEQ ID NOs: 159-266 and 268-375.

[0031] 18. The rAAV capsid protein of any one of the preceding embodiments, wherein the target tissue with the improved targeting is skeletal muscle or cardiac muscle.

[0032] 19. The rAAV capsid protein of embodiment 18, wherein an rAAV vector prepared from said capsid protein exhibits at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold greater transduction of skeletal muscle and / or cardiac muscle than an rAAV vector prepared from said capsid protein without said peptide insert or said reference capsid protein.

[0033] 20. The rAAV capsid protein of any one of the preceding embodiments, wherein the target tissue is a CNS neuron.

[0034] 21. The rAAV capsid protein of embodiment 20, wherein an rAAV vector prepared from said capsid protein has at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold greater transduction of CNS neurons than an rAAV vector prepared from said capsid protein without said peptide insert or said reference capsid protein.

[0035] 22. The rAAV capsid protein of any one of the preceding embodiments, wherein an rAAV vector prepared from said capsid protein has reduced transduction of liver, heart, or astrocytes compared to an rAAV vector prepared from said capsid protein without said peptide insert or said reference capsid protein.

[0036] 23. The rAAV capsid protein of embodiment 22, wherein the rAAV vector prepared from the capsid protein has liver transduction that is at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, or 40-fold lower than that of an rAAV vector prepared from the capsid protein without the peptide insert or the reference capsid protein.

[0037] 24. The rAAV capsid protein of any one of embodiments 19, 21, 22, or 23, wherein the reference capsid protein is an AAV9 capsid or an AAVhu.32 capsid.

[0038] 25. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to any one of the preceding embodiments or embodiments 42-48, or encoding an amino acid sequence sharing at least 80% identity therewith.

[0039] 26. The nucleic acid of embodiment 25, encoding the rAAV capsid protein of any one of the preceding embodiments.

[0040] 27. A packaging cell capable of expressing the nucleic acid of embodiment 25 or 26 to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

[0041] 28. An rAAV vector comprising a capsid protein according to any one of embodiments 1 to 24 or embodiments 42 to 48.

[0042] 29. The rAAV vector of embodiment 28, further comprising an rAAV genome comprising a transgene flanked by AAV ITR sequences.

[0043] 30. A pharmaceutical composition comprising the rAAV vector of embodiment 28 or 29 and a pharmaceutically acceptable carrier.

[0044] 31. A method for delivering a transgene to a cell, said method comprising contacting said cell with an rAAV vector of embodiment 28 or 29, or an rAAV vector of embodiment 28 or 29 for use in delivering a transgene to a cell, wherein said cell is contacted with said vector.

[0045] 32. A method for delivering a transgene to a target tissue in a subject in need thereof, said method comprising administering to said subject an rAAV vector of embodiment 28 or 29, or an rAAV vector of embodiment 28 or 29 for use in delivering a transgene to a target tissue in a subject in need thereof, wherein said vector is administered to said subject.

[0046] 33. The method or use of embodiment 32, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, intravitreally, via lumbar puncture, or via the cisterna magna.

[0047] 34. The rAAV vector for the method or use according to embodiment 32 or embodiment 33, wherein the target tissue is muscle, retina, or CNS.

[0048] 35. A method for producing a recombinant AAV (rAAV) vector library, comprising: (i) generating a starting plasmid having a gene expression cassette comprising a nucleic acid sequence encoding an AAV capsid protein, wherein a stop codon is positioned at a target insertion site within the nucleic acid sequence encoding the AAV capsid protein; (ii) providing a population of nucleic acids encoding a repertoire of peptides to generate a peptide library; (iii) creating individual plasmids based on the starting plasmids, each of which comprises the nucleic acid encoding the AAV capsid with a nucleic acid encoding a random peptide from the peptide library inserted into the target insertion site of the nucleic acid encoding the capsid protein, thereby replacing the stop codon, and 2) each of which further contains a nucleic acid encoding a barcode for identifying the nucleic acid encoding the capsid, and a peptide insert positioned before the 5' end or after the 3' end of the nucleic acid encoding the capsid; (iv) collecting the individual plasmids; and (v) transfecting the cell population with a collection of individual plasmids and one or more plasmids carrying the rAAV genome containing the transgene and required genes; (vi) culturing the transfected cell population under appropriate conditions to produce a collection of rAAV vectors, each comprising the AAV capsid with the peptide insert encapsulating the rAAV genome containing the transgene; (vii) harvesting the rAAV vector library.

[0049] 36. The method of embodiment 35, wherein the capsid gene encodes an AAV1 capsid protein (SEQ ID NO: 139), an AAV4 capsid protein (SEQ ID NO: 142), an AAV5 capsid protein (SEQ ID NO: 143), an AAV8 capsid protein (SEQ ID NO: 146), an AAV9 capsid protein (SEQ ID NO: 151), an AAV9.AAA capsid protein (SEQ ID NO: 158), an AAVhu.32 (SEQ ID NO: 148), an AAVhu.32.AAA (SEQ ID NO: 267), an AAV3B capsid protein (SEQ ID NO: 154), or an AAVhu37 capsid protein (SEQ ID NO: 153).

[0050] 37. The method of any one of embodiments 34 to 36, wherein the target insertion site is at or after an amino acid residue within VR-4 or VR-8 of the capsid protein.

[0051] 38. The method of embodiment 37, wherein the target insertion site is after the amino acid corresponding to S454 of AAV9 (sequence number 151).

[0052] 39. The method of any one of embodiments 34 to 38, wherein the peptide is a peptide of 7 amino acids.

[0053] 40. A rAAV vector library produced by the method of any one of embodiments 34 to 39.

[0054] 41. The rAAV vector library of embodiment 40, wherein at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the rAAV vectors in the library have capsids with peptide inserts.

[0055] 42. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insert of at least 4 and up to 7 consecutive amino acids selected from the group consisting of SEQ ID NOs: 1-138, wherein the peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle; the capsid protein is a wild-type capsid protein or a variant capsid protein having up to three amino acid substitutions; The recombinant adeno-associated virus (rAAV) capsid protein, wherein an rAAV vector prepared from the capsid protein containing the peptide insert provides improved target tissue targeting compared to an rAAV vector prepared from a capsid protein without the peptide insert or a reference capsid protein.

[0056] 43. The rAAV capsid protein of embodiment 42, wherein the capsid protein is serotype 5 (AAV5) (SEQ ID NO: 143), or a capsid protein having 90%, 95%, or 99% sequence identity thereto.

[0057] 44. The rAAV capsid protein of embodiment 42 or 43, wherein the peptide insertion occurs immediately after one of amino acid residues 574 to 584 of the AAV5 capsid protein.

[0058] 45. The rAAV capsid protein of any one of embodiments 42 to 44, wherein the peptide insertion occurs immediately after amino acid residue 578 of the AAV capsid protein.

[0059] 46. ​​The rAAV capsid protein of any one of embodiments 42 to 45, wherein the peptide insert is RHVSASD (sequence number 113), VRSDRDQ (sequence number 114), or TVVTSIN (sequence number 115).

[0060] 47. The rAAV capsid protein of any one of embodiments 42 to 46, wherein the reference capsid protein is AAV5, AAV9, AAV9.AAA, AAVhu32, or AAVhu32.AAA.

[0061] 48. The rAAV capsid protein of any one of embodiments 42 to 47, having the amino acid sequence of SEQ ID NO: 376, 377, or 378. [Brief explanation of the drawings]

[0062] 4. Brief description of the drawings [Figure 1-1] An alignment of the AAV1-9, hu31, hu32, and rh10 capsid sequences is shown, highlighting the VR-IV insertion sites for these capsids (see VR4, which corresponds to amino acids 451-461 of the AAV9 capsid protein, and VR8, which corresponds to amino acids 585-593 of the AAV9 capsid protein). A clustal multiple sequence alignment of AAV capsids was performed to demonstrate that amino acid substitutions (shown in bold on the bottom line) can be made into AAV9, AAVhu.32, or other capsids by "recruiting" amino acid residues from corresponding positions in other aligned AAV capsids. Sequences shown in gray are hypervariable regions (HVRs). The amino acid sequences of the AAV capsids are assigned SEQ ID NO: 139, AAV2 is SEQ ID NO: 140, AAV3 is SEQ ID NO: 141, AAV4 is SEQ ID NO: 142, AAV5 is SEQ ID NO: 143, AAV6 is SEQ ID NO: 144, AAV7 is SEQ ID NO: 145, AAV8 is SEQ ID NO: 146, AAV9 is SEQ ID NO: 151, AAVrh10 is SEQ ID NO: 152, hu31 is SEQ ID NO: 147, and hu32 is SEQ ID NO: 148. [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 1-4] Same as above [Figure 1-5] Same as above [Figure 2] 1 shows protein models of variable regions 4 and 8 of adeno-associated virus type 9 (AAV9 VR-IV and AAV9 VR-VIII, respectively). [Figure 3]A representative genomic construct of a capsid gene for use in constructing an rAAV library is shown, with, from 5' to 3': the 5' inverted terminal repeat (ITR), the CMV enhancer-promoter, the Rep intron, the AAV Cap gene of interest, a polyA sequence, and the 3'-ITR. The diagram shows the insertion of a random peptide library (see arrow) in place of the stop codon inserted into the Cap gene variable region prior to library construction (to reduce expression of wild-type sequences in the library). [Figure 4] Shown are PCR amplification products obtained from libraries A1 to G1 used for NGS analysis of library diversity. [Figure 5] Figure 1 shows graphed results of % wild-type and % stop codon sequences from NGS analysis of plasmid and vector libraries. A shows a library with high parental vector levels (% vector weight) after vector generation compared to the percent of parental plasmid in the initial plasmid preparation of the library (% plasmid weight). B shows NGS analysis of a library in which a stop codon was inserted into the template plasmid prior to plasmid library construction. Results show a reduction in stop parental sequences in the vector library (% vector stop) compared to the initial plasmid library (% plasmid stop). [Figure 6] Liquid chromatography-mass spectrometry (LC-MS) of VP3 protein after generation of AAV5 vector libraries with or without stop codons in the template. [Figure 7] Peptides analyzed by NGS in various tissues are shown, with enrichment scores represented by different shading. SEQ ID NOs: 1-24 (top to bottom) are listed to the left of the density plot. [Figure 8] The relative abundance (RA), expressed as fold change, is shown for a given rAAV vector with a peptide insert (e.g., 24 = peptide 24 = SEQ ID NO: 24) relative to the control parent vector, AAV9.AAA. RA comparisons are made in heart (A), skeletal muscle (B), and liver (C) tissues. [Figure 9]Shown are nRAAFI in NHPs for selected rAAV vectors (NVG01-NVG14) with peptide inserts compared to AAV9, AAVhu32, and AAV5 in skeletal muscle (A) and heart (B). [Figure 10] 1A-B are bar graphs showing mRNA / DNA in NHPs for selected rAAV vectors (NVG01-NVG14) with peptide inserts compared to AAV9, AAVhu32, and AAV5 in skeletal muscle (A) and heart (B). [Figure 11] 1 is a bar graph showing nRAAFI of mRNA (A) and DNA (B) in the livers of NHPs injected with pooled AAVs (NVG01-NVG14) with peptide inserts. [Figure 12] 1A-C are bar graphs showing the relative abundance (RA) of mRNA in skeletal muscle (A) liver (B) or the ratio of mRNA in muscle / liver (C) in individual NHPs (numbered 1001, 1002, 1003, 4001, and 4003) injected with either AAV9 or AAV9.AAA.NVG07 (SEQ ID NO: 8). [Figure 13]

[0033] Figure 1 is a bar graph showing RNA (TdTom / TBP) or DNA (TdTom GC / diploid genome) in CB57B16 mice injected with a 1E14 GC / kg IV dose of AAV (AAVhu.32, AAV9.AAA.NVG07, or AAVhu.32.AAA.NVG09) expressing the CAG.TdTomato transgene at 3 weeks. RNA and DNA were measured in the heart, gastrocnemius, quadriceps, biceps, tibialis anterior (TA), brain, liver, and diaphragm. DETAILED DESCRIPTION OF THE INVENTION

[0063] 5. MODE FOR CARRYING OUT THE INVENTION Recombinant adeno-associated viruses (rAAVs) are provided having capsid proteins modified to contain amino acid sequences that confer and / or enhance desired properties, such as tissue targeting, transduction, and integration of the rAAV genome. In particular, modified capsid proteins are provided that contain peptide inserts of 4 to 7 consecutive amino acids from a random peptide library inserted within or near variable region IV (VR-IV) or VR-VIII of the viral capsid, such that the peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle. Libraries of recombinant capsid proteins and individual rAAVs containing peptides that target specific tissues and / or enhance rAAV cellular uptake, transduction, and / or transgene expression are also provided; see, for example, Table 17.

[0064] 5.1.Definition The term "AAV" or "adeno-associated virus" refers to Dependoparvovirus, a virus in the Parvovirus genus of viruses. AAV can be derived from a naturally occurring "wild-type" virus, derived from a rAAV genome packaged in a capsid containing capsid proteins encoded by a naturally occurring cap gene, and / or derived from a rAAV genome packaged in a capsid containing capsid proteins encoded by a non-naturally occurring capsid cap gene. Examples of the latter include rAAVs with capsid proteins containing peptide insertions within the amino acid sequence of the naturally occurring capsid.

[0065] The term "rAAV" refers to "recombinant AAV." In some embodiments, a recombinant AAV has an AAV genome in which some or all of the rep and cap genes have been replaced with heterologous sequences.

[0066] The term "rep-cap helper plasmid" refers to a plasmid that provides viral rep and cap gene functions and assists in the production of AAV from rAAV genomes that lack functional rep and / or cap gene sequences.

[0067] The term "cap gene" refers to a nucleic acid sequence that encodes a capsid protein that forms or helps form the capsid coat of a virus. In the case of AAV, the capsid protein can be VP1, VP2, or VP3.

[0068] The term "rep gene" refers to a nucleic acid sequence that encodes a nonstructural protein necessary for viral replication and production.

[0069] The terms "nucleic acid" and "nucleotide sequence" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA / RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated using nucleotide analogs, including, but not limited to, inosine or tritylated bases. Such analogs can also include DNA or RNA molecules containing modified backbones that confer beneficial attributes on the molecule, such as, for example, increased nuclease resistance or ability to cross cell membranes. A nucleic acid or nucleotide sequence can be single-stranded, double-stranded, or contain both single- and double-stranded portions, or triple-stranded portions, but is preferably double-stranded DNA.

[0070] As used herein, the terms "subject," "host," and "patient" are used interchangeably. As used herein, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), or in certain embodiments, a human.

[0071] A "library" or "libraries" generally refers to a repertoire of rAAV vectors generated from capsid genes (each unique and typically recombinantly placed in a vehicle such as a plasmid) or unique capsids.

[0072] As used herein, the term "conservative amino acid substitution" refers to substitutions made in accordance with Tables A and B. [Table 1] [Table 2]

[0073] It is understood that one way to define variants and derivatives of the capsid proteins disclosed herein is to define them in terms of homology / identity to specific known sequences. Specifically disclosed are variants of the capsids disclosed herein that have at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the capsid sequences specifically recited herein. Those skilled in the art will readily understand how to determine the identity of two proteins. The capsid variants described herein can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid substitutions, including conservative amino acid substitutions. Variants of the 4-7 amino acid peptides described herein include peptides with 1, 2, or 3 amino acid substitutions, including conservative amino acid substitutions.

[0074] The term "therapeutic agent" refers to any agent that can be used in the treatment, management, or amelioration of symptoms associated with a disease or disorder, where the disease or disorder is related to the function provided by the transgene. As used herein, a "prophylactically effective amount" refers to an amount of agent (e.g., the amount of product expressed by the transgene) that, when administered to a subject suffering from the target disease or disorder, provides at least one therapeutic benefit in the treatment or management of the target disease or disorder in preventing or delaying the disease or disorder. Furthermore, a therapeutically effective amount with respect to an agent of the present invention means that the amount of agent, alone or in combination with other therapeutic agents, provides at least one therapeutic benefit in the treatment or management of the disease or disorder.

[0075] As used herein, the term "prophylactic agent" refers to any agent that can be used in preventing, delaying, or slowing the progression of a disease or disorder, where the disease or disorder is related to the function provided by the transgene. As used herein, a "prophylactically effective amount" refers to the amount of a prophylactic agent (e.g., the amount of a product expressed by a transgene) that provides at least one prophylactic benefit in preventing or delaying a target disease or disorder when administered to a subject predisposed thereto. A prophylactically effective amount can also refer to the amount of agent sufficient to prevent or delay the onset of the target disease or disorder or slow the progression of the target disease or disorder, to delay or minimize the onset of the target disease or disorder, or to prevent or delay their recurrence or spread. A prophylactically effective amount can also refer to the amount of agent sufficient to prevent or delay the worsening of symptoms of the target disease or disorder. Furthermore, a prophylactically effective amount with respect to a prophylactic agent of the present invention means that the amount of prophylactic agent, alone or in combination with other therapeutic agents, provides at least one prophylactic benefit in preventing or delaying a disease or disorder.

[0076] The preventive agent of the present invention can be administered to a subject who is "predisposed" to the target disease or disorder. A subject who is "predisposed" to a disease or disorder is a subject who shows symptoms related to the onset of the disease or disorder, or who has a genetic makeup, environmental exposure, or other risk factors for such a disease or disorder, but whose symptoms have not yet reached the level required for diagnosis as a disease or disorder. For example, a patient who has a family history of a disease associated with a defective gene (provided by the transgene) may be considered to be predisposed to it.

[0077] 5.2. Recombinant AAV Capsids and Vectors One aspect relates to a capsid protein library and its recombinant adeno-associated virus (rAAV) vectors, in which capsid proteins in the library are modified to contain peptide inserts from a random peptide library, where the peptides are not AAV proteins or peptide fragments thereof, and the peptide inserts are surface-exposed when packaged as AAV particles. In some embodiments, the peptide inserts occur within variable region IV (VR-IV) of the AAV9 capsid or AAV9.AAA capsid, or a corresponding region of another type of AAV capsid (i.e., between two amino acids without deleting any capsid amino acids) (see alignment in Figure 1). In some embodiments, the peptide inserts occur within variable region VIII (VR-VIII) of the AAV9 capsid or AAV9.AAA capsid, or a corresponding region of another type of AAV capsid (i.e., between two amino acids without deleting any capsid amino acids) (see alignment in Figure 1). In some embodiments, the peptide insert is derived from a heterologous protein or domain (other than an AAV capsid protein or domain) that targets the rAAV particle to a target tissue and / or facilitates rAAV uptake, transduction, and / or transgene expression. Also provided are nucleic acids encoding the modified capsid proteins and variants thereof, packaging cells for expressing nucleic acids to generate rAAV vectors, rAAV vectors further comprising a transgene, and pharmaceutical compositions of the rAAV vectors, as well as methods of using the rAAV vectors to deliver a transgene to a target cell type or tissue in a subject in need thereof.

[0078] In various embodiments, the target tissue may be muscle tissue, such as skeletal or cardiac muscle, nervous tissue, bone, kidney, eye / retina, or endothelial tissue, and the capsid with the peptide insert specifically recognizes and / or binds to and / or homes to that tissue or one or more particular cell types, for example, within the target tissue or its cellular matrix. In particular, peptides that can target rAAV to muscle tissue, including skeletal muscle and heart, may be useful for delivering therapeutic agents to treat muscle disorders. In embodiments, rAAV capsids containing modified capsid proteins with peptide inserts have increased uptake, transduction, and / or transgene expression over parental or reference capsids, including AAV9, AAV9.AAA, AAVhu.32, AAVhu.32.AAA, or AAV5. The modified capsids may further have reduced transduction in tissues such as liver, heart, or astrocytes, or other tissues relative to the target tissue or relative to transduction with a parent or reference capsid, including AAV9, AAV9.AAA, AAVhu.32, AAVhu.32.AAA, or AAV5.

[0079] In various embodiments, the target tissue can be neural tissue, particularly neurons in the brain. In embodiments, the peptide insert preferentially targets the rAAV to neurons over astrocytes, compared to an rAAV without the peptide insert.

[0080] In embodiments, the parent capsid and / or peptide insert detargets rAAV vectors prepared from the modified capsid proteins from one or more tissue types, including the liver.

[0081] 5.2.1 rAAV Vectors with Peptide Inserts A peptide insertion described as being inserted "at" a given site refers to an insertion immediately following (i.e., with a peptide bond to its carboxy group) the residue normally found at that site in wild-type virus. For example, an insertion at S454 in AAV9 means that the peptide insertion occurs between S454 and the consecutive amino acid (G455) in the AAV9 wild-type capsid protein sequence (SEQ ID NO: 151). In embodiments, there is no deletion of amino acid residues at or near the insertion point (within 5, 10, 15 residues or within the structural loop that is the site of the insertion).

[0082] In embodiments, the capsid protein is an AAV9 capsid protein (SEQ ID NO: 151, or a capsid protein having an amino acid sequence 90%, 95%, or 99% identical to SEQ ID NO: 151) or an AAV9.AAA capsid protein, and the insertion occurs immediately after at least one of amino acid residues 451-461. In embodiments, the peptide insertion occurs immediately after amino acid 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid (amino acid sequence SEQ ID NO: 151). In some embodiments, the peptide is inserted between residues S454 and G455 of the AAV9 capsid protein, or between residues corresponding to S454 and G455 of an AAV capsid protein other than the AAV9 capsid protein (amino acid SEQ ID NO: 151). In embodiments, the capsid protein is an AAV9.AAA capsid protein, and the insertion occurs immediately after at least one of amino acid residues 451 to 461. In embodiments, the peptide insertion occurs immediately after amino acid 1451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9.AAA capsid (amino acid sequence SEQ ID NO: 123). In certain embodiments, the peptide is inserted between residues S454 and G455 of the AAV9.AAA capsid protein, or between the corresponding residues of S454 and G455 of an AAV.AAA capsid protein other than the AAV9 capsid protein (amino acid sequence SEQ ID NO: 123).

[0083] In embodiments, the capsid protein is a hu.32 capsid protein (SEQ ID NO: 148, or a capsid protein having an amino acid sequence 90%, 95%, or 99% identical to SEQ ID NO: 148) or an AAVhu.32.AAA capsid protein, and the insertion occurs immediately after at least one of amino acid residues 451-461. In embodiments, the peptide insertion occurs immediately after amino acid I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAVhu.32 capsid (amino acid sequence SEQ ID NO: 148). In certain embodiments, the peptide is inserted between residues S454 and G455 of the AAVhu.32 capsid protein. In embodiments, the capsid protein is an AAVhu.32.AAA capsid protein, and the insertion occurs immediately after at least one of amino acid residues 451 to 461. In embodiments, the peptide insertion occurs immediately after amino acid I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAVhu.32.AAA capsid (amino acid sequence SEQ ID NO: 148). In certain embodiments, the peptide is inserted between residues S454 and G455 of the AAVhu.32.AAA capsid protein.

[0084] In embodiments, the capsid protein is an AAV5 capsid protein (SEQ ID NO: 143, or a capsid protein having an amino acid sequence 90%, 95%, or 99% identical to SEQ ID NO: 143), and the insertion occurs immediately after at least one of amino acid residues 442-447. In embodiments, the peptide insertion occurs immediately after amino acids N442, N443, T444, G445, G446, V447 of the AAV5 capsid (amino acid sequence SEQ ID NO: 143). In embodiments, the peptide is inserted after a residue within VR8 of the AAV5 capsid protein. In embodiments, the peptide is inserted after residue 578 of the AAV5 capsid protein.

[0085] In other embodiments, the capsid protein is selected from AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu37 (AAVhu37), serotype hu40 (AAVhu40), serotype hu50 (AAVhu50), serotype hu60 (AAVhu60), serotype hu70 (AAVhu70), serotype hu8 (AAVhu70), serotype hu90 (AAVhu9 ... The capsids are derived from at least one AAV type selected from AAVhu.37, serotype rh74 (AAVrh74, versions 1 and 2) (see FIG. 1), or capsids having an amino acid sequence at least 90%, 95%, or 99% identical to the amino acid sequence of the aforementioned capsid proteins VP1, VP2, or VP3, and the insertion occurs immediately after at least one corresponding amino acid residue among amino acid residues 451-461 of AAV9. Alignments of these different AAV serotypes, as shown in FIG. 1, show "corresponding" amino acid residues in the different capsid amino acid sequences such that the "corresponding" amino acid residues are aligned in the same positions in the alignment as residues in the reference sequence. In some embodiments, the peptide insert is located at positions 450-459 of the AAV1 capsid (SEQ ID NO: 139), 449-458 of the AAV2 capsid (SEQ ID NO: 140), 449-459 of the AAV3 capsid (SEQ ID NO: 141), 443-453 of the AAV4 capsid (SEQ ID NO: 142), 442-445 of the AAV5 capsid (SEQ ID NO: 143), 450-459 of the AAV6 capsid (SEQ ID NO: 144), 451-461 of the AAV7 capsid (SEQ ID NO: 145), or 452-463 of the AAV8 capsid (SEQ ID NO: 146), as shown in FIG. 451-461 of the AAV9 capsid (SEQ ID NO: 151), 451-461 of the AAVhu.32 capsid (SEQ ID NO: 148), 452-461 of the AAVrh10 capsid (SEQ ID NO: 152), 452-461 of the AAVrh20 capsid (SEQ ID NO: 155), 452-461 of the AAVhu.37 (SEQ ID NO: 149), 452-461 of the AAVrh74 (SEQ ID NO: 156 or SEQ ID NO: 157), or 452-461 of the AAVrh39 (SEQ ID NO: 150).In certain embodiments, the rAAV capsid protein comprises a peptide insert immediately (i.e., C-terminal to) amino acid 588 of the AAV9 capsid protein (having the amino acid sequence of SEQ ID NO:151; see FIG. 1 ) or AAV9.AAA (having SEQ ID NO:158), such that the peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle. In other embodiments, the rAAV capsid protein has a peptide insert that is not immediately after amino acid 588 of AAV9 or AAV9.AAA, or corresponding to amino acid 588 of AAV9.

[0086] Also provided are AAV vectors comprising modified capsids. In some embodiments, the AAV vectors are non-replicative and do not contain nucleotide sequences encoding rep or cap proteins (which are provided by packaging cells in the production of rAAV vectors). In some embodiments, the AAV-based vectors comprise components from one or more serotypes of AAV. In some embodiments, the AAV-based vectors provided herein include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, and / or AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more thereof.In some embodiments, the AAV-based vectors provided herein are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB , AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or other rAAV particles, or a combination of two or more serotypes.In some embodiments, the rAAV particles may be any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4 AAV capsid proteins that are at least 80% identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or derivatives, modifications, or pseudotypes thereof. These modified AAV vectors can contain a genome that includes a transgene encoding a therapeutic protein.

[0087] In embodiments, the recombinant AAV for use in the compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6):1056-1068, incorporated herein by reference in its entirety). In embodiments, the recombinant AAV for use in the compositions and methods herein is AAV.7m8 (including variants thereof) (see, e.g., US9,193,956, US9,458,517, US9,587,282, US2016 / 0376323, and WO2018 / 075798, each of which is incorporated herein by reference in its entirety). In embodiments, the AAV for use in the compositions and methods herein is any AAV disclosed in US9,585,971, such as AAV-PHP.B. In embodiments, the AAV for use in the compositions and methods herein is an AAV2 / Rec2 or AAV2 / Rec3 vector, which has a hybrid capsid sequence derived from AAV8 and serotypes cy5, rh20, or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4):e60361, which is incorporated herein by reference for a description of these vectors). In embodiments, the AAV for use in the compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US 7,282,199, US 7,906,111, US 8,524,446, US 8,999,678, US 8,628,966, US 8,927,514, US 8,734,809, US 9,284,357, US 9,4 09,953, US9,169,299, US9,193,956, US9,458,517, US9,587,282, US2015 / 0374803, US2015 / 0126588, US2017 / 0067908, US2013 / 0224836, US2016 / 0215024, US2017 / 0051257, PCT / US2015 / 034799, and PCT / EP2015 / 053335.In some embodiments, the rAAV particles have capsid proteins that are at least 80% identical or greater, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences of the AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: U.S. Pat. Nos. 7,282,199, 7,906,111, 8,524,446, 8,999,678, and 7,906,111. Nos. 8,628,966, 8,927,514, 8,734,809, 9,284,357, 9,409,953, 9,169,299, 9,193,956, 9,458,517, and 9,587,282, U.S. Patent Application Publication No. 2015 / 0374 Nos. 803, 2015 / 0126588, 2017 / 0067908, 2013 / 0224836, 2016 / 0215024, and 2017 / 0051257, as well as International Patent Application Nos. PCT / US2015 / 034799 and PCT / EP2015 / 053335.

[0088] In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in U.S. Patent No. 9,840,719 and WO 2015 / 013313, e.g., AAV.Rh74 and RHM4-1 (each of which is incorporated by reference in its entirety). In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO 2014 / 172669, such as AAV rh.74 (each of which is incorporated by reference in its entirety). In some embodiments, the rAAV particles comprise the AAV2 / 5 capsid described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450 (each of which is incorporated by reference in its entirety). In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO2017 / 070491, such as AAV2tYF, which are incorporated by reference in their entirety. In some embodiments, the rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418, which are incorporated by reference in their entirety. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Pat. Nos. 8,628,966, 8,927,514, 9,923,120, and WO2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of which is incorporated by reference in its entirety).

[0089] In some embodiments, the rAAV particles are prepared using the methods described in International Application Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO: 2 of the '051 publication), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of the '321 publication), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of the '888 publication), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of the '689 publication), WO200 and WO 2015 / 191508 (see, e.g., SEQ ID NOS: 80-294 of the '508 publication), as well as U.S. Application Publication No. 2015 / 0023924 (see, e.g., SEQ ID NOS: 1, 5-10 of the '924 publication), the contents of each of which are incorporated herein by reference in their entirety.In some embodiments, the rAAV particles are prepared using methods described in International Application Publication Nos. WO2003 / 052051 (see, e.g., SEQ ID NO: 2 of the '051 publication), WO2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of the '321 publication), WO03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of the '888 publication), WO2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of the '689 publication), WO2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31 of the '964 publication), and WO 2010 / 127097 (see, e.g., SEQ ID NOS: 5-38 of the '097 publication), WO 2010 / 127097 (see, e.g., SEQ ID NOS: 80-294 of the '508 publication), and WO 2015 / 191508 (see, e.g., SEQ ID NOS: 80-294 of the '508 publication), and U.S. Application Publication No. 2015 / 0023924 (see, e.g., SEQ ID NOS: 1, 5-10 of the '924 publication), and have capsid proteins that are at least 80% identical, e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

[0090] In additional embodiments, the rAAV particles comprise pseudotyped AAV capsids. In some embodiments, the pseudotyped AAV capsids are rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0091] In certain embodiments, single-stranded AAV (ssAAV) may be used. In certain embodiments, self-complementary vectors, such as scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, 8(16):1248-1254; US 6,596,535; US 7,125,717; and US 7,456,683, each of which is incorporated by reference in its entirety).

[0092] Typically, a peptide insert is a sequence of consecutive amino acids from a heterologous protein or domain thereof. The inserted peptide is typically long enough to retain the specific biological function, property, or characteristic of the protein or domain from which it is derived. The inserted peptide is typically short enough to allow the capsid protein to form a coat similar to or substantially similar to the native capsid protein without the insert. In preferred embodiments, the peptide insert is about 4 to about 30 amino acid residues in length, about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length. The peptide sequence for insertion is at least 4 amino acids in length and can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide sequence is 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide is 7 amino acids or less, 10 amino acids or less, or 12 amino acids or less in length.

[0093] A "peptide insert from a heterologous protein" in an AAV capsid protein refers to an amino acid sequence that has been introduced into the capsid protein and that is not native to the AAV serotype capsid into which it is inserted.

[0094] As used herein, the terms "homing" and "targeting" are used interchangeably. These peptides may also, or alternatively, promote cellular uptake, transduction, and / or genomic integration of rAAV in cells of the target tissue.

[0095] Exemplary peptides for use as peptide inserts in any of the AAV capsid sites described herein are set forth in Tables 4, 5, 14, 15, and 16 below in the Examples (including peptides having the amino acid sequences of SEQ ID NOS: 1-138), including at least a four amino acid contiguous portion thereof, or a seven amino acid contiguous portion thereof (including variants having one, two, or three amino acid substitutions, including conservative amino acid substitutions), where the functional attribute of the inserted peptide alters the properties of the capsid, particularly its tropism. In certain embodiments, recombinant AAV capsids and AAV vectors are modified to include a peptide from any of Tables 4, 5, 14, 15, and 16 below (including peptides having the amino acid sequences of SEQ ID NOS: 1-138), or at least a four, five, six, or seven amino acid contiguous portion thereof, inserted into the AAV capsid sequence in such a manner that the peptide insert is indicated. In other embodiments, the peptide is inserted after amino acid residues 138, 262-273, 451-461, or 585-593 of the amino acid sequence of the AAV9 capsid (SEQ ID NO: 151) or AAV9.AAA capsid (SEQ ID NO: 158), or the corresponding position in any other AAV serotype (see Figure 1 for capsid sequence alignment).

[0096] In another aspect, a heterologous peptide insert library is provided. A heterologous peptide insert library refers to a collection of rAAV vectors carrying the same random peptide inserts at the same insertion site in the viral capsid to generate a specific library, for example, at a position within a given variable region of the capsid. Generally, the capsid proteins used include an AAV genome containing modified rep and cap sequences to prevent viral replication under conditions that would normally allow replication (co-infection of mammalian cells with a helper virus such as adenovirus). Members of the peptide insert library can then be assayed for functional presentation of peptides on the rAAV surface, tissue targeting, and / or gene transduction. Improved or desired properties can be evaluated by comparing them to the parent capsid from which the insert library was generated.

[0097] Peptide insert libraries and methods for making these libraries are provided. An exemplary method for generating a capsid library with peptide inserts is described in Example 2 herein, and such libraries are screened herein. In such libraries, the nucleic acid encoding the parent capsid has a stop codon at the target insertion site of the population of peptide-encoding nucleic acids, such that the capsid protein is expressed only if the peptide insert is present; otherwise, translation terminates prematurely. Generation of a population of rAAV from the expression construct results in an rAAV population with a high percentage of capsids with peptide inserts (including 80%, 85%, 90%, 95%, 98%, 99%, or 100%) (see, e.g., Figures 5A, 5B, 6A, and 6B).

[0098] Also provided is a method for generating a capsid library with variable peptide inserts. In embodiments, the method includes: (1) providing a starting plasmid containing a gene expression cassette comprising a nucleic acid sequence encoding an AAV capsid, wherein a stop codon is positioned at a target insertion site within the capsid gene; (2) providing a repertoire of nucleic acids encoding randomized peptides to generate a peptide library; and (3) generating a peptide library comprising: a) a nucleic acid encoding a random peptide from the peptide library that is inserted into the target insertion site of the capsid gene, thereby replacing the stop codon; and b) a barcode for identifying capsid genes with an insertion positioned before the 5' end or after the 3' end of the capsid gene. (3) creating individual plasmids based on the expression plasmid, (4) collecting the individual plasmids to form a population or collection of plasmids encoding capsids with the peptide inserts, and transfecting cells with the collection of plasmids and with the plasmid encoding a recombinant rAAV genome containing a construct with a transgene and required genes, including a detectable one, to produce a collection of rAAV vectors that encapsidate the rAAV genome containing the transgene under appropriate conditions, wherein the rAAV vectors have capsids with encoded capsid proteins containing the library peptide inserts. In embodiments, the parent AAV is AAV9, AAV9.AAA, AAVhu.32, AAVhu.32.AAA, or AAV5, or any other suitable AAV serotype. The insertion site may be within VR-IV, including immediately after or corresponding to one of amino acids 451-461 of AAV9, or within VR-VIII, including immediately after amino acid 588 of AAV9 or corresponding to that position in different AAV capsid types (see Figure 1 for alignment). Peptides may be 4, 5, 6, or 7 amino acids in length.

[0099] In an embodiment, the rep gene is on a separate expression plasmid from the plasmid encoding the cap gene with the insert.

[0100] A library of modified capsids is harvested from these cells. In embodiments, the generated rAAV library population has high levels of capsids with peptide inserts, including 85%, 90%, 95%, or 98%, 99%, or even 100%.

[0101] 5.2.2 Capsids Containing Muscle-Homing and Other Targeting Peptides The present inventors have also surprisingly discovered that peptides inserted into rAAV vectors can "retarget" or improve the targeting properties of such AAV vectors to specific tissues, organs, or cells, and in particular provide peptides that target rAAV vectors to other target tissues of interest, such as target muscle tissue and / or retinal tissue, or cross the blood-brain barrier and target neural tissues of the CNS. This can provide improved transport of rAAV particles encapsulating transgenes to optimize distribution of the vectors upon administration to the body. Such peptides and modified vectors are described below.

[0102] Another aspect of the present invention relates to capsid proteins selected from random peptide insert libraries containing peptide inserts selected to confer or enhance muscle-homing properties, or "muscle tropism," including homing to muscle tissue, muscle cells, or muscle cell matrix. Also included are capsids and rAAV vectors having capsids containing these peptide-containing capsid proteins. In other aspects, peptides may target other tissues, such as the CNS, including the retina, preferentially containing neurons within the CNS, and may detarget tissues such as the liver, heart, or astrocytes.

[0103] In certain embodiments, the peptide insert consists of 4 to 7 consecutive amino acids (i.e., 4, 5, 6, or 7 consecutive amino acids) of the peptide sequences in Tables 4, 5, 14, 15, and 16 (SEQ ID NOS: 1-138). The peptides disclosed herein are identified by screening libraries of peptides inserted into AAV capsids, which are screened for properties such as tropism for muscle tissue (skeletal and / or cardiac), retinal tissue, CNS neural tissue, and other tissues, e.g., via mouse and NHP biodistribution studies described in the Examples. In embodiments, capsids with peptide inserts have increased tropism for target tissues, including muscle, retinal tissue, and CNS, including neuronal tissue, relative to a parent or reference capsid (having capsid proteins without the peptide insert), such as AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA. Capsids with peptide inserts may also distribute to liver tissue at lower levels than target tissues such as muscle, retina, CNS or other target tissues, and / or at lower levels than reference capsids such as AAV9 or AAVhu.32.

[0104] In embodiments, peptides that can be inserted into the capsid protein include those listed in Tables 4, 5, 14, 15, and 16, including peptides having an amino acid sequence of 4, 5, 6, or 7 consecutive amino acids of one of the amino acid sequences of SEQ ID NOs: 1 to 134. In embodiments, the peptide is a 4, 5, 6, or 7 consecutive amino acid sequence of one of SGTVIRS (SEQ ID NO: 1), TRQYVPG (SEQ ID NO: 4), VQVGRTS (SEQ ID NO: 8), RGAVQKV (SEQ ID NO: 13), GQVHQAR (SEQ ID NO: 32), TSGGQIR (SEQ ID NO: 38), HMGHSGK (SEQ ID NO: 88), MRAVSQL (SEQ ID NO: 109), PRQYVPG (SEQ ID NO: 110), RSSSGR (SEQ ID NO: 111), VVKSTKS (SEQ ID NO: 112), RHVSASD (SEQ ID NO: 113), VRSDRDQ (SEQ ID NO: 114), or TVVTSIN (SEQ ID NO: 115). In embodiments, the peptide is inserted into VR-IV (including immediately following one of amino acids 451-461 or 454 of AAV9, AAV9.AAA, AAVhu.32, AAVhu.32.AAA).

[0105] In embodiments, the peptide is a 4, 5, 6, or 7 consecutive amino acid sequence of one of AQVGRAS (SEQ ID NO:24), SVTSVRV (SEQ ID NO:37), SIAKNSA (SEQ ID NO:76), DGRRIGV (SEQ ID NO:116), TSGERRG (SEQ ID NO:117), PSSVQHR (SEQ ID NO:118), SSSVQHR (SEQ ID NO:119), SGMQERR (SEQ ID NO:120), LERGNLE (SEQ ID NO:121), YRDVRQT (SEQ ID NO:122), PSAVQHR (SEQ ID NO:123), YVGGRAV (SEQ ID NO:124), IGSRGVA (SEQ ID NO:125), SDVSRPR (SEQ ID NO:126), GSVRQAA (SEQ ID NO:127), QSPHTSQ (SEQ ID NO:128), or ASQAYHG (SEQ ID NO:128). In embodiments, the peptide is inserted into VR-IV (including immediately following one of amino acids 451-461 or 454 of AAV9, AAV9.AAA, AAVhu.32, AAVhu.32.AAA).

[0106] In embodiments, capsids having the amino acid sequences of SEQ ID NOs: 159-266 or 268-375 are provided (see Table 17).

[0107] In other embodiments, the peptide is a variant of one of the amino acid sequences of SEQ ID NOs: 1-134 having one, two, or three amino acid substitutions, including conservative amino acid substitutions, while the peptide retains its biological activity when inserted into a capsid protein.

[0108] The inventors also identified predetermined consensus sequences of peptides that, when inserted into capsid proteins (e.g., into VR-IV of AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA), exhibit increased muscle tropism, including AAV uptake, transduction, and / or transgene expression, relative to parental or reference capsids such as AAV9, AAVhu.32, AAV9.AAA, or AAVhu.32.AAA. The peptides analyzed included those set forth in SEQ ID NOs: 5, 8, 24, 36, 44, 130, 131, 132, 133, or 134 (see Table 15, above), all of which exhibited significantly increased muscle tropism compared to the AAV9.AAA capsid. Thus, capsids having a peptide insert are provided, wherein the peptide is a 4, 5, 6, or 7 consecutive amino acid sequence of SEQ ID NO: 135, which is X1-QV-X2-X3-X4-X5, wherein X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S, G, V, or A. In embodiments, capsids having a peptide insert are provided, wherein the peptide is a 4, 5, 6, or 7 consecutive amino acid sequence of SEQ ID NO: 136, which is X1-QV-X2-X3-X4-X5, wherein X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S or A. In embodiments, capsids having a peptide insert are provided, wherein the peptide is a sequence of 4, 5, 6, or 7 consecutive amino acids of SEQ ID NO: 137, and is X1-QV-X2-X3-X4-X5, wherein X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S, G, V, or A. In embodiments, capsids having a peptide insert are provided, wherein the peptide is a sequence of 4, 5, 6, or 7 consecutive amino acids of SEQ ID NO: 138, and is X1-QV-X2-X3-X4-X5, wherein X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S or A.In embodiments, the peptide is a 4, 5, 6, or 7 contiguous amino acid sequence of one of SEQ ID NOs: 5, 8, 24, 36, 44, 130, 131, 132, 133, or 134. In embodiments, the peptide is inserted into region VR-IV of one of the capsids of AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA (immediately following one of positions 451-461, inclusive, immediately following or corresponding to 454 of AAV9). In embodiments, the capsid has the amino acid sequence of SEQ ID NOs: 263-266 (see Table 17).

[0109] As detailed herein, the peptide may be inserted into the wild-type or variant capsid protein amino acid sequence at a site that allows for surface exposure of the peptide when the capsid protein is incorporated into an AAV capsid, such as, for example, within a variable surface-exposed loop, and more typically, at a site described herein corresponding to VR-I, VR-IV, or VR-VIII, or after the first amino acid of VP2, e.g., after amino acid 137 (AAV4, AAV4-4, and AAV5) or at amino acid 138 (AAV1, AAV2, AAV3, AAV3-3, AAV6, AAV7, AAV8, AAV9, AAV9e, rh.10, rh.20, rh.39, rh.74v1, rh.74v2, AAVhu.32, and hu.37) (Figure 1). In embodiments, the capsid protein is an AAV9 capsid protein or an AAV9.AAA capsid protein (or a capsid protein having 90%, 95%, or 99% amino acid sequence identity to AAV9 or AAV9.AAA), and the peptide insertion occurs immediately after (or corresponds to) at least one of amino acid residues 451-461 of the AAV9 capsid. In embodiments, the capsid protein is an AAVhu.32 capsid protein or an AAVhu.32.AAA capsid protein (or a capsid protein having 90%, 95%, or 99% amino acid sequence identity to AAVhu.32 or AAVhu.32.AAA), and the peptide insertion occurs immediately after (or corresponds to) at least one of amino acid residues 451-461 of the AAVhu.32 capsid.In other embodiments, the capsid protein is derived from at least one AAV type selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrh10, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, and AAVrh74 (versions 1 and 2) (see, e.g., Figure 1). or a capsid protein having 90%, 95%, or 99% amino acid identity to the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV9e, AAVrh10, AAVrh20, AAVhu.31, AAVhu.32, AAVhu.37, AAVrh39, and AAVrh74 (versions 1 and 2). In embodiments, the peptide insertion occurs immediately after amino acid residues corresponding to at least one of amino acid residues 451-461 of AAV9 (SEQ ID NO: 151), or alternatively, amino acid residue 588 of AAV9. In embodiments, the peptide insertion occurs immediately after amino acid residue corresponding to 578 of AAV5 (SEQ ID NO: 143). As shown in Figure 1, an alignment of different AAV serotypes shows corresponding amino acid residues in the different amino acid sequences.

[0110] In embodiments, the capsid protein having the peptide insert is one of the capsid proteins in Table 17 having amino acid SEQ ID NOs: 159-266 and 268-375. In embodiments, the capsid protein is 90%, 95%, or 99% identical to SEQ ID NOs: 159-266 and 268-375, except that it is identical with respect to the peptide insert and retains its biological activity.

[0111] In certain embodiments, the peptide is inserted immediately after one of amino acids 574-584 (or at 582), including immediately after position 578, within VR-VIII of AAV5 (SEQ ID NO: 143) (see Figure 1 for alignment). The peptide includes any of the peptides having the amino acid sequence of SEQ ID NOs: 1-134, and may include one of the peptides having the amino acid sequence of SEQ ID NOs: 113, 114, or 115. In embodiments, the capsid has the amino acid sequence of SEQ ID NO: 376, 377, or 378.

[0112] In embodiments, capsids with peptide inserts described herein have increased tropism for target tissues such as muscle, retina, CNS, and other target tissues relative to a parent capsid, or a reference capsid, which may include AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA, i.e., containing identical capsid proteins except for the absence of the peptide insert, or other capsids of interest that do not contain the peptide insert. In embodiments, the modified capsids may have reduced tropism, including for target tissues including muscle, retina, CNS, or others, and / or relative to a parent or reference capsid, which may include AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAA, or are non-targeted to tissues such as the liver. In embodiments, the reference capsid does not contain the 496NNN / AAA498 amino acid substitution - for example, if the parent capsid is AAV9.AAA, liver tropism / detargeting can be relative to AAV9 without the peptide insert.

[0113] For example, as described in the Examples herein, tissue tropism can be assessed by introducing an rAAV vector having a modified capsid and a genome with a detectable transgene into a test animal such as a mouse or NHP at an appropriate dosage (e.g., 1E12, 1E13, or 1E14 vg / kg), e.g., by systemic, intravenous, intramuscular, intrathecal, subcutaneous, ocular, or other administration, and then after an appropriate period of time harvesting the animal's tissues to assess the presence of the vector genome, mRNA transcribed from the genome, the ratio of mRNA to vector DNA, the transgene protein product, or activity.

[0114] Thus, provided are capsids described herein that, when administered (e.g., IV, IM, subcutaneously) to animals, including mice or NHPs, exhibit at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, or 25-fold or greater tropism for muscle, including skeletal or cardiac, or other tissues, such as retina or CNS tissue, relative to a parent capsid or reference, such as AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAV, as measured by vector genomic DNA, transgene mRNA, mRNA to vector genomic DNA ratio, and transgene protein product, including protein product activity. In embodiments, the capsid transduces neurons preferentially over astrocytes or other CNS tissues in the CNS of the animal. Also provided in embodiments are capsids described herein that, when administered (e.g., IV, IM, subcutaneously) to an animal, including a mouse or NHP, exhibit at least 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 40-fold, or 50-fold less tropism for target tissues such as muscle (skeletal and / or cardiac), retina, or CNS (including target tissue to liver ratios), and / or for liver, either relative to a parental capsid or reference, such as AAV9, AAV9.AAA, AAVhu.32, or AAVhu.32.AAV, as measured by the amount of vector genomic DNA, transgene mRNA, mRNA to vector genomic DNA ratio, transgene protein product, including protein product activity.

[0115] 5.2.3 Additional AAV capsid insertion sites The following summarizes the insertion sites for the peptides described herein, including the peptides in Tables 4, 5, 14, 15, and 16 below (see also Figure 1): AAV1: 138, 262-272, 450-459, 595-593, and in an embodiment, between 453 and 454 (SEQ ID NO: 139). AAV2: 138, 262-272, 449-458, 584-592, and in an embodiment, between 452 and 453 (SEQ ID NO: 140). AAV3: 138, 262-272, 449-459, 585-593, and in an embodiment, between 452 and 453 (SEQ ID NO: 141). AAV4: 137, 256-262, 443-453, 583-591, and in an embodiment, between 446 and 447 (SEQ ID NO: 142). AAV5: 137, 252-262, 442-445, 574-582, and in an embodiment, between 445 and 446 (SEQ ID NO: 143). AAV6: 138, 262-272, 450-459, 585-593, and in an embodiment, between 452 and 453 (SEQ ID NO: 144). AAV7: 138, 263-273, 451-461, 586-594, and in an embodiment, between 453 and 454 (SEQ ID NO: 145). AAV8: 138, 263-274, 451-461, 587-595, and in an embodiment, between 453 and 454 (SEQ ID NO: 146). AAV9: 138, 262-273, 452-461, 585-593, and in an embodiment, between 454 and 455 (SEQ ID NO: 151). AAV9.AAA: 138, 262-273, 452-461, 585-593, and in an embodiment, between 454 and 455 (SEQ ID NO: 158). AAVrh10: 138, 263-274, 452-461, 587-595, and in an embodiment, between 454 and 455 (SEQ ID NO: 152). AAVrh20: 138, 263-274, 452-461, 587-595, and in an embodiment, between 454 and 455 (SEQ ID NO: 155). AAVrh74: 138, 263-274, 452-461, 587-595, and in an embodiment, between 454 and 455 (SEQ ID NO: 156 or SEQ ID NO: 157). AAVhu.32:138, 262-273, 452-461, 585-593, and in embodiments, between 454 and 455 (SEQ ID NO: 148). AAVhu.37:138, 263-274, 452-461, 587-595, and in embodiments, between 454 and 455 (SEQ ID NO: 153).

[0116] In embodiments, the peptide insertion occurs between amino acid residues 588-589 of the AAV9 capsid, or between the corresponding residues of another AAV type capsid as determined by amino acid sequence alignment (e.g., as in Figure 1). In embodiments, the peptide insertion occurs immediately after amino acid residues I451-L461, S268, and Q588 of the AAV9 capsid sequence, or immediately after the corresponding residues of another AAV capsid sequence (Figure 1).

[0117] In some embodiments, the capsid may be selected and / or further modified to reduce recognition of the AAV particle by the subject's immune system, e.g., by evasion of pre-existing antibodies in the subject. In some embodiments, the capsid may be selected and / or further modified to enhance desired tropism / targeting.

[0118] 5.2.4 Generation of engineered capsids In some embodiments, AAV capsids were modified by introducing selected single or multiple amino acid substitutions that increase effective gene delivery to the CNS, detarget the liver, and / or reduce neutralizing antibody immune responses prior to generating rAAV libraries with random peptide inserts.

[0119] If intravenously administered rAAV vectors must cross the blood-brain barrier, some modifications to the parent rAAV vector (capsid protein) prior to rAAV library generation may be effective for gene delivery to the CNS. A key cluster of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice was recently reported. Specifically, AAVrh.10-derived amino acids N262, G263, T264, S265, G267, S268, T269, and T273 were identified as critical residues that facilitate crossing the BBB (Albright et al., 2018, Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier). Amino acid substitutions in capsids, such as the AAV8 and AAV9 capsids, have been identified that facilitate rAAV passage across the blood-brain barrier, transduction, liver detargeting, and / or reduced immune responses.

[0120] In some embodiments, capsids are provided that have one or more amino acid substitutions that further enhance the transduction and / or tissue tropism of rAAVs with modified capsids. In embodiments, capsids are provided that have a single mutation at amino acid 269 of the AAV8 capsid (A269S), replacing alanine with serine (see FIG. 1), and capsids with amino acid substitutions at corresponding positions in other AAV types. In some embodiments, capsids are provided that have multiple substitutions at amino acids 263, 269, and 273 of the AAV9 capsid, resulting in the following substitutions: S263G, S269T, and A273T (see FIG. 1), or substitutions corresponding to these positions in other AAV types.

[0121] Exposure to the AAV capsid can generate a neutralizing antibody immune response. One approach to overcoming this response is to map AAV-specific neutralizing epitopes and rationally design AAV capsids that can evade neutralization. A monoclonal antibody specific for intact AAV9 capsids with high neutralizing potency was recently reported (Giles et al., 2018, Mapping an Adeno-associated Virus 9-Specific Neutralizing Epitope To Develop Next-Generation Gene Delivery Vectors). The epitope was mapped to the three-fold symmetry axis on the capsid, specifically, residues 496-NNN-498 and 588-QAQAQT-592 of AAV9, SEQ ID NO: 151. Capsid mutagenesis demonstrated that a single amino acid substitution within this epitope significantly reduced binding and neutralization. Additionally, in vivo studies have shown that mutations in the epitope conferred a "liver-detargeted" phenotype to mutant vectors, suggesting that the same residues are also responsible for AAV9 tropism. Liver detargeting has also been linked to a substitution of amino acid 503, replacing tryptophan with arginine. The presence of the W503R mutation in the AAV9 capsid was associated with lower glycan-binding affinity (Shen et al., 2012, Glycan Binding Avidity Determines the Systemic Fate of Adeno-Associated Virus Type 9).

[0122] In some embodiments, capsids are provided that are further modified by substituting alanine for asparagine at amino acid positions 498, 499, and 500 (such as AAV8.AAA) or 496, 497, and 498 (referred to herein as AAV9.AAA, SEQ ID NO: 158). In some embodiments, an AAVrhlO capsid can also be modified by substituting alanine for the three asparagine at amino acid positions 498, 499, and 500 (AAVrhlO.AAA). In some embodiments, an AAVhu32 capsid can also be modified by substituting alanine for the three asparagine at amino acid positions 496, 497, and 498 (referred to herein as AAVhu.32.AAA, SEQ ID NO: 148).

[0123] In some embodiments, capsids are provided in which three asparagines are replaced with alanine at amino acid positions 496, 497, and 498 of the AAV9 capsid, and a tryptophan is replaced with arginine at amino acid 503 of the AAV9 capsid, or capsids with substitutions corresponding to these positions in other AAV types. In some embodiments, capsids are provided in which a glutamine at amino acid position 474 of the AAV9 capsid is replaced with alanine, or capsids with substitutions corresponding to this position in other AAV types.

[0124] In some embodiments, the rAAV described herein increases tissue-specific (such as, but not limited to, muscle) cell transduction in a subject (human, non-human primate, or mouse subject) or cell culture compared to an rAAV that does not contain a peptide insert. In some embodiments, the increase in tissue-specific cell transduction is at least 2, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100-fold greater than that without the peptide insert. For example, in some embodiments, tissue-specific cell transduction increases 50-80-fold compared to transduction with the same AAV type without the peptide insert. Increased transduction can be assessed using methods described in the Examples herein and known in the art.

[0125] 5.3. Methods for producing rAAV molecules Another aspect of the invention involves producing the molecules disclosed herein. In some embodiments, the molecules of the invention are produced by providing nucleotides comprising a nucleic acid sequence encoding any of the capsid protein molecules described herein and using a packaging cell system to prepare corresponding rAAV particles having a capsid coat composed of the capsid proteins. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the sequence of a capsid protein molecule described herein and retains (or substantially retains) the biological function of the capsid protein and an inserted peptide from a heterologous protein or domain thereof. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity to the AAV9 capsid protein (SEQ ID NO: 151; see FIG. 1 ), while retaining (or substantially retaining) the biological function of the AAV9 capsid protein and the inserted peptide.

[0126] Capsid proteins, coats, and rAAV particles can be produced by techniques known in the art. In some embodiments, the viral genome contains at least one terminal inverted repeat to enable packaging into a vector. In some embodiments, the viral genome further contains a cap gene and / or a rep gene for expression and splicing of the cap gene. In other embodiments, the cap and rep genes are provided by the packaging cell and are not present in the viral genome.

[0127] In some embodiments, the nucleic acid encoding the modified capsid protein is cloned into an AAV Rep-Cap helper plasmid in place of the existing capsid gene. When introduced into a host cell together, this plasmid helps package the rAAV genome into the modified capsid protein as the capsid coat. Packaging cells can be any cell type that harbors the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Non-limiting examples include 293 cells or their derivatives, HELA cells, or insect cells.

[0128] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids are taught, for example, in US 7,282,199, US 7,790,449, US 8,318,480, US 8,962,332 and PCT / EP2014 / 076466, each of which is incorporated by reference in its entirety.

[0129] In some embodiments, rAAV provides a transgene delivery vector that can be used in therapeutic and prophylactic applications, as discussed in more detail below. In some embodiments, the rAAV vector also contains regulatory control elements known to those skilled in the art to affect expression of the RNA and / or protein product encoded by the nucleic acid (transgene) in target cells of a subject. The regulatory control elements can be tissue-specific, i.e., active only (or substantially more active or significantly more active) in the target cells / tissue. In certain embodiments, the AAV vector contains a regulatory sequence, e.g., a promoter operably linked to the transgene, that enables expression in the target tissue. The promoter can be a constitutive promoter, e.g., a CB7 promoter. Additional promoters include the cytomegalovirus (CMV) promoter, the Rous sarcoma virus (RSV) promoter, the MMT promoter, the EF-1 alpha promoter, the UB6 promoter, the chicken beta-actin promoter, the CAG promoter, the RPE65 promoter, the opsin promoter, the TBG (thyroxine-binding globulin) promoter, the APOA2 promoter, the SERPINA1 (hAAT) promoter, or the MIR122 promoter. In some embodiments, particularly when it is desirable to turn off transgene expression, an inducible promoter is used, such as a hypoxia-inducible or rapamycin-inducible promoter.

[0130] In embodiments, AAV9 vectors are provided that comprise a viral genome under the control of regulatory elements and comprising an expression cassette for expression of a transgene that is flanked by ITRs and a modified viral capsid described herein, or that is at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of an AAV9 capsid protein (see FIG. 1 ) while retaining the biological function of the modified AAV9 capsid. In certain embodiments, the encoded AAV9 capsid has a peptide insert described herein and further has the sequence of wild-type AAV9 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to the wild-type AAV sequence, and retains the biological function of the AAV9 capsid. Also provided are modified AAV vectors other than AAV9 vectors, such as modified AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9e, AAVrhlO, AAVrh20, AAVhu.37, AAVrh39, or AAVrh74 vectors that have the peptide inserts described herein and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions relative to the wild-type or unmodified sequence of that AAV type and that retain their biological function.

[0131] Recombinant adenoviruses can be first-generation vectors with an E1 deletion, with or without an E3 deletion, and with an expression cassette inserted into either deleted region. Recombinant adenoviruses can be second-generation vectors containing complete or partial deletions of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenoviral terminal inverted repeats and packaging signal (phi). The transgene is usually inserted between the packaging signal and the 3' ITR, with or without a stuffer sequence to maintain a genome close to the wild-type size of approximately 36 kb. An exemplary protocol for generating adenoviral vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy," Gene Therapy 12:S18-S27 (incorporated herein by reference in its entirety).

[0132] The rAAV vector for delivering a transgene to a target tissue, cell, or organ has tropism for that particular target tissue, cell, or organ. Tissue-specific promoters can also be used. The construct may further comprise expression control elements (e.g., introns, e.g., chicken β-actin intron, minute virus of mouse (MVM) intron, human factor IX intron (e.g., FIX cleavage intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron, and hybrid adenovirus splice donor / IgG splice acceptor intron and polyA signals, e.g., rabbit β-globin polyA signal, human growth hormone (hGH) polyA signal, SV40 late polyA signal, synthetic polyA (SPA) signal, and bovine growth hormone (bGH) polyA signal) that enhance expression of the transgene driven by the vector. See, e.g., Powell and See Rivera-Soto, 2015, Discov. Med., 19(102):49-57.

[0133] In certain embodiments, the nucleic acid sequences disclosed herein may be codon optimized, for example, via any codon optimization technique known to those of skill in the art (see, e.g., the review by Quax et al., 2015, Mol Cell 59:149-161).

[0134] In certain embodiments, the constructs described herein comprise the following components: (1) AAV9 inverted repeats flanking an expression cassette, (2) regulatory elements comprising a) a CB7 promoter comprising a CMV enhancer / chicken β-actin promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal, and (3) a transgene providing (e.g., encoding) a nucleic acid or protein product of interest. In certain embodiments, the constructs described herein comprise the following components: (1) AAV9 inverted repeats flanking an expression cassette, (2) regulatory elements comprising a) a hypoxia-inducible promoter, b) a chicken β-actin intron, and c) a rabbit β-globin polyA signal, and (3) a transgene providing (e.g., encoding) a nucleic acid or protein product of interest.

[0135] The viral vectors provided herein can be produced using mammalian host cells, including host cells from humans, monkeys, mice, rats, rabbits, or hamsters. Non-limiting examples include A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. Typically, host cells are stably transformed with sequences encoding the transgene and related elements (i.e., the vector genome), as well as genetic components for producing virus within the host cell, such as replication and capsid genes (e.g., the AAV rep and cap genes). For methods of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the detailed description in U.S. Patent No. 7,282,199 B2, the entire contents of which are incorporated herein by reference. The genome copy titer of the vector can be determined, for example, by TAQMAN® analysis. Virions can be recovered, for example, by CsCl2 sedimentation. Alternatively, AAV vectors can be produced using a baculovirus expression system in insect cells. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102:1045-1054 (incorporated herein by reference in its entirety for manufacturing techniques).

[0136] In vitro assays, e.g., cell culture assays, can be used to measure transgene expression from the vectors described herein and thus, for example, indicate vector efficacy. For example, the PER.C6® cell line (Lonza), a cell line derived from human embryonic retinal cells, or retinal pigment epithelial cells, e.g., the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to assess transgene expression. Alternatively, cell lines derived from liver or other cell types, such as, but not limited to, HuH-7, HEK293, fibrosarcoma HT-1080, HKB-11, and CAP cells, can be used. Upon expression, the expressed product (i.e., the transgene product) can be characterized, including determining glycosylation and tyrosine sulfation patterns, using assays known in the art.

[0137] 5.4. Therapeutic and prophylactic uses Another aspect relates to a therapeutic method involving administering a transgene via an rAAV vector according to the present invention to a subject in need thereof to delay, prevent, treat, and / or manage a disease or disorder and / or ameliorate one or more symptoms associated therewith. Subjects in need thereof include those suffering from or predisposed to a disease or disorder, e.g., those at risk of developing or experiencing a recurrence of the disease or disorder. Generally, an rAAV carrying a particular transgene will be used for a given disease or disorder in a subject in which the subject's native gene corresponding to the transgene is defective in providing the correct gene product, or the correct amount of gene product. The transgene can then provide a copy of the defective gene in the subject.

[0138] Generally, the transgene comprises a cDNA that restores protein function to a subject with a genetic mutation(s) in the corresponding native gene. In some embodiments, the cDNA comprises associated RNA for performing genome engineering, such as genome editing via homologous recombination. In some embodiments, the transgene encodes a therapeutic RNA, such as an shRNA, an artificial miRNA, or an element that affects splicing.

[0139] Tables 1A-1B below provide an exemplary list of transgenes that can be used in any of the rAAV vectors described herein, particularly to treat or prevent muscle-related diseases. [Table 3] [Table 4-1] [Table 4-2] [Table 4-3]

[0140] For example, an rAAV vector containing a transgene encoding microdystrophin finds use in treating / preventing / managing Duchenne muscular dystrophy. The microdystrophin can be, for example, the microdystrophin found in WO2021 / 108755. In other embodiments, the microdystrophin has the amino acid sequence of SEQ ID NO: 133 (human MD1(R4-R23 / ΔCT), SEQ ID NO: 134 (microdystrophin), SEQ ID NO: 135 (Dys3978), SEQ ID NO: 136 (MD3), or SEQ ID NO: 137 (MD4) described in WO2023 / 004331. In other embodiments, the microdystrophin is SEQ ID NO: 7 in WO2017 / 181015A1. Typically, rAAV vectors are administered systemically. For example, the rAAV vector can be provided by intravenous, intramuscular, intranasal, and / or intraperitoneal administration.

[0141] In some embodiments, the rAAV of the present invention finds application in delivery to target cell types, including target tissues or cell matrices associated with the disorder or disease being treated / prevented. A disease or disorder associated with a particular tissue or cell type is one that significantly affects that tissue or cell type compared to other tissues in the body, or one in which the effects or symptoms of the disorder manifest in a particular tissue or cell type. A method for delivering a transgene to a target tissue in a subject in need thereof includes administering to the subject an rAAV containing a homing peptide as a peptide insert. For example, in the case of dystrophinopathy, an rAAV vector containing a peptide insert that directs the rAAV to neural tissue can be used; specifically, the peptide insert facilitates high-efficiency transduction of muscle cells, including satellite cells, by rAAV, while still resulting in low transduction efficiency of liver cells.

[0142] In the case of muscle-related diseases or disorders, rAAV vectors can be selected from the libraries herein that contain peptide inserts that direct muscle transduction compared to parent rAAV vectors that do not have the peptide insert.

[0143] The rAAV vectors of the present invention can also facilitate the delivery, particularly targeted delivery, of oligonucleotides, drugs, contrast agents, inorganic nanoparticles, liposomes, and antibodies to target cells or tissues. The rAAV vectors can also facilitate the delivery, particularly targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.

[0144] The agents can be provided in pharmaceutically acceptable compositions known in the art and / or described herein, and the rAAV molecules of the invention can be administered alone or in combination with other prophylactic and / or therapeutic agents.

[0145] The dosages and frequency of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. Dosage and frequency will typically vary according to factors particular to each patient, depending on the particular therapeutic or prophylactic agent being administered, the severity and type of disease, the route of administration, and the patient's age, weight, response, and past medical history, and should be determined according to the judgment of the practitioner and each patient's circumstances. Suitable regimens will take such factors into account and will be determined according to the judgment of the practitioner and each patient's circumstances, as reported in the literature, for example, in the Physician's Desk Reference (56 th The dosages can be selected by one skilled in the art by following the dosages recommended in the "Prophylactic and / or therapeutic agents" section of ...

[0146] The amount of an agent of the present invention that will be effective can be determined by standard clinical techniques. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the methods of the present invention, the therapeutically effective dose can be initially estimated from cell culture assays. Doses can be determined based on the IC20 determined in cell culture. 50The test compound may be formulated in animal models to achieve a circulating plasma concentration range that includes the therapeutically effective dose (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0147] Prophylactic and / or therapeutic agents, and combinations thereof, can be tested in suitable animal model systems before use in humans. Such animal model systems include, but are not limited to, rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system known in the art can be used. Such model systems are widely used and familiar to those skilled in the art. In some embodiments, animal model systems for CNS conditions based on rats, mice, or other small mammals other than primates are used.

[0148] Once the prophylactic and / or therapeutic agents of the present invention have been tested in animal models, they can be tested in clinical trials to establish their efficacy. The establishment of clinical trials can be carried out according to general methodologies known to those skilled in the art to establish the optimal dosage and administration route of the agents of the present invention, as well as the toxicity profile. For example, clinical trials can be designed to test the efficacy and toxicity of the rAAV molecules of the present invention in human patients.

[0149] The toxicity and efficacy of the prophylactic and / or therapeutic agents of the present invention can be evaluated, for example, by LD 50 (lethal dose for 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of a population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50The therapeutic index can be expressed as a ratio of . Prophylactic and / or therapeutic agents that exhibit large therapeutic indices are preferred. Prophylactic and / or therapeutic agents that exhibit toxic side effects may be used, although care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0150] The rAAV molecules of the present invention are generally administered for a time and in an amount effective to achieve the desired therapeutic and / or prophylactic benefit. Data obtained from cell culture assays and animal studies can be used in designing ranges and / or schedules for the dosage of prophylactic and / or therapeutic agents for use in humans. Dosages of such agents are administered at an ED level with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0151] The therapeutically effective dosage of rAAV vectors for patients is typically about 1 x 10 9 ~Approx. 1×10 16 genomic rAAV vector, or approximately 1 x 10 10 ~Approx. 1×10 15 , about 1×10 12 ~Approx. 1×10 16 , or approximately 1 × 10 14 ~Approx. 1×10 16 It is a solution of about 0.1 ml to about 100 ml containing the AAV genome. By monitoring the expression level of the introduced gene, the dosage, frequency, scheduling, etc. can be determined / adjusted.

[0152] Treatment of a subject with a therapeutically or prophylactically effective amount of the agent of the present invention can include a single treatment or a series of treatments.For example, pharmaceutical compositions containing the agent of the present invention can be administered once a day, twice a day, or three times a day.In some embodiments, the agent can be administered once a day, every other day, once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once a year.It is also understood that the effective dosage of a given agent, for example, the effective dosage of an agent containing a double antigen-binding molecule of the present invention, can increase or decrease over the course of treatment.

[0153] In some embodiments, ongoing treatment is indicated, e.g., on a long-term basis, such as in the ongoing treatment and / or management of a chronic disease or disorder. For example, in embodiments, an agent of the invention is administered for a period of time, e.g., at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, or for the remainder of the life of the subject in need thereof.

[0154] The rAAV molecules of the present invention can be administered alone or in combination with other prophylactic and / or therapeutic agents. Each prophylactic or therapeutic agent can be administered sequentially in any order at the same or different times; however, if not administered at the same time, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered separately in any appropriate form and by any suitable route.

[0155] In various embodiments, different prophylactic and / or therapeutic agents are administered less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, within 24 hours apart, or within 48 hours apart. In certain embodiments, two or more agents are administered during the same patient visit.

[0156] Methods of administering agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous, including infusion or bolus injection), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal and intestinal mucosa, etc.). In embodiments, such as when the transgene is intended to be expressed in the CNS, the vector is administered via lumbar puncture or via the cisterna magna.

[0157] In certain embodiments, the agents of the present invention are administered intravenously and may be administered together with other biologically active agents.

[0158] In another specific embodiment, the agents of the present invention can be delivered in a sustained-release formulation, for example, where the formulation provides extended release and therefore an extended half-life of the administered agent. Suitable controlled-release systems include, but are not limited to, diffusion-controlled, solvent-controlled, and chemical-controlled systems. Diffusion-controlled systems include, for example, reservoir devices in which the molecules of the present invention are enclosed within a device such that release of the molecules is controlled by permeation through a diffusion barrier. Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers. Monolithic (matrix) devices are a second type of diffusion-controlled system in which the dual antigen-binding molecules are dispersed or dissolved in a rate-controlling matrix (e.g., a polymer matrix). The agents of the present invention can be homogeneously dispersed throughout the rate-controlling matrix, and the rate of release is controlled by diffusion through the matrix. Suitable polymers for use in monolithic matrix devices include natural polymers, synthetic polymers, and synthetically modified natural polymers, as well as polymer derivatives.

[0159] Any technique known to those skilled in the art can be used to prepare sustained release formulations containing one or more of the agents described herein. See, for example, U.S. Pat. No. 4,526,938, PCT Publication No. WO 91 / 05548, PCT Publication No. WO 96 / 20698, Ning et al., "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology, 39:179-189, 1996, Song et al., "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology, 50:372-397, 1995, Cleek et al., "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Proc. Intl. Symp. Control. Rel. Bioact. Mater., 24:853-854, 1997, and Lam et al., "Microencapsulation of Recombinant Humanized See, "Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759 760, 1997 (each of which is incorporated herein by reference in its entirety). In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321:574, 1989).In another embodiment, polymeric materials may be used to achieve controlled release of a drug comprising a dual antigen-binding molecule or an antigen-binding fragment thereof (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NY (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; also see Levy et al., Science, 228:190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al., J. Neurosurg., 7:141, 1989). 1:105, 1989), U.S. Patent Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463, 5,128,326, PCT Publication Nos. WO 99 / 15154, and WO 99 / 20253. In yet another embodiment, a controlled-release system can be placed near the therapeutic target (e.g., an affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems are discussed in the review by Langer, Science, 249:1527-1533, 1990.

[0160] rAAVs can also be used for scientific research, such as in vivo delivery of transgenes for optogenetics, gene knockdown with miRNA, recombinase delivery for conditional gene deletion, gene editing with CRISPR, etc.

[0161] 5.5. Pharmaceutical Compositions and Kits The present invention further provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an agent of the invention, wherein the agent comprises an rAAV molecule of the invention. In some embodiments, the pharmaceutical composition comprises an rAAV combined with a pharmaceutically acceptable carrier for administration to a subject. In one embodiment, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, more specifically in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvants), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a common carrier when pharmaceutical compositions are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; proteins such as low molecular weight polypeptides, serum albumin, and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), PLURONICS™, and the like, as known in the art.The pharmaceutical compositions of the present invention may also contain, in addition to the above ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, and preservatives. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0162] In certain embodiments of the invention, pharmaceutical compositions are provided for use in accordance with the methods of the invention, the pharmaceutical compositions comprising a therapeutically and / or prophylactically effective amount of an agent of the invention together with a pharmaceutically acceptable carrier.

[0163] In certain embodiments, the agents of the present invention are substantially pure (i.e., substantially free from substances that limit their effectiveness or cause undesirable side effects). In certain embodiments, the host or subject is an animal, e.g., a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkeys, e.g., cynomolgus monkeys, and humans). In certain embodiments, the host is a human.

[0164] The present invention further provides a kit that can be used in the above-mentioned method.In one embodiment, the kit includes, for example, one or more agents of the present invention in one or more containers.In another embodiment, the kit further includes one or more other preventive or therapeutic agents useful for treating the condition in one or more containers.

[0165] The present invention also provides pharmaceutical agents of the present invention packaged in a sealed container, such as an ampoule or sachet, indicating the quantity of drug and active agent. In one embodiment, the drug is supplied as a dry, sterile, lyophilized powder or anhydrous concentrate in a sealed container, which can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. Typically, the drug is supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dosage of at least 5 mg, more often at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized material should be stored in its original container at 2-8°C, and the drug should be administered within 12 hours, usually within 6 hours, 5 hours, 3 hours, or 1 hour, after reconstitution. In an alternative embodiment, the pharmaceutical agents of the present invention are supplied in liquid form in a sealed container indicating the quantity and concentration of drug or active agent. Typically, the liquid form of the drug is supplied in a sealed container of at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / kg, or at least 25 mg / ml.

[0166] The compositions of the present invention include bulk drug compositions (e.g., impure or non-sterile compositions) useful in the manufacture of pharmaceutical compositions, as well as pharmaceutical compositions (i.e., compositions suitable for administration to a subject or patient). Bulk drug compositions can be used to prepare unit dosage forms, for example, comprising prophylactically or therapeutically effective amounts of agents disclosed herein, or combinations of those agents with pharmaceutically acceptable carriers.

[0167] The present invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the present invention. Additionally, one or more other prophylactic or therapeutic agents useful for treating the target disease or disorder may also be included in the pharmaceutical pack or kit. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Optionally associated with such container(s) may be a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency for manufacture, use, or sale for human administration.

[0168] Generally, the components of the compositions of the present invention are supplied separately or mixed together in unit dosage form, for example, as dry lyophilized powder or anhydrous concentrate in a sealed container such as an ampoule or sachet indicating the amount of drug or active agent.When the composition is administered by injection, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration. [Example]

[0169] 6. Working Example The following examples report methods for generating rAAV libraries containing multiple rAAV capsids with surface-exposed peptides inserted into designated insertion sites in the capsid. Recombinantly modified capsids are screened to identify candidates for specific properties, such as tissue tropism. The present invention is illustrated by way of example and describes the construction of rAAV capsids modified to contain a 7-mer peptide (or a 4-, 5-, or 6-mer peptide). The method is designed to eliminate bias in parent capsid formation during production, thereby artificially increasing its abundance and, therefore, representation in the library. Several libraries of peptide insertion mutants were constructed, and pooled mutants were screened for viable capsid assembly, titer, and biodistribution efficacy. Top candidates were then further evaluated for use in retargeting rAAV vectors to tissues of interest. Additional examples demonstrate increased transduction and tissue tropism for certain modified AAV capsids described herein.

[0170] 6.1. Example 1 - AAV9 Capsid Insertion Site or Capsid with Corresponding Site for Insertion Figures 1 and 2 show an analysis and protein model (Figure 2) of variable region 4 of adeno-associated virus type 9 (AAV9 VR-IV) with amino acid sequence comparison to other AAV VR-IVs (Figure 1). As can be seen, AAV9 VR-IV is surface-exposed at the tip or outer surface of the three-fold spike. AAV9 VR-VIII is also surface-exposed. Further analysis showed that there are several polar interactions between VR-IV and VR-V and that the sequence and structure of VR-IV are variable among AAV serotypes and further suggest that there is the potential to interfere with commonly targeted neutralizing antibody epitopes, thereby reducing the immunogenicity of modified capsids. (See PCT International Publication No. WO 2020 / 206189 A1, which is incorporated herein by reference in its entirety.)

[0171] 6.2. Example 2 - rAAV Library Construction For the generation of rAAV libraries, we created custom Rep-only trans-plasmids in which the Cap sequence was removed. We created custom cis-plasmids in which the AAV Cap gene of interest was preceded by a CMV enhancer-promoter and Rep intron, followed by an RBG polyA. A stop codon was inserted into the Cap gene variable region into which the peptide library was later inserted to reduce expression of wild-type (or parental) sequences in the library and provide capsids that allow for "intelligent, guided adaptation" (Novel AAV Vector Intelligent Guided Adaptation Through Evolution = NAVIGATE).

[0172] AAV peptide insert libraries were created by synthesizing DNA fragments incorporating randomized 21-nucleotide sequences synthesized using column-based Trimer-19 oligos (Genscript). Library fragments were cloned into the desired AAV variable region of the library cis plasmid, eliminating the stop codon inserted into the intended parent capsid. After transformation of E. coli, library size was quantified by dilution plate colony counting. Library sizes generally exceeded 10^7 variants. Next-generation sequencing of the library was used to characterize library diversity and wild-type fraction. Plasmid maxipreps were used to generate 0.5–1 mg of library cis plasmid.

[0173] To generate the library vectors, we used custom triple transfection of suspension-adapted HEK293 cells (NAVXCell™, REGENXBIO Inc.), limiting the library cis plasmid to 100 copies per viable cell (typically approximately 3,000) to reduce the possibility of variant cross-packaging and capsid mosaicism. Libraries were generated using 20 L or 8 L scale transfections. Three days after transfection, cells were harvested, lysed, clarified, PEG precipitated, and purified using iodixanol gradient ultracentrifugation.

[0174] Vector titers were quantified using digital PCR with polyA primers / probes. Library variant diversity and parental / wild-type fractions after AAV packaging were characterized by next-generation sequencing (NGS) and LC-MS of the VP3 protein. Several libraries were generated starting from different parental capsids, and evaluation showed a high level of diversity in each capsid library. See, for example, Table 2.

[0175] Each library was generated at a 20L scale by the REGENXBIO Inc. Vector Core Group, as described above. Library diversity and titer measurements determined a diversity ranging from 1E7 to 1E8 in such production lots, with final BDS titers ranging from 1E12 to 1E13 (Table 2). Using deep NGS technology (NovaSeq™, Illumina) to understand input variant distribution, NGS testing yielded approximately 300 million reads per library. [Table 5]

[0176] NGS analysis of the initial vector library showed high parental vector levels, as indicated by the greater representation of parental vectors after generation compared to the parental plasmid percentage in the initial preparation of the library (Figure 5A). However, the stop codon inserted into the template plasmid prevented wild-type (parental) vector generation from carryover template present in the production culture after library cloning. The abundance level of the stop codon in the drop-in vector indicates selection against cross-packaging (Figure 5B).

[0177] The parental fraction after AAV packaging was further characterized by LC-MS of the VP3 protein. After generating an AAV5 vector library lacking a stop codon in the template, the theoretical wild-type mass of the parental (AAV5) VP3 was 59,551.64 Da, while the AAV5 VP3 with peptide inserts ranged from 59,951 to 60,855 Da. Figures 6A and 6B show (by LC-MS analysis of the VP3 protein in each version of the library) that the addition of a stop codon (Figure 6B) significantly reduced packaging of the parental vector.

[0178] 6.3. Example 3 - Evaluation and Selection of Capsids from Various Libraries Samples of the vector library generated according to the above methods are listed in Table 3 and were administered accordingly to non-human primates (NHPs, cynomolgus monkeys) approximately 2-3 years old on the study start date. Thus, as shown in Table 3, three NHPs per study were dosed at 9.3e10 to 2.9e13 GC / kg by different routes of administration under ketamine sedation. [Table 6]

[0179] Blood was collected immediately prior to dosing, and serum was collected and processed. 21 days after dosing, animals were euthanized, and blood and target tissues were collected. Tissues were examined, weighed, and then placed in RNAse / DNase-free cryotubes and flash-frozen in liquid nitrogen. Based on the size of the designated tissue / region, up to five samples of approximately 50–100 mg per sample were collected from each location. Tissue samples were collected using aseptic technique and RNAse-free instruments and workspace, ensuring care was taken to avoid cross-contamination between tissues. Tissues were flash-frozen in liquid nitrogen and then maintained on dry ice before being stored at -70 to -90°C. DNA and RNA were extracted from all tissues using standard techniques, and next-generation sequencing (NGS) and quantitative PCR (mRNA transcript expression) were performed, respectively. A custom bioinformatics platform utilizing open-source software packages and R-scripts was used to analyze cDNA counts and RNA-seq data. For example, merge_counts.R was used to merge peptide counts from all samples into a single data frame, based on methods well known in the art and available at github.com. Analysis of vector repertoires in multiple tissues revealed that 1795 peptides in the AAV9.AAA.VR4 library were detected in one or more muscle tissue samples. A pool of the top hits from this library was further evaluated in a similar NHP study.

[0180] 6.4. Example 4 - Evaluation and Selection of Muscle-Transducing Capsids from the AAV9.AAA Library The 496NNN498 to AAA mutation in AAV9 (SEQ ID NO: 151) results in a greater than 100-fold reduction in liver transduction. The AAV9.AAA parent capsid was used as the starting template to generate a vector library with random peptide inserts after amino acid residue S454 using methods described in the Examples above, generating the AAV9.AAA.VR4 vector library.

[0181] Following analysis of the vector repertoire containing a diverse set of peptides targeted to one or more muscle tissue samples in the first round of animal testing, a pool from AAV9.AAA.VR4 was further analyzed by dosing NHPs at 1.52e13 GC / kg. Bioinformatics tools were used to determine vector performance rankings based on readouts such as relative abundance (RA), ES count (e.g., using Dunnett's multiple comparison test p-values), and enrichment scores. A representative list of 134 hits (peptides) and four consensus sequences emerged, as shown in Tables 4, 5, 14, 15, and 16, based on selection in muscle, for example, compared to the parent spike-in vector control (AAV9.AAA). The top 24 hits (peptides) analyzed by NGS in various tissues are shown in Figure 7. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0182] Skeletal muscle and heart tissue data were compared to liver as in Figures 8A-C and expressed as fold change R relative to the control parental vector. Vectors containing capsids with peptide 01 (SEQ ID NO: 1) showed a 32-fold increase in relative abundance (mRNA expression) in skeletal muscle and 16-fold in heart. Peptide 24 (SEQ ID NO: 24) inserts resulted in vectors that showed 55-fold increases in heart and 12-fold increases in skeletal muscle. Meanwhile, vectors with inserts of peptide 29 (SEQ ID NO: 29) or peptide 30 (SEQ ID NO: 29) showed 173-fold and 103-fold increased levels in liver, respectively, compared to the parental capsid.

[0183] Following analysis of the vector repertoire in the second round of animal testing, a third pool of AAV9.AAA.VR4 capsids (see Table 5) and capsids from the AAV5.VR8 library (containing peptides SEQ ID NO:118, SEQ ID NO:119, and SEQ ID NO:120 in AAV5) were further analyzed in NHPs. All study animals were screened for the presence of AAV2, AAV8, and AAV9 neutralizing antibodies in their serum by in vitro neutralizing antibody assay (medical precision), and only animals with titers below a defined threshold were enrolled in the study. Pre-dose blood collections were taken (after ketamine sedation) and placed into serum separator tubes. After a minimum of 15 minutes at ambient temperature, blood was centrifuged (2500 × g for 10 minutes) and serum was collected. [Table 8]

[0184] Test samples (pooled capsids) were administered IV (2e13 GC / kg) to each of three animals. Test samples were administered via IV bolus under ketamine sedation (ketamine hydrochloride (HCl) delivered by intramuscular (IM) injection), followed by at least a 5 mL saline flush. After 21 days, animals were anesthetized with ketamine HCl delivered by IM injection. Blood was collected and placed into serum separator tubes. After a minimum of 15 minutes at ambient temperature, blood was centrifuged (2500 x g for 10 minutes) and serum was collected. Collected tissues were examined, weighed, and then placed into RNAse / DNase-free cryotubes. Based on the size of the designated tissue / region, up to five samples of approximately 50-100 mg per sample were collected from each location. Tissue samples were collected using aseptic technique and RNAse-free equipment and workspace. Tissues were snap frozen in liquid nitrogen and then kept on dry ice before being stored at -70 to -90°C.

[0185] DNA (Kingfisher DNA) and mRNA (Dynabeads mRNA Direct) were extracted from tissues, and vector barcode sequences were amplified by PCR and then detected by next-generation sequencing on an Illumina Miseq. Units of "nRAAFI," normalized relative abundance adjusted for input, were calculated. Essentially, the relative abundance of each barcode was divided by the relative abundance of the input, then normalized to a single total in each sample to determine the nRAAFI for each barcoded transgene.

[0186] Figure 9 shows that AAV9.AAA.NVG07 mediated a 2-fold increase in mRNA expression in skeletal muscle (Figure 9A) and heart (Figure 9B) compared to AAV9. Figure 10 shows that AAV9.AAA.NVG07 provided a 10-fold higher mRNA / DNA ratio in heart (Figure 10A) and skeletal muscle (Figure 10B) compared to AAV9. Figure 11 shows the levels of transgene mRNA (A) compared to DNA (B) in liver. AAV9.AAA.NVG07 exhibited over 7-fold greater liver detargeting in mRNA compared to AAV9. Some capsids with peptide inserts (from the AAV9.AAA.VR4 or AAV5.VR8 vector libraries) showed good biodistribution for muscle versus liver transduction, while some capsids showed lower cardiac transduction, which may be desirable for certain transductions.

[0187] Data from this NHP study on NVG07 mRNA distribution in skeletal muscle and liver were selected and combined with NVG07 data from animals 4001 and 4003 from the study described in Example 6 below, in which the same capsid was tested. AAV9 data values ​​were also obtained from these NHP studies and normalized to 1 to show the fold difference of NVG07 compared to AAV9. Figure 12A (skeletal muscle) and Figure 12B (liver). For each animal, the ratio of muscle mRNA to liver mRNA was also calculated. As shown in Figure 12C, NVG07 transduction of muscle tissue relative to liver tissue is approximately 8-18 times better than AAV9.

[0188] 6.5. Example 5: Evaluation of Selected Capsids In Vitro and In Vivo Next-generation sequencing (NGS) and quantitative PCR will be used to further evaluate AAV capsid sequences modified by peptide inserts with or without additional substitutions in in vitro assays and for in vivo biodistribution in test animals.

[0189] Studies are conducted in which selected vectors from the library are generated by the methods described in these Examples or by standard methods of triple transfection using a Rep / cap trans plasmid (containing the modified capsid gene sequence), a cis plasmid encoding the transgene, and a helper gene plasmid. The modified vectors of interest are individually injected into test animals, such as NHPs, mice, or rats, and DNA and RNA determinations from extracted tissues are compared with those of the parent vector. Quantitative PCR (qPCR) can be performed using a primer-probe combination specific to the transgene using any standard method, such as QuantStudio5 (Life Technologies, Inc.) or ddPCR.

[0190] From studies in which individual vectors were injected into mice for characterization, formalin-fixed brains could be sectioned at a thickness of 40 μm, for example, on a vibrating blade microtome (VT1000S, Leica), and free-floating sections probed with antibodies against the transgene (or viewed with fluorescence in the case of fluorescent transgenes) to examine the cellular distribution of the delivered vector.

[0191] C57B16 mice (n=5 per test group) were administered AAV.hu32, AAV9.AAA.VQVGRTS (NVG07), or AAVhu.32.AAA.VQVGRTS capsids carrying the CAG.TdTomato transgene via the tail vein at 1E14GC / kg per animal. Three weeks later, animals were sacrificed and heart, gastrocnemius, quadriceps, biceps, brain, liver, and diaphragm tissues were collected.

[0192] TdTomato genome (cDNA) and transcript (mRNA) were detected by digital PCR and plotted against the reference gene, TATA-box binding protein (TBP). The results show that AAV9.AAA.VQVGRTS (NVG07) had lower expression in the liver. Figure 13A-B.

[0193] C57B16 mice (n=4 per treatment group) were administered AAV9 capsids carrying the CAG.TdTomato transgene at an IV dose of 1E13 or 1E14 GC / kg per animal. Three weeks later, animals were sacrificed and brain, liver, biceps, and gastrocnemius muscle tissues were collected.

[0194] Separately, C57B16 mice (n=5 per treatment group) were administered NVG07 capsids carrying the CAG.TdTomato transgene at an IV dose of 1E13 or 1E14 GC / kg per animal. After 3 weeks, animals were sacrificed and brain, liver, tibialis anterior (TA) quadriceps, biceps brachii, and gastrocnemius muscle tissues were collected.

[0195] Brain slices from cortical, hippocampal, and striatal tissues from both studies (AAV9 and NVG07) were also prepared for fluorescent imaging (tdTomato / DAPI). AAV9.AAA.NVG07 redirects targeting from astrocytes to neurons in the mouse brain (data not shown).

[0196] The TdTomato genome (cDNA) and transcript (mRNA) were detected by digital PCR and plotted against the reference gene TATA-box binding protein (TBP). The results are shown in Tables 6 to 13. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

[0197] In this study, at the high dose (1E14GC / kg) of a single vector administered to mice, NVG07 resulted in 40-fold less genomic DNA in the liver than AAV9, yet still had essentially equivalent, or slightly lower, observed DNA and RNA in skeletal muscle RNA than AAV9. Lower liver expression of a given AAV gene therapy transgene is desirable. Currently, some challenges with AAV therapeutics include potential adverse effects on patient liver function and immune response. For more systemic distribution, such as in muscle diseases, approved AAV therapeutics are typically administered at doses of 1 × 10 per kg of patient body weight. 13These drugs are administered intravenously at high doses exceeding 1E13 genome copies (vector genome (vg) = genome copies (GC)). For example, the delane dystrogen moxeparvovec is administered at 1.33E14 vector genomes (vg) / kg body weight. AAV therapeutics with poor liver distribution, where liver transduction is not guaranteed or may be the cause of adverse reactions, may prove beneficial to patients, especially if other properties of AAV therapy result in the intended efficacy. Based on the finding that NVG07 showed higher observable neuronal expression (by fluorescent imaging) than AAV9, which primarily transduced astrocytes, the NVG07 capsid also offers advantages in neuromuscular diseases where neuronal expression of AAV-delivered transgenes would be guaranteed.

[0198] 6.6. Example 6 - Evaluation and Selection of Capsids from the Library Another round of peptides was selected from the B4 pool and administered to NHPs as described above. [Table 17-1] [Table 17-2] [Table 17-3]

[0199] In the B4 study of pooled vectors, several vectors with peptide inserts (e.g., DGRRIGV, YVGGRAV, PSSVQHR, IGSRGVA, GSVRQAA, SDVSRPR, AQVGRAS) showed higher mRNA expression in skeletal muscle tissue (e.g., gastrocnemius) than AAV9.

[0200] 6.7. Example 7 - Evaluation of Consensus Sequences Highly similar sequences and motifs were found among peptides in the AAV9.AAA capsid library that efficiently transduced muscle while detargeting the liver.

[0201] The "fold difference" values ​​in the chart represent the fold difference in muscle tropism of capsids bearing the peptides compared to the parent. As shown in Table 15, AAV9.AAA capsids containing these peptides with similar amino acid motifs result in more than 2-fold, and up to 27-fold, transduction in muscle compared to AAV9.AAA capsids. [Table 18]

[0202] Consensus sequence SEQ ID NO: 135 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S, G, V, or A. Consensus sequence SEQ ID NO: 136 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S or A. Consensus sequence SEQ ID NO: 137 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S, G, V, or A. Consensus sequence SEQ ID NO: 138 is X1-QV-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S or A. [Table 19]

[0203] 6.7.1.1 Conclusion AAV capsid modifications performed by random peptide insertions in the surface-exposed loops of VR-IV were able to generate sufficient library titers in the production system described herein for analysis of transduction properties in NHP tissues. This method was shown to reduce carryover of parental plasmid after cloning of random inserts and to reduce overrepresentation of parental vector in library biodistribution and transduction studies.

[0204] Intravenous administration of AAV9.AAA.VR4 to NHPs resulted in several higher relative abundances of the modified vector in muscle, suitable for use as a gene therapy vector carrying a gene of interest to treat muscle disorders. [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] [Table 20-5] [Table 20-6] [Table 20-7] [Table 20-8] [Table 20-9]

Table 20-10

Table 20-11

Table 20-12

Table 20-13

Table 20-14

Table 20-15

Table 20-16

Table 20-17

Table 20-18

Table 20-19

Table 20-20

Table 20-21

Table 20-22

Table 20-23

Table 20-24

Table 20-25

Table 20-26

Table 20-27

Table 20-28

Table 20-29

Table 20-30

Table 20-31

Table 20-32

Table 20-33

Table 20-34

Table 20-35

Table 20-36

Table 20-37

Table 20-38

Table 20-39

Table 20-40

Table 20-41

Table 20-42

Table 20-43

Table 20-44

Table 20-45

Table 20-46

Table 20-47

Table 20-48

Table 20-49

Table 20-50

Table 20-51

Table 20-52

Table 20-53

Table 20-54

Table 20-55

Table 20-56

Table 20-57

Table 20-58

Table 20-59

Table 20-60

Table 20-61

Table 20-62

Table 20-63

Table 20-64

Table 20-65

Table 20-66

Table 20-67

Table 20-68

Table 20-69

Table 20-70

Table 20-71

Table 20-72

Table 20-73

Table 20-74

Table 20-75

Table 20-76

Table 20-77

[0205] 7. Equivalents Although the present invention has been described in detail with reference to specific embodiments thereof, it will be understood that functionally equivalent variants are within the scope of the present invention. Indeed, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

[0206] 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 in its entirety.

[0207] The discussion herein provides a better understanding of the nature of the problems facing the art and should not be construed in any manner as an admission regarding prior art, nor should the citation of any reference herein be construed as an admission that such reference constitutes "prior art" to the present application.

[0208] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety for all purposes, as if each individual publication or patent or patent application were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insert, wherein the peptide insert is immediately following an amino acid residue corresponding to one of amino acids 451 to 461 of an AAV9 capsid protein comprising the amino acid sequence described in SEQ ID NO: 151, and the peptide insert has an amino acid sequence of at least 4 to a maximum of 7 consecutive amino acids as described in SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO:

138. Sequence ID 135 is X1-Q-V-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S, G, V, or A; Sequence ID 136 is X1-Q-V-X2-X3-X4-X5, where X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is any amino acid, and X5 is S or A; Sequence ID 137 is X1-Q-V-X2-X3-X4-X5, where X1 is V or A, and X2 If X is S, G, V, or A, and X3 is R or H, and X4 is T, S, V, Y, A, or P, and X5 is S, G, V, or A, or if sequence number 138 is X1-Q-V-X2-X3-X4-X5, and X1 is V or A, X2 is S, G, V, or A, X3 is R or H, X4 is T, S, V, Y, A, or P, and X5 is S or A, The capsid protein is either a wild-type capsid protein or a variant capsid protein having up to three amino acid substitutions. The recombinant adeno-associated virus (rAAV) capsid protein, prepared from the capsid protein containing the peptide insert, has improved targeting of target tissues compared to an rAAV vector prepared from i) a capsid protein without the peptide insert or ii) a reference capsid protein.

2. The capsid protein is at least one AAV type selected from AAV1, serotype 2, serotype 3, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype rh8, serotype 9, serotype hu. 31, serotype hu. 32, serotype rh10, serotype rh20, serotype hu. 37, serotype rh39, and serotype rh74, or the variant capsid protein is AAV9. AAA or AAVhu.

32. The rAAV capsid protein according to claim 1, which is AAA or a variant capsid protein.

3. The peptide insert is (a) amino acid sequence 450-459 of AAV1 capsid (SEQ ID NO: 139), (b) 449-458 of the AAV2 capsid amino acid sequence (SEQ ID NO: 140), (c) AAV3 capsid amino acid sequence (SEQ ID NO: 141) 449-459, (d) AAV4 capsid amino acid sequence (SEQ ID NO: 142) 443-453, (e) AAV5 capsid amino acid sequence (SEQ ID NO: 143) 442-445, (f) AAV6 capsid amino acid sequence (SEQ ID NO: 144) 450-459, (g) AAV7 capsid amino acid sequence (SEQ ID NO: 145) 451-461, (h) AAV8 capsid amino acid sequence (SEQ ID NO: 146) 451-461, (i) amino acid sequences 451-461 of the AAV9 capsid (SEQ ID NO: 151), (j) AAVhu. 32 capsid amino acid sequence (SEQ ID NO: 148), 451-461 (k) AAVrh10 capsid amino acid sequence (SEQ ID NO: 152) 452-461, (l) AAVrh20 capsid amino acid sequence (SEQ ID NO: 155), 452-461 (m) AAVhu. 37 capsid amino acid sequence (SEQ ID NO: 153), 452-461 (n) AAVrh39 capsid amino acid sequence (SEQ ID NO: 150) 452-461, (o) AAVrh74 capsid amino acid sequence (SEQ ID NO: 156 or SEQ ID NO: 157) 452-461, (p) AAV9. AAA capsid amino acid sequence (SEQ ID NO: 158), 452-461, or (q) AAVhu.

32. AAA capsid amino acid sequence (SEQ ID NO: 267) 451-461 The rAAV capsid protein according to claim 1 or 2, which occurs immediately after one of the amino acid residues within it.

4. The rAAV capsid protein according to claim 3, wherein the peptide insertion occurs after an amino acid residue corresponding to one of the amino acids I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid protein or AAV9.AAA capsid protein.

5. The rAAV capsid protein according to claim 4, wherein the peptide insert is formed after the amino acid residue corresponding to amino acid S454 of the AAV9 capsid protein, the AAV.32 capsid protein, the AAV9.AAA capsid protein, the AAVhu.32 capsid protein, or the AAVhu.32.AAA capsid protein.

6. The rAAV capsid protein according to claim 1, wherein the peptide is seven amino acids.

7. The rAAV capsid protein according to any one of claims 1, 2, and 4 to 6, wherein the reference capsid protein is AAV9, AAV9.AAA, AAVhu32, or AAVhu32.AAA.

8. The rAAV capsid protein according to any one of claims 1, 2, and 4 to 6, wherein the peptide insert has the amino acid sequence of VQVGRAA (SEQ ID NO: 5), VQVGRTS (SEQ ID NO: 8), AQVGRAS (SEQ ID NO: 24), VQVGRVS (SEQ ID NO: 36), VQVGRSS (SEQ ID NO: 44), VQVGRYS (SEQ ID NO: 130), VQVGRAS (SEQ ID NO: 131), VQVGRPS (SEQ ID NO: 132), VQVVRPS (SEQ ID NO: 133), or VQVGHAS (SEQ ID NO: 134).

9. The rAAV capsid protein according to claim 8, having the amino acid sequence of SEQ ID NOs: 211, 212, 263, 264, 265, 266, 318, 319, 320, 321, 372, 373, 374, or 375.

10. a. The peptide insert contains an amino acid sequence of at least 4 to a maximum of 7 consecutive amino acids of SGTVIRS (SEQ ID NO: 1), TRQYVPG (SEQ ID NO: 4), VQVGRTS (SEQ ID NO: 8), RGAVQKV (SEQ ID NO: 13), GQVHQAR (SEQ ID NO: 32), TSGGQIR (SEQ ID NO: 38), HMGHSGK (SEQ ID NO: 88), MRAVSQL (SEQ ID NO: 109), PRQYVPG (SEQ ID NO: 110), RSSSGR (SEQ ID NO: 111), VVKSTKS (SEQ ID NO: 112), RHVSASD (SEQ ID NO: 113), VRSDRDQ (SEQ ID NO: 114), or TVVTSIN (SEQ ID NO: 115), or b. The peptide insert is SGTVIRS (SEQ ID NO: 1), TRQYVPG (SEQ ID NO: 4), VQVGRTS (SEQ ID NO: 8), RGAVQKV (SEQ ID NO: 13), GQVHQAR (SEQ ID NO: 32), TSGGQIR (SEQ ID NO: 38), HMGHSGK (SEQ ID NO: 88), MRAVSQL (SEQ ID NO: 109), PRQYVPG (SEQ ID NO: 110), RSSSGR (SEQ ID NO: 111), VVKSTKS (SEQ ID NO: 112), RHVSASD (SEQ ID NO: 113), VRSDRDQ (SEQ ID NO: 114), or TVVTSIN (SEQ ID NO: 115). The rAAV capsid protein according to claim 1 or 2.

11. The peptide insert contains an amino acid sequence of at least 4 to a maximum of 7 consecutive amino acids, such as AQVGRAS (SEQ ID NO: 24), SVTSVRV (SEQ ID NO: 37), SIAKNSA (SEQ ID NO: 76), DGRRIGV (SEQ ID NO: 116), TSGERRG (SEQ ID NO: 117), PSSVQHR (SEQ ID NO: 118), SSSVQHR (SEQ ID NO: 119), SGMQERR (SEQ ID NO: 120), LERGNLE (SEQ ID NO: 121), YRDVRQT (SEQ ID NO: 122), PSAVQHR (SEQ ID NO: 123), YVGGRAV (SEQ ID NO: 124), IGSRGVA (SEQ ID NO: 125), SDVSRRPR (SEQ ID NO: 126), GSVRQAA (SEQ ID NO: 127), QSPHTSQ (SEQ ID NO: 128), or ASQAYHG (SEQ ID NO: 129), or b. The peptide insert is AQVGRAS (SEQ ID NO: 24), SVTSVRV (SEQ ID NO: 37), SIAKNSA (SEQ ID NO: 76), DGRRIGV (SEQ ID NO: 116), TSGERRG (SEQ ID NO: 117), PSSVQHR (SEQ ID NO: 118), SSSVQHR (SEQ ID NO: 119), SGMQERR (SEQ ID NO: 120), LERGNLE (SEQ ID NO: 121), YRDVRQT (SEQ ID NO: 122), PSAVQHR (SEQ ID NO: 123), YVGGRAV (SEQ ID NO: 124), IGSRGVA (SEQ ID NO: 125), SDVSRRPR (SEQ ID NO: 126), GSVRQAA (SEQ ID NO: 127), QSPHTSQ (SEQ ID NO: 128), or ASQAYHG (SEQ ID NO: 129). The rAAV capsid protein according to claim 1.

12. The rAAV capsid protein according to claim 11, wherein the peptide insert is VQVGRTS (Sequence ID 8).

13. The rAAV capsid protein according to claim 9, having one amino acid sequence of any one of SEQ ID NOs: 163, 166, 182, 217, 220, 236, 272, 275, 291, 326, 329, or 345.

14. The rAAV capsid protein according to claim 1, 11, or 12, wherein the target tissue due to the improved directivity is skeletal muscle or cardiac muscle.

15. a. The rAAV vector prepared from the capsid protein exhibits transduction of skeletal muscle and / or cardiac muscle at least 2 times, 5 times, 10 times, 15 times, 20 times, or 25 times greater than the rAAV vector prepared from the capsid protein without the peptide insert or from the reference capsid protein. b. The rAAV vector prepared from the capsid protein exhibits transduction of CNS neurons at least 2 times, 5 times, 10 times, 15 times, 20 times, or 25 times greater than the rAAV vector prepared from the capsid protein without the peptide insert or from the reference capsid protein, and / or c. The rAAV vector prepared from the capsid protein has at least 1 / 2, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, or 1 / 40 of liver transduction compared to the rAAV vector prepared from the capsid protein without the peptide insert or from the reference capsid protein. The rAAV capsid protein according to claim 1, 11, or 12.

16. The rAAV capsid protein according to claim 11 or 12, wherein the rAAV vector prepared from the capsid protein has reduced transduction of liver, heart, or astrocytes compared to the rAAV vector prepared from the capsid protein without the peptide insert or from the reference capsid protein.

17. The rAAV capsid protein according to claim 16, wherein the reference capsid protein is AAV9 capsid or AAVhu.32 capsid.

18. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to claim 1, 11, or 12, or an amino acid sequence sharing at least 80% identity therewith.

19. A packaging cell capable of expressing the nucleic acid according to claim 18 in order to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

20. An rAAV vector comprising the capsid protein described in claim 11.

21. The rAAV vector according to claim 20, further comprising an rAAV genome containing a transgene adjacent to an AAV ITR sequence.

22. A pharmaceutical composition comprising the rAAV vector according to claim 21 and a pharmaceutically acceptable carrier.

23. A method for delivering a transgene to a cell, wherein the method comprises (a) contacting the cell with the rAAV vector according to claim 20 or 21 or the pharmaceutical composition according to claim 22, or the method, rAAV vector, or pharmaceutical composition for use in the delivery of a transgene to a cell, wherein the cell comes into contact with the vector.

24. A pharmaceutical composition according to claim 22 for use in the delivery of a transgene to a target tissue of a subject requiring delivery of the transgene, wherein the vector is administered to the subject.

25. The pharmaceutical composition according to claim 24, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, intravitreously, via lumbar puncture, or via the cisterna magna.

26. The pharmaceutical composition according to claim 25, wherein the target tissue is muscle, retina, or CNS.

27. A method for creating a recombinant AAV (rAAV) vector library, (a) Generating a starting plasmid having a gene expression cassette containing a nucleotide sequence encoding the AAV capsid protein, wherein the stop codon is located at a target insertion site within the nucleotide sequence encoding the AAV capsid protein, (b) To provide a collection of nucleotide sequences encoding a repertoire of peptides to generate a peptide library, (c) 1) each comprising the nucleotide sequence encoding the AAV capsid having a nucleotide sequence encoding a random peptide from the peptide library that is inserted into the target insertion site of the nucleotide sequence encoding the capsid protein and thus replaces the stop codon, and 2) each plasmid further comprising a nucleotide sequence encoding a barcode for identifying the nucleotide sequence encoding the capsid, and a peptide insert positioned before the 5' end or after the 3' end of the nucleotide sequence encoding the capsid, (d) Collecting the individual plasmids, (e) Transfecting a cell population with one or more plasmids carrying the rAAV genome, including individual plasmid collections, and the transgene and required genes, (f) Culturing the transfected cell population under appropriate conditions to produce a collection of rAAV vectors, each containing an AAV capsid having a peptide insert that encapsulates the rAAV genome containing the transgene, (g) The method comprising taking the rAAV vector library.

28. The method according to claim 27, wherein the nucleotide sequence encoding the capsid protein encodes AAV1 capsid protein (SEQ ID NO: 139), AAV4 capsid protein (SEQ ID NO: 142), AAV5 capsid protein (SEQ ID NO: 143), AAV8 capsid protein (SEQ ID NO: 146), AAV9 capsid protein (SEQ ID NO: 151), AAV9.AAA capsid protein (SEQ ID NO: 158), AAVhu.32 (SEQ ID NO: 148), AAVhu.32.AAA (SEQ ID NO: 267), AAV3B capsid protein (SEQ ID NO: 154), or AAVhu37 capsid protein (SEQ ID NO: 153).

29. The method according to claim 27 or 28, wherein the target insertion site is located at or after an amino acid residue in VR-4 or VR-8 of the capsid protein.

30. The method according to claim 29, wherein the target insertion site is after the amino acid corresponding to S454 of AAV9 (SEQ ID NO: 151).

31. The method according to claim 29, wherein the peptide is seven amino acids.