Redirection of tropism of AAV capsids

Abstract

To provide compositions, methods, and processes for the preparation, use, and / or formulation of adeno-associated virus capsid proteins, wherein the capsid proteins comprise targeting peptide insert fragments for enhanced tropism to a target tissue.SOLUTION: The disclosure relates to, at least in part, a composition of an AAV particle comprising an AAV capsid polypeptide, for example, an AAV capsid variant, and a method for the production and use thereof. In some embodiments, the AAV capsid variant has enhanced tropism for a tissue or a cell, for example, a CNS tissue, a CNS cell, a muscle tissue, or a muscle cell. The tropism is useful for delivering a payload to a cell or tissue for the treatment of a disorder, for example, a neurological or neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 023,927, filed May 13, 2020, and U.S. Provisional Application No. 63 / 122,300, filed December 7, 2020, the contents of which are incorporated herein by reference in their entireties.

[0002] Reference to sequence listing This application has been filed in electronic format along with a Sequence Listing, which is provided as a file created on March 31, 2021. The information in the electronic format of this Sequence Listing is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure relates to compositions, methods, and processes for the preparation, use, and / or formulation of adeno-associated virus capsid proteins that contain peptides, e.g., targeting peptide inserts, to enhance target cell or tissue tropism. [Background technology]

[0004] Gene delivery to the adult central nervous system (CNS) remains a significant challenge in gene therapy. Engineered adeno-associated virus (AAV) capsids with improved brain tropism represent an attractive solution to the limitations of CNS delivery.

[0005] AAV-derived vectors are promising tools for clinical gene transfer due to their nonpathogenic nature, low immunogenicity profile, low integration rate into the host genome, and long-term transgene expression in non-dividing cells. However, the transduction efficiency of natural AAV variants in certain organs is too low for clinical application, and capsid neutralization by pre-existing neutralizing antibodies can prevent treatment for most patients. For these reasons, considerable efforts have been made to obtain novel capsid variants with enhanced properties. Among the many approaches explored to date, significant progress has come from directed evolution of AAV capsids using in vitro or in vivo selection of capsid variants created by randomization of the capsid sequence using either error-prone PCR, mixing of various parent serotypes, or insertion of completely randomized short peptides at defined positions.

[0006] Attempts to provide AAV capsids with improved properties, such as improved target cell or tissue targeting upon systemic administration, have met with limited success. Thus, there is a need for improved methods of producing AAV capsids and generating AAV capsids for delivering a desired payload to target cells or tissues, such as cells or tissues of the CNS or muscle. Summary of the Invention

[0007] The present disclosure relates, at least in part, to compositions of AAV particles comprising AAV capsid polypeptides, e.g., AAV capsid variants, and methods for their production and use. In some embodiments, the AAV capsid variants have enhanced tropism for tissues or cells, e.g., CNS tissue, CNS cells, muscle tissue, or muscle cells. This tropism can be useful for delivering a payload, e.g., a payload described herein, to cells or tissues for the treatment of a disorder, e.g., a neurological or neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder.

[0008] Thus, in one aspect, the present disclosure provides an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; an amino acid sequence containing four or fewer modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, the amino acid sequence is located in loop VIII. In some embodiments, the amino acid sequence is located immediately after position 586, 588, or 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 3636-3647, or an amino acid sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0009] In another aspect, the disclosure provides an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising a parent amino acid sequence having an insert, wherein the insert comprises the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659, an amino acid sequence containing four or fewer modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, the parental sequence comprises an amino acid sequence that contains at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 138; and / or the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the insertion is present, e.g., inserted, in loop VIII of the parental amino acid sequence. In some embodiments, the insertion is present, e.g., inserted, immediately after position 586, 588, or 589 of the parental sequence. In some embodiments, the AAV capsid variant further comprises a deletion at position 587 and / or a deletion at position 588 of the parental amino acid sequence.

[0010] In another aspect, the present disclosure provides a peptide, e.g., a targeting peptide, comprising at least the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659, an amino acid sequence comprising four or fewer modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, the peptide is encoded by the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleic acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% sequence identity); or a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660-3671. In some embodiments, the nucleotide sequence encoding the peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleic acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% sequence identity), or a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660-3671.

[0011] In yet another aspect, the disclosure provides a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, wherein the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; an amino acid sequence that includes no more than four modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0012] In yet another aspect, the present disclosure provides an AAV particle comprising an AAV capsid polypeptide, such as an AAV capsid variant, described herein. In some embodiments, the AAV particle comprises a nucleic acid sequence encoding a payload. In some embodiments, the AAV particle further comprises a viral genome comprising a promoter operably linked to the nucleic acid encoding the payload.

[0013] In yet another aspect, the present disclosure provides a method for producing an AAV particle comprising an AAV capsid polypeptide, e.g., an AAV capsid variant, described herein, comprising providing a host cell comprising a viral genome and incubating the host cell under conditions suitable for packaging the viral genome into an AAV capsid variant, e.g., an AAV capsid variant described herein, thereby producing an AAV particle.

[0014] In yet another aspect, the present disclosure provides a method for delivering a payload to a cell or tissue (e.g., a CNS cell, CNS tissue, muscle cell, or muscle tissue), comprising administering an effective amount of an AAV particle comprising an AAV capsid variant described herein.

[0015] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with a neurological disorder, e.g., a neurodegenerative disorder, comprising administering an effective amount of AAV particles comprising an AAV capsid variant described herein.

[0016] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with a muscle disorder, e.g., a neuromuscular disorder, comprising administering an effective amount of AAV particles comprising an AAV capsid variant described herein.

[0017] In yet another aspect, the present disclosure provides a method of treating a subject having or diagnosed with a neuro-oncological disorder, the method comprising administering an effective amount of AAV particles comprising an AAV capsid variant described herein.

[0018] The present disclosure provides an AAV capsid protein comprising a parent amino acid sequence selected from any of SEQ ID NOs: 1-1724, optionally having inserted therein at least one peptide selected from any of the members of SEQ ID NOs: 1725-3622.

[0019] In certain embodiments, the inserted peptide is selected from SEQ ID NOs: 1725-3622 and is inserted into the parent amino acid sequence. In certain embodiments, the peptide is inserted at any amino acid position between amino acids 586 and 592, inclusive, of the parent amino acid sequence. In such embodiments, the peptide may be inserted between amino acids 588 and 589 of the parent amino acid sequence.

[0020] In some embodiments, the parent amino acid sequence may be SEQ ID NO:138 or SEQ ID NO:11. The present disclosure also provides a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1725-3622.

[0021] In another aspect, the present disclosure provides an AAV particle comprising the AAV capsid proteins disclosed herein and a viral genome. In such aspects, the viral genome may comprise a nucleic acid sequence encoding a payload. In some embodiments, the payload may be an RNAi agent, which may consist of dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, miRNA precursor, stRNA, lncRNA, piRNA, or snoRNA.

[0022] In additional embodiments, the payload may be a peptide, polypeptide, antibody, or antibody fragment. The present disclosure also provides a pharmaceutical composition comprising AAV particles and a pharmaceutically acceptable excipient, and a method of treating a disease in a subject by administering the pharmaceutical composition to the subject.

[0023] 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 which equivalents are intended to be encompassed by the following recited embodiments.

[0024] Enumerated Embodiments 1. AAV capsid polypeptides, e.g., AAV capsid variants, (a) an amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; (b) at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or (c) The AAV capsid polypeptide, e.g., an AAV capsid mutant, comprising an amino acid sequence containing four or fewer modifications, e.g., substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659.

[0025] 2. AAV capsid polypeptides, for example, AAV capsid variants, (a) an amino acid sequence of any one of SEQ ID NOs: 3648 to 3659; (b) at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 3648 to 3659; or (c) The AAV capsid polypeptide, for example, an AAV capsid mutant, comprising an amino acid sequence containing four or fewer modifications, for example, substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 3648 to 3659.

[0026] 3. An AAV capsid polypeptide, e.g., an AAV capsid variant, according to embodiment 1 or 2, comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 3648-3659.

[0027] 4. The AAV capsid polypeptide, e.g., an AAV capsid mutant, of any one of embodiments 1 to 3, wherein the three consecutive amino acids comprise PLN. 5. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-4, wherein the four consecutive amino acids comprise PLNG (SEQ ID NO: 3678).

[0028] 6. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., the AAV capsid variant, wherein the five consecutive amino acids comprise PLNGA (SEQ ID NO: 3679).

[0029] 7. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, wherein the six consecutive amino acids comprise PLNGAV (SEQ ID NO: 3680).

[0030] 8. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of the preceding embodiments, wherein the seven contiguous amino acids comprise PLNGAVH (SEQ ID NO: 3681).

[0031] 9. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of the preceding embodiments, wherein the eight contiguous amino acids comprise PLNGAVHL (SEQ ID NO: 3682).

[0032] 10. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., the AAV capsid variant, wherein the 9 contiguous amino acids comprise PLNGAVHLY (SEQ ID NO: 3648).

[0033] 11. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of embodiments 1 to 3, wherein the three consecutive amino acids comprise YST. 12. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 11, wherein the four consecutive amino acids comprise YSTD (SEQ ID NO: 3690).

[0034] 13. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 11-12, wherein the five consecutive amino acids comprise YSTDE (SEQ ID NO: 3691) or YSTDV (SEQ ID NO: 3700).

[0035] 14. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 11-13, wherein the six consecutive amino acids comprise YSTDER (SEQ ID NO: 3692) or YSTDVR (SEQ ID NO: 3701).

[0036] 15. The AAV capsid polypeptide, e.g., an AAV capsid mutant, of any one of embodiments 1 to 3, wherein the three consecutive amino acids comprise an IVM. 16. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 15, wherein the four consecutive amino acids comprise IVMN (SEQ ID NO: 3693).

[0037] 17. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 15-16, wherein the five consecutive amino acids comprise IVMNS (SEQ ID NO: 3694).

[0038] 18. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 15-17, wherein the six consecutive amino acids comprise IVMNSL (SEQ ID NO: 3695).

[0039] 19. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 15-18, wherein the seven consecutive amino acids comprise IVMNSLK (SEQ ID NO: 3651).

[0040] 20. The AAV capsid polypeptide of any one of embodiments 1 to 19, e.g., an AAV capsid variant, comprising an amino acid sequence containing at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 3648-3659.

[0041] 21. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 1 to 10 or 20, comprising the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) or an amino acid sequence having at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), and optionally an H at position 7.

[0042] 22. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of embodiments 1-3 or 20, comprising the amino acid sequence of RDSPKGW (SEQ ID NO: 3649) or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RDSPKGW (SEQ ID NO: 3649).

[0043] 23. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 15-20, comprising the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of IVMNSLK (SEQ ID NO: 3651).

[0044] 24. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of embodiments 1-3, 11-14, or 20, comprising the amino acid sequence of YSTDVRM (SEQ ID NO: 3650), or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of YSTDVRM (SEQ ID NO: 3650).

[0045] 25. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3 or 20, comprising the amino acid sequence of RESPRGL (SEQ ID NO: 3652) or a sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RESPRGL (SEQ ID NO: 3652).

[0046] 26. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising the amino acid sequence of any of SEQ ID NOs: 3648-3659.

[0047] 27. (i) an amino acid sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide described in any one of embodiments 1 to 26, e.g., an AAV capsid variant, comprising an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660 to 3671.

[0048] 28. The nucleotide sequence encoding the AAV capsid variant (i) the nucleotide sequence of any one of SEQ ID NOs: 3660 to 3671, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 1 to 27, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660 to 3671.

[0049] 29. (i) the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-10, 20-21, or 26-28, comprising an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3660, e.g., an AAV capsid variant.

[0050] 30. The nucleotide sequence encoding the AAV capsid variant is (i) the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-10, 20-21, or 26-29, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3660, e.g., an AAV capsid variant.

[0051] 31. (i) the nucleotide sequence of SEQ ID NO: 3661, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 1-3, 20, 22, or 26-28, comprising an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3661.

[0052] 32. The nucleotide sequence encoding the AAV capsid variant is (i) the nucleotide sequence of SEQ ID NO: 3661, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-3, 20, 22, 26-28, or 31, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3661, e.g., an AAV capsid variant.

[0053] 33. (i) the nucleotide sequence of SEQ ID NO: 3662, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-3, 11-14, 20, 24, or 26-28, comprising an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3662, e.g., an AAV capsid variant.

[0054] 34. The nucleotide sequence encoding the AAV capsid variant is (i) the nucleotide sequence of SEQ ID NO: 3662, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-3, 11-14, 20, 24, 26-28, or 33, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3662, e.g., an AAV capsid variant.

[0055] 35. (i) the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 1-3, 15-20, 23, or 26-28, comprising an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3663.

[0056] 36. The nucleotide sequence encoding the AAV capsid variant is (i) the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-3, 15-20, 23, 26-28, or 35, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3663, e.g., an AAV capsid variant.

[0057] 37. (i) the nucleotide sequence of SEQ ID NO: 3664, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 1-3, 20, or 25-28, comprising an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3664.

[0058] 38. The nucleotide sequence encoding the AAV capsid variant is (i) the nucleotide sequence of SEQ ID NO: 3664, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) An AAV capsid polypeptide of any one of embodiments 1-3, 20, 25-28, or 37, e.g., an AAV capsid variant, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3664.

[0059] 39. The AAV capsid polypeptide of any one of the preceding embodiments, such as an AAV capsid variant, wherein the amino acid sequence is present in loop VIII. 40. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-10, 20-21, 26-30, or 39, wherein the amino acid sequence is located immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0060] 41. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, or 39, wherein the amino acid sequence is located immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0061] 42. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3, 11-14, 20, 22, 24-28, 31-34, 37-38, or 39, wherein the amino acid sequence is located immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0062] 43. The AAV capsid polypeptide of any one of embodiments 1 to 42, e.g., an AAV capsid variant, comprising an amino acid residue other than "A" at position 587 and / or an amino acid residue other than "Q" at position 588, numbered according to SEQ ID NO: 138.

[0063] 44. The AAV capsid polypeptide of any one of embodiments 1 to 10, 20 to 21, 26 to 30, 39 to 40, or 43, e.g., an AAV capsid variant, comprising the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), wherein the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) is located immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0064] 45. The AAV capsid polypeptide of any one of embodiments 1 to 3, 20, 26 to 28, 39 to 40, or 43, e.g., an AAV capsid variant, comprising the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654), wherein the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654) is located immediately after position 586 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0065] 46. ​​The AAV capsid polypeptide of any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, 39 or 41, e.g., an AAV capsid variant, comprising the amino acid sequence of IVMNSLK (SEQ ID NO: 3651), wherein the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) is located immediately after position 588 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0066] 47. The AAV capsid polypeptide of any one of embodiments 1-3, 11-14, 20, 22, 24-28, 31-34, 37-38, 39, or 42, e.g., an AAV capsid variant, comprising the amino acid sequence of any of SEQ ID NOs: 3649, 3650, 3652, 3653, or 3655-3659, wherein said amino acid sequence of any of said sequences occurs immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0067] 48. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., the AAV capsid variant, further comprising an amino acid substitution of K449R numbered according to SEQ ID NO: 138.

[0068] 49. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of the preceding embodiments, further comprising a modification, e.g., an insertion, substitution, and / or deletion, in loop I, II, IV and / or VI.

[0069] 50. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising an amino acid sequence having at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, of the amino acid sequence of SEQ ID NO: 138.

[0070] 51. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0071] 52. The AAV capsid polypeptide of any one of the preceding embodiments, such as an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 138. 53. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0072] 54. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of the preceding embodiments, wherein the nucleotide sequence encoding the capsid variant comprises the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0073] 55. The AAV capsid polypeptide of any one of the preceding embodiments, such as an AAV capsid variant, comprising a VP1 protein, a VP2 protein, a VP3 protein, or a combination thereof.

[0074] 56. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of the preceding embodiments, comprising an amino acid sequence corresponding to positions 138 to 743 of any one of SEQ ID NOs: 3636 to 3647, e.g., VP2, or a sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0075] 57. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of the preceding embodiments, comprising an amino acid sequence corresponding to positions 203 to 743 of any one of SEQ ID NOs: 3636 to 3647, e.g., VP3, or a sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0076] 58. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising the amino acid sequence of any one of SEQ ID NOs: 3636-3647, or an amino acid sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0077] 59. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising an amino acid sequence having at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, of the amino acid sequence of any one of SEQ ID NOs: 3636-3647.

[0078] 60. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, comprising an amino acid sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0079] 61. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of the preceding embodiments, wherein the nucleotide sequence encoding the capsid variant comprises the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0080] 62. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 1-10, 20-21, 26-30, 39-40, 43-44, or 48-61, wherein the nucleotide sequence encoding the capsid variant comprises the nucleotide sequence of SEQ ID NO: 3623 or a nucleotide sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0081] 63. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, 39, 41, 46, or 48-61, wherein the nucleotide sequence encoding the capsid variant comprises the nucleotide sequence of SEQ ID NO: 3627 or a nucleotide sequence having at least 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity thereto.

[0082] 64. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of the preceding embodiments, wherein the nucleotide sequence encoding the capsid variant is codon-optimized.

[0083] 65. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising an amino acid sequence according to any one of embodiments 1-10, 20-21, 26-30, 39-40, 43-44, 48-62, or 64, and further comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3636.

[0084] 66. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3636. 67. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising an amino acid sequence according to any one of embodiments 1-3, 20, 22, 26-28, 31-32, 39, 42, 47-61, or 64, and further comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3637.

[0085] 68. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3637. 69. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising an amino acid sequence according to any one of embodiments 1-3, 11-12, 20, 24, 26-28, 33-34, 39, 42, 47-61, or 64, and further comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3638.

[0086] 70. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3638. 71. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising an amino acid sequence according to any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, 39, 41, 46, 48-61, or 63-64, and further comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3639.

[0087] 72. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3639. 73. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising an amino acid sequence according to any one of embodiments 1-3, 20, 25-28, 37-39, 42, 47-61, or 64, and further comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 3640.

[0088] 74. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3640. 75. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3641.

[0089] 76. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3642. 77. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3643.

[0090] 78. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3644. 79. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3645.

[0091] 80. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3646. 81. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3647.

[0092] 82. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising an amino acid sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence at least 95% identical thereto.

[0093] 83. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising a parent amino acid sequence with an insert, e.g., a targeting peptide, wherein the insert is: (a) an amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; (b) at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or (c) The AAV capsid polypeptide, e.g., an AAV capsid mutant, comprising an amino acid sequence containing four or fewer modifications, e.g., substitutions, relative to the amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659.

[0094] 84. The parent sequence: (i) the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); and / or (ii) An AAV capsid polypeptide, e.g., an AAV capsid variant, described in embodiment 83, comprising an amino acid sequence containing at least one, two, or three modifications, but not more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 138.

[0095] 85. The AAV capsid polypeptide, e.g., the AAV capsid variant, of embodiment 83 or 84, wherein the parent sequence further comprises a substitution at position K449, e.g., a K449R substitution.

[0096] 86. An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 83 to 85, wherein the parent sequence comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0097] 87. An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 83 to 86, wherein the nucleotide sequence encoding the parent sequence comprises the nucleotide sequence of SEQ ID NO: 137 or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0098] 88. The parent sequence: (i) the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); and / or (ii) an amino acid sequence that includes at least one, two, or three modifications, but not more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 11; An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 83 to 87, optionally with the proviso that position 449 of SEQ ID NO: 11 is not K, but e.g., R.

[0099] 89. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, wherein the insert comprises the amino acid sequence of PLNGAVHLY (sequence number 3648).

[0100] 90. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, wherein the insert comprises the amino acid sequence of GGTLAVVSL (sequence number 3654).

[0101] 91. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, wherein the insert comprises the amino acid sequence of IVMNSLK (sequence number 3651).

[0102] 92. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, wherein the insert comprises an amino acid sequence selected from RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656).

[0103] 93. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-92, wherein the insert is in loop VIII of the parent amino acid sequence.

[0104] 94. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-90 or 93, wherein the inserted fragment is located immediately after position 586 in the parent amino acid sequence.

[0105] 95. An AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 83 to 89 or 93 to 94, wherein the insert comprises the amino acid sequence of PLNGAVHLY (sequence number 3648) and is inserted immediately after position 586 in the parent amino acid sequence.

[0106] 96. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, 90, or 93-94, wherein the insert comprises the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654) and is inserted immediately after position 586 in the parent amino acid sequence.

[0107] 97. The AAV capsid polypeptide of any one of embodiments 83-90, 93-96, e.g., an AAV capsid variant, comprising an amino acid other than "A" at position 587 and / or an amino acid other than "Q" at position 588 of the parent sequence.

[0108] 98. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 83 to 90, 93 to 97, further comprising a deletion of the amino acid "A" at position 587 and / or a deletion of the amino acid "Q" at position 588 of the parent amino acid sequence.

[0109] 99. (i) an insert comprising the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), inserted immediately after position 586 of the parent amino acid sequence; and (ii) an AAV capsid polypeptide, e.g., an AAV capsid mutant, described in any one of embodiments 83 to 89, 93 to 95, or 97 to 98, comprising a deletion of amino acids "AQ" at positions 587 and 588 of the parent amino acid sequence.

[0110] 100. (i) an insert comprising the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654), inserted immediately after position 586 of the parent amino acid sequence; (ii) an AAV capsid polypeptide, e.g., an AAV capsid variant, described in any one of embodiments 83-88, 90, 93-94, or 96-98, comprising a deletion of amino acids "AQ" at positions 587 and 588 of the parent amino acid sequence.

[0111] 101. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, 91, or 93, wherein the insert is inserted immediately after position 588 in the parent amino acid sequence.

[0112] 102. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, 91, 93, or 101, wherein the insert comprises the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) and is inserted immediately after position 588 in the parent amino acid sequence.

[0113] 103. The AAV capsid polypeptide, e.g., an AAV capsid variant, of embodiments 83-88 or 92-93, wherein the inserted fragment is inserted immediately after position 589 in the parent amino acid sequence.

[0114] 104. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 83-88, 92-93, or 103, wherein the insert comprises an amino acid sequence selected from RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656), and is inserted immediately after position 589 in the parent amino acid sequence.

[0115] 105. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., an AAV capsid variant, which does not include an insert sequence having at least five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138 of any of SEQ ID NOs: 1-1724 immediately following position 586, 588, or 589 numbered relative to SEQ ID NO: 138, as set forth in Table 6.

[0116] 106. The AAV capsid polypeptide of any one of the preceding embodiments, e.g., the AAV capsid variant, which does not include the amino acid sequence of TLAVPFK (SEQ ID NO: 1262), which is located immediately after position 588 numbered according to SEQ ID NO: 138.

[0117] 107. The AAV capsid polypeptide, e.g., the AAV capsid variant, of any one of the preceding embodiments, having increased tropism for cells or tissues of the CNS, e.g., brain cells, brain tissue, spinal cord cells, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138.

[0118] 108. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 1-12, 15-44, 46-74, 82-89, 91-95, 97-99, 101-107, which transduces a brain region selected from the dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, and putamen, and optionally wherein the level of transduction is at least 5, 10, 50, 100, 200, 500, 1,000, 2,000, 5,000, or 10,000-fold greater than the reference sequence of SEQ ID NO: 138, as measured, e.g., by an assay such as that described in Example 5, e.g., an immunohistochemistry assay, a qRT-PCR, or an RT-ddPCR assay.

[0119] 109. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of the preceding embodiments, which is at least about 5, 6, 7, 8, 9, or 10-fold enriched in the brain compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay such as that described in Example 4.

[0120] 110. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-14, 20-22, 24-34, 39-40, 42-44, 47-62, 64-70, 79-80, 82-89, 92-95, 97-99, or 103-109, which is enriched in the brain by at least about 20, 30, 40, or 50 times as compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay such as that described in Example 4.

[0121] 111. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-10, 20-22, 26-32, 39-40, 42-44, 47-62, 64-68, 82-89, 92-95, 97-99, or 103-110, which is enriched in the brain by at least about 100, 200, 300, or 400 times as compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay such as that described in Example 4.

[0122] 112. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-10, 20-21, 26-30, 39-40, 43-44, 48-62, 64-66, 82-89, 93-95, 97-99, or 105-111, wherein high levels of viral genomes are delivered to a brain region, and optionally wherein the level of viral genomes is increased by at least 5, 10, 20, 30, 40, or 50-fold compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR or RT-ddPCR assay (e.g., as described in Example 5).

[0123] 113. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-12, 15-44, 46-74, 82-89, 91-95, 97-99, 101-112, wherein the AAV capsid polypeptide delivers high levels of payload to a brain region, and optionally wherein the level of payload is increased by at least 5, 10, 50, 100, 200, 500, 1,000, 2,000, 5,000, or 10,000-fold compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR or RT-ddPCR assay (e.g., as described in Example 5).

[0124] 114. The AAV capsid polypeptide, e.g., an AAV capsid mutant, described in embodiment 113, wherein the brain region comprises the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0125] 115. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-12, 15-44, 46-74, 82-89, 91-95, 97-99, 101-114, which delivers high levels of payload to the spinal cord region, optionally wherein the level of payload is increased by at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900-fold compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR assay (e.g., as described in Example 5).

[0126] 116. The AAV capsid polypeptide, e.g., an AAV capsid mutant, of embodiment 115, wherein the spinal region comprises the cervical, thoracic, and / or lumbar regions. 117. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, 39, 41, 46, 48-61, 63-64, 71-72, 83-88, 91, 93, 101-102, 105-109, or 113-116, which exhibits preferential transduction in brain regions compared to transduction in dorsal root ganglia (DRG).

[0127] 118. The capsid variant is: (i) is at least about 300-fold, or 400-fold, enriched in the brain relative to the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 4; (ii) transduces a brain region selected from, for example, the dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, and putamen, wherein the level of transduction is at least 500, 1,000, 2,000, 5,000, or 10,000-fold greater than the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 5, e.g., an immunohistochemistry assay, a qRT-PCR, or an RT-ddPCR assay; (iii) delivers elevated levels of payload to brain regions, optionally wherein the levels of payload are at least 500, 1,000, 2,000, 5,000, or 10,000 times higher compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR or an RT-ddPCR assay (e.g., as described in Example 5), and optionally the brain regions include the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus; (iv) delivering elevated levels of payload to spinal cord regions, optionally wherein said levels of payload are at least 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 times higher compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR assay (e.g., as described in Example 5), and optionally said spinal cord regions include the cervical, thoracic, and / or lumbar regions; and / or (v) the AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 1-10, 20-21, 26-30, 39-40, 43-44, 48-62, 64-66, 82-89, 93-95, 97-99, or 105-116, delivers elevated levels of viral genomes to a brain region, optionally wherein the level of viral genomes is increased by at least 5, 10, 20, 30, 40, or 50 fold compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR or an RT-ddPCR assay (e.g., as described in Example 5), and optionally wherein the brain region comprises the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0128] 119. An AAV capsid polypeptide, e.g., an AAV capsid variant, comprising: (a) the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); (b) an amino acid sequence containing at least one, two, or three modifications, but not more than four modifications, e.g., substitutions, relative to the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); or (c) at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); (i) is at least about 300-fold, or 400-fold, enriched in the brain relative to the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 4; (ii) transduces a brain region selected from, for example, the dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, and putamen, wherein the level of transduction is at least 500, 1,000, 2,000, 5,000, or 10,000-fold greater than the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 5, e.g., an immunohistochemistry assay, a qRT-PCR, or an RT-ddPCR assay; (iii) delivers elevated levels of payload to brain regions, wherein the levels of payload are at least 500, 1,000, 2,000, 5,000, or 10,000 times higher than the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR or an RT-ddPCR assay (e.g., as described in Example 5), and optionally, the brain regions include the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus; (iv) delivering elevated levels of payload to spinal cord regions, optionally wherein said levels of payload are at least 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 times higher compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR assay (e.g., as described in Example 5), and optionally said spinal cord regions include the cervical, thoracic, and / or lumbar regions; and / or (v) the AAV capsid polypeptide, e.g., an AAV capsid variant, delivers elevated levels of viral genomes to brain regions, optionally wherein the level of viral genomes is increased by at least 5, 10, 20, 30, 40, or 50-fold compared to the reference sequence of SEQ ID NO: 138, as measured by an assay, e.g., a qRT-PCR or an RT-ddPCR assay (e.g., as described in Example 5), and optionally wherein the brain regions include the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0129] 120. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, 39, 41, 46, 48-61, 63-64, 71-72, 83-88, 91, 93, 101-102, 105-109, or 113-117, wherein the AAV capsid variant has increased tropism for muscle cells or muscle tissue, e.g., cardiac tissue, compared to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138.

[0130] 121. The AAV capsid polypeptide, e.g., an AAV capsid variant, of any one of embodiments 1-3, 15-20, 23, 26-28, 35-36, 39, 41, 46, 48-61, 63-64, 71-72, 83-88, 91, 93, 101-102, 105-109, 113-117, or 120, which delivers elevated levels of payload to muscle regions, optionally wherein the levels of payload are increased by at least 10, 15, 20, 30, or 40 fold compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay, e.g., an IHC assay or an RT-ddPCR assay (e.g., as described in Example 5).

[0131] 122. The AAV capsid polypeptide, e.g., an AAV capsid mutant, of embodiment 120 or 121, wherein the muscle region comprises a region of the cardiac muscle, quadriceps muscle, and / or diaphragm muscle.

[0132] 123. An AAV capsid polypeptide, e.g., an AAV capsid mutant, described in any one of embodiments 120 to 122, wherein the muscle region comprises a myocardial region, e.g., an atrial muscle region or a ventricular muscle region.

[0133] 124. The AAV capsid polypeptide, e.g., AAV capsid variant, of any one of the preceding embodiments, which is isolated, e.g., recombinant. 125. A polynucleotide encoding a polypeptide, such as an AAV capsid variant according to any one of embodiments 1 to 124.

[0134] 126. (i) a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660 to 3671; or (ii) The polynucleotide of embodiment 125, comprising the nucleotide sequence of any one of SEQ ID NOs: 3660 to 3671, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0135] 127. The polynucleotide of embodiment 125 or 126, comprising the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0136] 128. The polynucleotide according to any one of embodiments 125 to 127, comprising a nucleotide sequence that is codon-optimized. 129. Peptides, for example targeting peptides, (a) an amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; (b) an amino acid sequence containing four or fewer modifications, e.g., substitutions, relative to any of the amino acid sequences of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; or (c) A peptide, e.g., a targeting peptide, comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725 to 3622 or 3648 to 3659.

[0137] 130. A peptide, such as a targeting peptide, comprising an amino acid sequence according to any one of embodiments 1 to 106. 131. Peptides, for example targeting peptides, (i) the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); or (iii) A peptide, e.g., a targeting peptide, comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648).

[0138] 132. Peptides, for example targeting peptides, (i) the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The peptide, e.g., a targeting peptide, encoded by a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3660.

[0139] 133. A peptide, for example a targeting peptide, wherein the nucleotide sequence encoding the peptide is: (i) the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) a peptide, e.g., a targeting peptide, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3660.

[0140] 134. Peptides, for example targeting peptides, (i) the amino acid sequence of IVMNSLK (SEQ ID NO: 3651); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of IVMNSLK (SEQ ID NO: 3651); or (iii) A peptide, e.g., a targeting peptide, comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of IVMNSLK (SEQ ID NO: 3651).

[0141] 135. Peptides, for example targeting peptides, (i) the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The peptide, e.g., a targeting peptide, encoded by a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3663.

[0142] 136. A peptide, for example a targeting peptide, wherein the nucleotide sequence encoding the peptide is: (i) the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) a peptide, e.g., a targeting peptide, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3663.

[0143] 137. Peptides, for example targeting peptides, (i) the amino acid sequence of RDSPKGW (SEQ ID NO: 3649); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RDSPKGW (SEQ ID NO: 3649); or (iii) A peptide, e.g., a targeting peptide, comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of RDSPKGW (SEQ ID NO: 3649).

[0144] 138. Peptides, for example targeting peptides, (i) the nucleotide sequence of SEQ ID NO: 3661, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The peptide, e.g., a targeting peptide, encoded by a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3661.

[0145] 139. A peptide, for example a targeting peptide, wherein the nucleotide sequence encoding the peptide is: (i) the nucleotide sequence of SEQ ID NO: 3661, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) a peptide, e.g., a targeting peptide, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3661.

[0146] 140. Peptides, for example targeting peptides, (i) the amino acid sequence of YSTDVRM (SEQ ID NO: 3650); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of YSTDVRM (SEQ ID NO: 3650); or (iii) A peptide, e.g., a targeting peptide, comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of YSTDVRM (SEQ ID NO: 3650).

[0147] 141. Peptides, for example targeting peptides, (i) the nucleotide sequence of SEQ ID NO: 3662, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The peptide, e.g., a targeting peptide, encoded by a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3662.

[0148] 142. A peptide, for example a targeting peptide, wherein the nucleotide sequence encoding the peptide is: (i) the nucleotide sequence of SEQ ID NO: 3662, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) a peptide, e.g., a targeting peptide, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven, but not more than ten, modifications of the nucleotide sequence of SEQ ID NO: 3662.

[0149] 143. Peptides, for example targeting peptides, (i) the amino acid sequence of RESPRGL (SEQ ID NO: 3652); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RESPRGL (SEQ ID NO: 3652); or (iii) The peptide, e.g., a targeting peptide, comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of RESPRGL (SEQ ID NO: 3652).

[0150] 144. Peptides, for example targeting peptides, (i) the nucleotide sequence of SEQ ID NO: 3664, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The peptide, e.g., a targeting peptide, encoded by a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3664.

[0151] 145. A peptide, for example a targeting peptide, wherein the nucleotide sequence encoding the peptide is: (i) the nucleotide sequence of SEQ ID NO: 3664, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) a peptide, e.g., a targeting peptide, comprising a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of the nucleotide sequence of SEQ ID NO: 3664.

[0152] 146. An AAV capsid polypeptide, such as an AAV capsid mutant, comprising a peptide, such as a targeting peptide, according to any one of embodiments 129 to 145. 147. A polynucleotide encoding a peptide, such as a targeting peptide, according to any one of embodiments 129 to 145.

[0153] 148. A polynucleotide encoding an AAV capsid polypeptide, for example, an AAV capsid variant, (a) an amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; (b) an amino acid sequence containing four or fewer modifications, e.g., substitutions, relative to any of the amino acid sequences of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; or (c) containing at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any one of SEQ ID NOs: 1725 to 3622 or 3648 to 3659; Optionally, the amino acid sequence of (a), (b) and / or (c) is located immediately after position 586, 588 or 589 of the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0154] 149. A polynucleotide encoding an AAV capsid polypeptide, for example, an AAV capsid variant, wherein the AAV capsid variant is: (i) the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); or (iii) contains at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); Optionally, the amino acid sequence of (i), (ii) and / or (iii) is located immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0155] 150. (i) the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The polynucleotide of embodiment 149, comprising a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but not more than ten, modifications of the nucleotide sequence of SEQ ID NO: 3660.

[0156] 151. A polynucleotide encoding an AAV capsid polypeptide, for example, an AAV capsid variant, wherein the AAV capsid variant is (i) the amino acid sequence of IVMNSLK (SEQ ID NO: 3651); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of IVMNSLK (SEQ ID NO: 3651); or (iii) comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of IVMNSLK (SEQ ID NO: 3651); Optionally, the amino acid sequence of (i), (ii) and / or (iii) is located immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0157] 152. (i) the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The polynucleotide of embodiment 151, comprising a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but not more than ten, modifications of the nucleotide sequence of SEQ ID NO: 3663.

[0158] 153. A polynucleotide encoding an AAV capsid polypeptide, for example, an AAV capsid variant, wherein the AAV capsid variant is (i) the amino acid sequence of RDSPKGW (SEQ ID NO: 3649); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RDSPKGW (SEQ ID NO: 3649); or (iii) comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of RDSPKGW (SEQ ID NO: 3649); Optionally, the amino acid sequence of (i), (ii) and / or (iii) is located immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0159] 154. (i) the nucleotide sequence of SEQ ID NO: 3661, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The polynucleotide of embodiment 153, comprising a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but not more than ten, modifications of the nucleotide sequence of SEQ ID NO: 3661.

[0160] 155. A polynucleotide encoding an AAV capsid polypeptide, for example, an AAV capsid variant, wherein the AAV capsid variant is (i) the amino acid sequence of YSTDVRM (SEQ ID NO: 3650); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of YSTDVRM (SEQ ID NO: 3650); or (iii) comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of YSTDVRM (SEQ ID NO: 3650); Optionally, the amino acid sequence of (i), (ii) and / or (iii) is located immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0161] 156. (i) the nucleotide sequence of SEQ ID NO: 3662, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The polynucleotide of embodiment 155, comprising a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but not more than ten, modifications of the nucleotide sequence of SEQ ID NO: 3662.

[0162] 157. A polynucleotide encoding an AAV capsid polypeptide, for example, an AAV capsid variant, wherein the AAV capsid variant is (i) the amino acid sequence of RESPRGL (SEQ ID NO: 3652); (ii) an amino acid sequence containing at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RESPRGL (SEQ ID NO: 3652); or (iii) at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of RESPRGL (SEQ ID NO: 3652); Optionally, the amino acid sequence of (i), (ii) and / or (iii) is located immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0163] 158. (i) the nucleotide sequence of SEQ ID NO: 3664, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or (ii) The polynucleotide of embodiment 157, comprising a nucleotide sequence comprising at least one, two, three, four, five, six, or seven, but not more than ten, modifications of the nucleotide sequence of SEQ ID NO: 3664.

[0164] 159. The AAV capsid variant is: (i) an amino acid sequence of any one of SEQ ID NOs: 3636 to 3647, or an amino acid sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto; or (ii) A polynucleotide according to any one of embodiments 147 to 158, comprising an amino acid sequence having at least one, two, or three modifications, but not more than 30, 20, or 10 modifications, of the amino acid sequence of any one of SEQ ID NOs: 3636 to 3647.

[0165] 160. The polynucleotide of any one of embodiments 147-159, comprising the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0166] 161. A polynucleotide, peptide, AAV capsid polypeptide, such as an AAV capsid mutant, according to any one of embodiments 125 to 160, which is isolated, e.g., recombinant.

[0167] 162. An AAV particle comprising an AAV capsid polypeptide, e.g., an AAV capsid mutant, according to any one of embodiments 1 to 124 or 146. 163. An AAV particle according to embodiment 162, comprising a nucleotide sequence encoding a payload.

[0168] 164. The AAV particle of embodiment 163, wherein the encoded payload comprises a therapeutic protein or a functional variant thereof; an antibody or antibody fragment; an enzyme; a component of a gene editing system; an RNAi agent (e.g., dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA); or a combination thereof.

[0169] 165. The AAV particle of embodiment 164, wherein the therapeutic protein or a functional variant thereof, e.g., a recombinant protein, is associated with (e.g., is abnormally expressed in) a neurological or neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder.

[0170] 166. The AAV particle of embodiment 164 or 165, wherein the therapeutic protein or functional variant thereof is selected from apolipoprotein E (APOE) (e.g., ApoE2, ApoE3 and / or ApoE4); human survival of motor neuron (SMN)1 or SMN2; glucocerebrosidase (GBA1); aromatic L-amino acid decarboxylase (AADC); aspartoacylase (ASPA); tripeptidyl peptidase I (CLN2); beta-galactosidase (GLB1); N-sulfoglucosamine sulfohydrolase (SGSH); N-acetyl-alpha-glucosaminidase (NAGLU); iduronate 2-sulfatase (IDS); intracellular cholesterol transporter (NPC1); gigaxonin (GAN); or a combination thereof.

[0171] 167. The antibody or antibody-binding fragment thereof (i) CNS-related targets, e.g., antigens associated with neurological or neurodegenerative disorders, e.g., β-amyloid, APOE, tau, SOD1, TDP-43, huntingtin (HTT), and / or synuclein; (ii) a muscle- or neuromuscular-associated target, e.g., an antigen associated with a muscle disorder or a neuromuscular disorder; or (iii) An AAV particle described in embodiment 164, which binds to a neuro-oncology-associated target, e.g., an antigen associated with a neuro-oncology disorder, e.g., HER2, or EGFR (e.g., EGFRvIII).

[0172] 168. The AAV particle of embodiment 164, wherein the enzyme comprises a meganuclease, zinc finger nuclease, TALEN, recombinase, integrase, base editor, Cas9, or a fragment thereof.

[0173] 169. An AAV particle described in embodiment 164, wherein the components of the gene editing system include one or more components of a CRISPR-Cas system. 170. The one or more components of the CRISPR-Cas system include Cas9, e.g., a Cas9 ortholog or Cpf1, and a single guide RNA (sgRNA), optionally wherein: (i) the sgRNA is located upstream (5') of the cas9 enzyme; or (ii) the AAV particle of embodiment 164 or 169, wherein the sgRNA is located downstream (3') of the cas9 enzyme.

[0174] 171. The AAV particle of embodiment 164, wherein the RNAi agent (e.g., dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA) regulates, e.g., inhibits, the expression of a CNS-related gene, mRNA, and / or protein.

[0175] 172. The AAV particle of embodiment 171, wherein the CNS-related gene is selected from SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, SCN8A-SCN11A, or a combination thereof.

[0176] 173. An AAV particle described in any one of embodiments 162 to 172, comprising a viral genome comprising a promoter operably linked to the nucleic acid sequence encoding the payload.

[0177] 174. The promoter is selected from the group consisting of human elongation factor 1 α-subunit (EF1α), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC), neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), intercellular adhesion molecule 2 (ICAM-2), synapsin (Syn), methyl-CpG-binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase II (CaMKII), and metabotropic glutamate receptor 2. (mGluR2), neurofilament light chain (NFL) or heavy chain (NFH), beta-globin minigene nβ2, preproenkephalin (PPE), enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2), glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), cardiovascular promoters (e.g., αMHC, cTnT, and CMV-MLC2k), liver promoters (e.g., hAAT, TBG), skeletal muscle promoters (e.g., desmin, MCK, C512), or fragments, e.g., truncated forms, or functional variants thereof.

[0178] 175. An AAV particle described in any one of embodiments 173 or 174, wherein the viral genome further comprises a polyA signal sequence. 176. An AAV particle described in any one of embodiments 173 to 175, wherein the viral genome further comprises an inverted terminal repeat (ITR) sequence.

[0179] 177. An AAV particle described in any one of embodiments 173 to 176, wherein the viral genome comprises an ITR sequence located 5' to the encoded payload. 178. An AAV particle described in any one of embodiments 173 to 177, wherein the viral genome comprises an ITR sequence located 3' to the encoded payload.

[0180] 179. An AAV particle described in any one of embodiments 173 to 178, wherein the viral genome comprises an ITR sequence located 5' to the encoded payload and an ITR sequence located 3' to the encoded payload.

[0181] 180. An AAV particle described in any one of embodiments 173 to 179, wherein the viral genome further comprises an enhancer, a Kozak sequence, an intron region, and / or an exon region.

[0182] 181. An AAV particle described in any one of embodiments 173 to 180, wherein the viral genome further comprises an miR binding site, e.g., an miR binding site that regulates, e.g., reduces, expression of the payload encoded by the viral genome in cells or tissues in which the corresponding miRNA is expressed.

[0183] 182. An AAV particle described in any one of embodiments 173 to 181, wherein the viral genome comprises at least 1 to 5 copies, for example at least 1, 2, 3, 4, or 5 copies, of the miR binding site.

[0184] 183. An AAV particle described in any one of embodiments 173 to 182, wherein the viral genome comprises at least three copies of the miR binding site, optionally wherein all three copies comprise the same miR binding site, or wherein at least one, two, or all copies comprise different miR binding sites.

[0185] 184. An AAV particle described in any one of embodiments 173 to 182, wherein the viral genome comprises at least four copies of the miR binding site, optionally wherein all four copies comprise the same miR binding site, or wherein at least one, two, three or all copies comprise different miR binding sites.

[0186] 185. The miR binding site comprises a miR122 binding site, a miR183 binding site, a miR-142-3p, or a combination thereof, optionally wherein: (i) the miR122-binding site comprises the nucleotide sequence of SEQ ID NO: 3672, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of SEQ ID NO: 3672; (ii) the miR183-binding site comprises the nucleotide sequence of SEQ ID NO: 3675, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of SEQ ID NO: 3675; and / or (iii) An AAV particle described in any one of embodiments 181 to 184, wherein the miR-142-3p binding site comprises a nucleotide sequence of SEQ ID NO: 3674 or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, but not more than ten modifications, of SEQ ID NO: 3674.

[0187] 186. An AAV particle described in any one of embodiments 173 to 185, wherein the viral genome is single-stranded. 187. An AAV particle described in any one of embodiments 173 to 186, wherein the viral genome further comprises a nucleotide sequence encoding a Rep protein, e.g., a nonstructural protein, and the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein.

[0188] 188. The AAV particle of embodiment 187, wherein the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein are encoded by at least one Rep gene.

[0189] 189. An AAV particle described in any one of embodiments 173 to 188, wherein the viral genome further comprises a nucleic acid sequence encoding an AAV capsid variant described in any one of embodiments 1 to 124 or 146.

[0190] 190. An AAV particle according to any one of embodiments 162 to 189, which is isolated, e.g., recombinant. 191. A polypeptide comprising a parent amino acid sequence of any of SEQ ID NOs: 1-1724, and having one or more targeting peptide inserts inserted therein, wherein the targeting peptide inserts individually comprise contiguous amino acid sequence regions of 2 to 9 amino acids selected from any of the targeting peptides of SEQ ID NOs: 1725-3622.

[0191] 192. The polypeptide of embodiment 191, wherein the polypeptide has a first targeting peptide insert, and the first targeting peptide insert has a contiguous amino acid region of at least five amino acids selected from any of the targeting peptides of SEQ ID NOs: 1725-3622.

[0192] 193. An AAV capsid comprising VP1, VP2, and VP3 proteins, wherein each of the VP1, VP2, and VP3 proteins comprises a polypeptide of any of embodiments 191 or 192.

[0193] 194. The polypeptide of embodiment 191, wherein the parent amino acid sequence is SEQ ID NO: 138. 195. The polypeptide of embodiment 194, wherein the parent amino acid sequence has the substitution K449R.

[0194] 196. The polypeptide of embodiment 194 or 195, wherein the first targeting peptide insert comprises the amino acid sequence PLNGAVHLY (SEQ ID NO: 3648), inserted immediately after amino acid 586 of the parent amino acid sequence.

[0195] 197. The polypeptide of embodiment 196, further comprising a deletion of amino acids "AQ" at positions 587-588 of the parent amino acid sequence. 198. The polypeptide of embodiment 194 or 195, wherein the first targeting peptide insert comprises the amino acid sequence GGTLAVVSL (SEQ ID NO: 3654), inserted immediately after amino acid 586 of the parent amino acid sequence.

[0196] 199. The polypeptide of embodiment 198, further comprising a deletion of amino acids "AQ" at positions 587-588 of the parent amino acid sequence. 200. The polypeptide of embodiment 194 or 195, wherein the first targeting peptide insert comprises a 7 amino acid sequence selected from RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656), inserted immediately after amino acid 589 of the parent amino acid sequence.

[0197] 201. The polypeptide of embodiment 194 or 195, wherein the first targeting peptide insert comprises the 7 amino acid sequence IVMNSLK (SEQ ID NO: 3651) inserted immediately after amino acid 588 of the parent amino acid sequence.

[0198] 202. The polypeptide of embodiment 191, wherein the parent amino acid sequence is SEQ ID NO:5. 203. The polypeptide of embodiment 202, wherein the parent amino acid sequence has the substitution K449R.

[0199] 204. An AAV capsid comprising VP1, VP2, and VP3 proteins, wherein each of the VP1, VP2, and VP3 proteins comprises a polypeptide of any of embodiments 194 to 203.

[0200] 205. A polynucleotide encoding the polypeptide or capsid protein of any of embodiments 191-204. 206. The polynucleotide of embodiment 205, which is DNA, and the sequence of said DNA is codon-optimized.

[0201] 207. An AAV particle comprising a capsid according to any of embodiments 193 or 205 and a vector genome encoding a therapeutic payload. 208. The AAV particle of embodiment 207, wherein the therapeutic payload is a gene of interest.

[0202] 209. The AAV particle of embodiment 208, wherein the therapeutic payload encodes a therapeutic RNA. 210. A VP1 capsid of an AAV selected from the group consisting of any of SEQ ID NOs: 3636 to 3647.

[0203] 211. A polynucleotide encoding any of the VP1 capsid proteins of AAV described in embodiment 210. 212. A vector comprising a polynucleotide encoding an AAV capsid variant according to any one of embodiments 1 to 124 or 146, a polynucleotide according to any one of embodiments 126 to 128, 147 to 161, 205 to 206, or 211, a polynucleotide encoding a peptide, e.g., a targeting peptide, according to any one of embodiments 129 to 145 or 161, or a polynucleotide encoding a polypeptide according to any one of embodiments 191 to 192 or 194 to 203.

[0204] 213. A cell, e.g., a host cell, comprising an AAV capsid variant according to any one of embodiments 1 to 124 or 146, a polynucleotide according to any one of embodiments 126 to 128, 147 to 161, 205 to 206, or 211, a peptide according to any one of embodiments 129 to 145 or 161, a polypeptide according to any one of embodiments 191 to 192 or 194 to 203, an AAV particle according to any one of embodiments 162 to 190 or 207 to 209, or a vector according to embodiment 212.

[0205] 214. The cell according to embodiment 213, wherein the cell is a mammalian cell or an insect cell. 215. The cell of embodiment 213 or 214, wherein the cell is a cell of a brain region or a spinal cord region, optionally a cell of the frontal cortex, sensory cortex, motor cortex, caudate nucleus, dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, putamen, cervical spinal cord region, thoracic spinal cord region, and / or lumbar spinal cord region.

[0206] 216. The cell of embodiment 213 or 214, wherein the cell is a neuron, sensory neuron, motor neuron, astrocyte, or muscle cell (e.g., a cardiac, diaphragm, or quadriceps cell).

[0207] 217. A method for producing AAV particles, comprising: (i) providing a host cell containing a viral genome; (ii) incubating a host cell under conditions suitable for encapsulating the viral genome into an AAV capsid variant of any one of embodiments 1 to 124 or 146, or an AAV capsid variant encoded by a polynucleotide of any one of embodiments 125 to 128 or 147 to 161; This method thereby produces AAV particles.

[0208] 218. The method of embodiment 217, further comprising, prior to step (i), introducing into said host cell a first nucleic acid molecule comprising said viral genome. 219. The method of embodiment 217 or 218, wherein the host cell comprises a second nucleic acid encoding the capsid variant.

[0209] 220. The method of any one of embodiments 217-219, wherein the second nucleic acid molecule is introduced into the host cell before, simultaneously with, or after the first nucleic acid molecule. 221. A pharmaceutical composition comprising an AAV particle according to any one of embodiments 162 to 190, an AAV particle comprising a capsid variant according to any one of embodiments 1 to 124 or 146, an AAV particle comprising a peptide according to any one of embodiments 129 to 145 or 161, or an AAV particle comprising a polypeptide according to any one of embodiments 191 to 192 or 194 to 203, and a pharmaceutically acceptable excipient.

[0210] 222. A method for delivering a payload to a cell or tissue (e.g., a CNS cell, CNS tissue, muscle cell, or muscle tissue), comprising administering an effective amount of the pharmaceutical composition of embodiment 221, an AAV particle of any one of embodiments 162 to 190, an AAV particle comprising a capsid variant of any one of embodiments 1 to 124 or 146, an AAV particle comprising a peptide of any one of embodiments 129 to 145 or 161, or an AAV particle comprising a polypeptide of any one of embodiments 191 to 192 or 194 to 203.

[0211] 223. The method of embodiment 222, wherein the cell is a cell of a brain region or a spinal cord region, optionally a cell of the frontal cortex, sensory cortex, motor cortex, caudate nucleus, dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, putamen, cervical spinal cord region, thoracic spinal cord region, and / or lumbar spinal cord region.

[0212] 224. The method of embodiment 222 or 223, wherein the cell is a neuron, sensory neuron, motor neuron, astrocyte, or muscle cell (e.g., a cardiac, diaphragm, or quadriceps cell).

[0213] 225. The method of any one of embodiments 222-224, wherein the cell or tissue is in a subject. 226. The method of embodiment 225, wherein the subject has, has been diagnosed with, or is at risk of having a neurological disorder, e.g., a neurodegenerative disorder.

[0214] 227. The method of embodiment 225, wherein the subject has, has been diagnosed with, or is at risk of having a muscle disorder, e.g., a neuromuscular disorder. 228. The method of embodiment 225, wherein the subject has, has been diagnosed with, or is at risk of having a neuro-oncological disorder.

[0215] 229. A method for treating a subject having or diagnosed as having a neurological disorder, e.g., a neurodegenerative disorder, comprising administering to the subject an effective amount of a pharmaceutical composition described in embodiment 221, an AAV particle described in any one of embodiments 162 to 190, an AAV particle comprising a capsid variant described in any one of embodiments 1 to 124 or 146, an AAV particle comprising a peptide described in any one of embodiments 129 to 145 or 161, or an AAV particle comprising a polypeptide described in any one of embodiments 191 to 192 or 194 to 203.

[0216] 230. A method for treating a subject having or diagnosed as having a muscle or neuromuscular disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 221, an AAV particle of any one of embodiments 162 to 190, an AAV particle comprising a capsid variant of any one of embodiments 1 to 124 or 146, an AAV particle comprising a peptide of any one of embodiments 129 to 145 or 161, or an AAV particle comprising a polypeptide of any one of embodiments 191 to 192 or 194 to 203.

[0217] 231. A method for treating a subject having or diagnosed as having a neuro-oncological disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 221, the AAV particle of any one of embodiments 162 to 190, the AAV particle comprising a capsid variant of any one of embodiments 1 to 124 or 146, the AAV particle comprising a peptide of any one of embodiments 129 to 145 or 161, or the AAV particle comprising a polypeptide of any one of embodiments 191 to 192 or 194 to 203.

[0218] 232. The method of any one of embodiments 229-231, wherein treating comprises preventing the progression of said disease or disorder in said subject. 233. The method of embodiments 225-232, wherein the subject is a human.

[0219] 234. The method of any one of embodiments 225 to 233, wherein the AAV particles are administered to the subject intramuscularly, intravenously, intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, or via intracisternal injection (ICM).

[0220] 235. The method of any one of embodiments 225 to 233, wherein the AAV particles are administered to the subject via focused ultrasound (FUS), for example, focused ultrasound in conjunction with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS in conjunction with intravenous administration.

[0221] 236. The method of any one of embodiments 225 to 235, wherein the AAV particles are administered to the subject intravenously. 237. The method of any one of embodiments 222 to 236, wherein administration of the AAV particles results in a decrease in the presence, level, and / or activity of a gene, mRNA, protein, or a combination thereof.

[0222] 238. The method of any one of embodiments 222 to 148, wherein administration of the AAV particles results in an increase in the presence, level, and / or activity of a gene, mRNA, protein, or a combination thereof.

[0223] 239. A pharmaceutical composition according to embodiment 221, an AAV particle according to any one of embodiments 162 to 190, an AAV particle comprising a capsid variant according to any one of embodiments 1 to 124 or 146, an AAV particle comprising a polypeptide according to any one of embodiments 129 to 145 or 161, or an AAV particle comprising a polypeptide according to any one of embodiments 191 to 192 or 194 to 203, for use in a method for delivering a payload to a cell or tissue.

[0224] 240. The pharmaceutical composition of embodiment 221, the AAV particle of any one of embodiments 162 to 190, the AAV particle comprising a capsid variant of any one of embodiments 1 to 124 or 196, the AAV particle comprising a polypeptide of any one of embodiments 129 to 145 or 161, or the AAV particle comprising a polypeptide of any one of embodiments 191 to 192 or 194 to 203 for use in a method for treating a neurological, neurodegenerative, muscular, neuromuscular or neuro-oncological disorder.

[0225] 241. A pharmaceutical composition according to embodiment 221, an AAV particle according to any one of embodiments 162 to 190, an AAV particle comprising a capsid variant according to any one of embodiments 1 to 124 or 146, an AAV particle comprising a peptide according to any one of embodiments 129 to 145 or 161, or an AAV particle comprising a polypeptide according to any one of embodiments 191 to 192 or 194 to 203 for use in the manufacture of a medicament.

[0226] 242. Use of the pharmaceutical composition according to embodiment 221, the AAV particle according to any one of embodiments 162 to 190, the AAV particle comprising a capsid variant according to any one of embodiments 1 to 124 or 146, the AAV particle comprising a peptide according to any one of embodiments 129 to 145 or 161, or the AAV particle comprising a polypeptide according to any one of embodiments 191 to 192 or 194 to 203 in the manufacture of a medicament for treating a neurological, neurodegenerative, muscular, neuromuscular or neuro-oncological disorder.

[0227] The above and other objects, features, and advantages will become apparent from the following description of specific embodiments of the present disclosure, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present disclosure. [Brief explanation of the drawings]

[0228] [Figure 1A] 1 shows a diagram of the identification and design of the non-human primate (NHP) TRACER AAV capsid library. [Figure 1B] 1 shows a diagram of the identification and design of the non-human primate (NHP) TRACER AAV capsid library. [Figure 2] A diagram of the orthogonal evolution of the TRACER AAV capsid library is shown. [Figure 3] 1 shows a diagram of high-throughput screening by next-generation sequencing in non-human primates. [Figure 4] FIG. 1 shows a diagram of an exemplary TRACER AAV library design (SEQ ID NOs: 3696-3699). [Figure 5] 1 shows a diagram of an alternative TRACER backbone construct. [Figure 6A] Quantification of mRNA for AAV particle transduction in NHP tissues using qRT-PCR. [Figure 6B] Quantification of mRNA for AAV particle transduction in NHP tissues using qRT-PCR. [Figure 6C] Quantification of mRNA for AAV particle transduction in NHP tissues using qRT-PCR. [Figure 7] Quantification of mRNA for AAV particle transduction in NHP tissues using ddPCR. [Figure 8] Quantification of mRNA transduction by AAV particles in the spinal cord and dorsal root ganglia of NHPs is shown. [Figure 9A] Quantification of viral genomes in NHP tissues. [Figure 9B] Quantification of viral genomes in NHP tissues. [Figure 9C] Quantification of viral genomes in NHP tissues. [Figure 9D] Quantification of viral genomes in NHP tissues. [Figure 10A] Quantification of payload-HA in peripheral tissues after transduction of AAV particles as fold over TBP transcripts is shown. [Figure 10B] Quantification of payload-HA in peripheral tissues after transduction of AAV particles as a fold increase relative to AAV9 is shown. [Figure 11A] Transgene mRNA expression (RT-ddPCR) in the brain is shown as fold increase relative to TBP (housekeeping gene). [Figure 11B] Transgene mRNA expression (RT-ddPCR) in the brain is shown as fold increase relative to TBP (housekeeping gene). [Figure 12A] Biodistribution of viral DNA in the brain as vector genomes per cell (ddPCR) is shown. [Figure 12B] Biodistribution of viral DNA in the brain as vector genomes per cell (ddPCR) is shown. [Figure 13A] Transgene mRNA expression in the brain (RT-ddPCR) is shown as fold increase relative to AAV9. [Figure 13B] Transgene mRNA expression in the brain (RT-ddPCR) is shown as fold increase relative to AAV9. [Figure 14A]Shown is the biodistribution of viral DNA in the brain (ddPCR) as a fold increase relative to AAV9. [Figure 14B] Shown is the biodistribution of viral DNA in the brain (ddPCR) as a fold increase relative to AAV9. [Figure 15A] Spinal cord transgene mRNA expression is shown as fold over TATA box binding protein. [Figure 15B] Transgene mRNA expression in DRGs is shown as fold over TATA box-binding protein. [Figure 16A] Biodistribution of viral genomes in the spinal cord is shown as vector genomes per cell. [Figure 16B] Biodistribution of viral genomes in DRGs is shown as vector genomes per cell. [Figure 17A] Spinal cord mRNA expression is shown as fold increase relative to AAV9. [Figure 17B] DRG mRNA expression is shown as fold over AAV9. [Figure 18A] Biodistribution of viral genome in the spinal cord is shown as a fold increase relative to AAV9. [Figure 18B] Biodistribution of viral genome in DRG is shown as a fold increase relative to AAV9. [Figure 19A] 1 shows images of the brain transduction profiles of TTD-001 and AAV9 as determined by immunohistochemical analysis of the dentate nucleus. [Figure 19B] 1 shows images of the brain transduction profiles of TTD-001 and AAV9 as determined by immunohistochemical analysis of the cerebellar cortex. [Figure 19C] Images of the brain transduction profiles of TTD-001 and AAV9 as determined by immunohistochemical analysis of the cortex (FIG. 19C) are shown. [Figure 19D] Images of the brain transduction profiles of TTD-001 and AAV9 as determined by immunohistochemical analysis of the brainstem, hippocampus, thalamus, and putamen are shown. [Figure 19E]1 shows images of the brain transduction profiles of TTD-001 and AAV9 as determined by immunohistochemical analysis of dorsal root ganglia. [Figure 20A] Immunohistochemistry images of detargeting in DRG characteristic of capsid mutant TTD-004 compared to AAV9 are shown. [Figure 20B] Immunohistochemistry images of detargeting in DRG characteristic of capsid mutant TTD-004 compared to AAV9 are shown. [Figure 21A] Biodistribution of viral genomes in peripheral tissues quantified as vector genomes per cell is shown. [Figure 21B] Biodistribution of viral genomes in peripheral tissues quantified as vector genomes per cell is shown. [Figure 22A] Immunohistochemistry images of the heart of a female NHP 14 days after intravenous administration of AAV particles containing the TTD-001 capsid mutant, the TTD-004 capsid mutant, or the wild-type AAV9 control capsid polypeptide are shown. A series of overall views of the myocardium are shown. For each series of images (A-C), the upper left panel shows staining after administration of AAV particles containing the TTD-001 capsid mutant, the upper right panel shows staining after administration of AAV particles containing the TTD-004 capsid mutant, and the lower panel shows staining after administration of AAV particles containing the wild-type AAV9 control capsid mutant. [Figure 22B] Immunohistochemistry images of the heart of a female NHP 14 days after intravenous administration of AAV particles containing the TTD-001 capsid mutant, the TTD-004 capsid mutant, or the wild-type AAV9 control capsid polypeptide are shown. A series of images of the left ventricle of the heart are shown. For each series of images (A-C), the upper left panel shows staining after administration of AAV particles containing the TTD-001 capsid mutant, the upper right panel shows staining after administration of AAV particles containing the TTD-004 capsid mutant, and the lower panel shows staining after administration of AAV particles containing the wild-type AAV9 control capsid mutant. [Figure 22C]Immunohistochemistry images of the heart of a female NHP 14 days after intravenous administration of AAV particles containing the TTD-001 capsid mutant, the TTD-004 capsid mutant, or the wild-type AAV9 control capsid polypeptide are shown. A series of images of the right ventricle of the heart are shown. For each series of images (A-C), the upper left panel shows staining after administration of AAV particles containing the TTD-001 capsid mutant, the upper right panel shows staining after administration of AAV particles containing the TTD-004 capsid mutant, and the lower panel shows staining after administration of AAV particles containing the wild-type AAV9 control capsid mutant. DETAILED DESCRIPTION OF THE INVENTION

[0229] The details of one or more embodiments of the present disclosure are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are now described. Other features, objects, and advantages of the present disclosure will become apparent from the description. In the description, the singular forms include the plural forms unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the case of conflict, the present description shall control. Certain terms are defined in the definitions section and throughout.

[0230] Described herein are, among other things, compositions comprising AAV capsid polypeptides, e.g., AAV capsid variants, e.g., the AAV capsid variants described herein, as well as methods of making and using the same. Generally, the AAV capsid variants have enhanced tropism for cells or tissues, e.g., CNS tissue, CNS cells, muscle cells, or muscle tissue, for delivery of a payload to the cells or tissues.

[0231] As demonstrated in the Examples herein below, certain AAV capsid variants described herein exhibit multiple advantages over wild-type AAV9, including (i) increased penetrance across the blood-brain barrier following intravenous administration, (ii) broader distribution throughout multiple brain regions, e.g., the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus, and / or (iii) increased payload expression in multiple brain regions. Without wishing to be bound by theory, it is believed that these advantages may be due, in part, to the spread of the AAV capsid variants through the cerebral vasculature. In some embodiments, the AAV capsids described herein enhance payload delivery to multiple brain regions, including, e.g., the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0232] The present disclosure provides AAV particles with enhanced tropism for target tissues (e.g., the CNS), as well as related processes for their targeting, preparation, formulation, and use. Peptides, e.g., targeting peptides, and nucleic acid sequences encoding peptides, e.g., targeting peptides, are also provided. These peptides, e.g., targeting peptides, may be inserted into AAV capsid protein sequences to alter tropism for specific cell types, tissues, organs, or organisms in vivo, ex vivo, or in vitro.

[0233] Several approaches have previously been used to generate AAV capsids with enhanced tropism for cells or tissues, such as CNS cells or tissues. One approach used co-infection of cultured cells (Grimm et al., 2008, the contents of which are incorporated herein by reference in their entirety) or animal tissues in situ (Lisowski et al., 2014, the contents of which are incorporated herein by reference in their entirety) with adenovirus to induce exponential replication of infectious AAV DNA. Another approach involved using cell-specific CRE transgenic mice (Deverman et al., 2016, the contents of which are incorporated herein by reference in their entirety) to enable viral DNA recombination specifically in astrocytes, followed by recovery of CRE-recombined capsid variants. Both approaches met with limited success.

[0234] The transgenic CRE system used by Deverman et al. (2016) has limited utility in other animal species, and AAV variants selected by directed evolution in mouse tissues do not exhibit similar properties in larger animals. The previously described transduction-specific approach is not suitable for large animal studies because: 1) many tissues of interest (e.g., CNS) are not readily accessible for adenoviral co-infection; 2) specific adenoviral tropism itself biases the library distribution; and 3) large animals are typically not amenable to transfection or genetic engineering to express CRE recombinase in defined cell types.

[0235] To address these limitations, a screening platform for broadly applicable functional AAV capsid libraries for cell-type-specific biopanning in non-transgenic animals has been developed and is described in the accompanying Examples. In the TRACER (Tropism Redirection of AAV by Cell Type-Specific Expression of RNA) platform system, the capsid gene is placed under the control of a cell-type-specific promoter, driving capsid mRNA expression in the absence of helper virus co-infection. Without wishing to be bound by theory, this RNA-driven selection is thought to increase selective pressure favoring capsid variants that transduce specific cell types. The TRACER platform enables the generation of AAV capsid libraries without the need for transgenic animals or helper virus co-infection, thereby achieving specific recovery and subcloning of capsid mRNA expressed in transduced cells. Without wishing to be bound by theory, because mRNA transcription is a hallmark of complete transduction, it is believed that the method disclosed herein allows for the identification of fully infectious AAV capsid mutants, and in addition to its increased stringency, this method allows for the identification of capsids with enhanced tropism for specific cell types using libraries designed to express CAP mRNA under the control of any cell-specific promoter, such as, but not limited to, the synapsin-1 promoter (neurons), the GFAP promoter (astrocytes), the TBG promoter (liver), the CAMK promoter (skeletal muscle), or the MYH6 promoter (cardiomyocytes). Described herein are novel AAV capsid mutants generated using the TRACER method that exhibit enhanced tropism for, for example, CNS cells, CNS tissue, muscle cells, or muscle tissue.

[0236] The AAV particles and payloads of the present disclosure may be delivered to one or more target cells, tissues, organs, or organisms. In some embodiments, the AAV particles of the present disclosure exhibit enhanced tropism for target cell types, tissues, or organs. As a non-limiting example, the AAV particles may have enhanced tropism for cells and tissues of the central or peripheral nervous system (CNS and PNS, respectively), or muscle cells and tissues. The AAV particles of the present disclosure may additionally or alternatively have reduced tropism for undesirable target cell types, tissues, or organs.

[0237] In some embodiments, the AAV comprises a small, non-enveloped, icosahedral-capsid virus of the Parvoviridae family, characterized by a single-stranded DNA viral genome. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. The Parvoviridae family includes the genus Dependovirus, which contains AAVs capable of replicating in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and ovine species.

[0238] Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY. (3d Ed. 1996), the contents of which are incorporated herein by reference in their entirety.

[0239] In some embodiments, AAVs are used as biological tools due to their relatively simple structure, their ability to infect a wide range of cells (including quiescent and dividing cells) without integrating into the host genome and replicating, and their relatively benign immunogenic profile. The viral genome may be engineered to contain minimal components for the assembly of functional recombinant viruses or viral particles that are targeted to specific tissues and loaded or engineered to express or deliver a desired payload.

[0240] In some embodiments, the AAV is a naturally occurring (e.g., wild-type) AAV or a recombinant AAV. In some embodiments, the wild-type AAV vector genome is a linear single-stranded DNA (ssDNA) molecule approximately 5,000 nucleotides (nt) in length. In some embodiments, inverted terminal repeats (ITRs) cap the viral genome at both the 5' and 3' ends and provide origins of replication for the viral genome. In some embodiments, the AAV viral genome typically contains two ITR sequences. These ITRs have a characteristic T-shaped hairpin structure defined by self-complementary regions (145 nt in wild-type AAV) at the 5' and 3' ends of the ssDNA that form an energetically stable double-stranded region. The double-stranded hairpin structure has multiple functions, including, but not limited to, acting as an origin of DNA replication by serving as a primer for the endogenous DNA polymerase complex of the host viral replicating cell.

[0241] In some embodiments, the wild-type AAV viral genome further comprises nucleotide sequences for two open reading frames, one for four nonstructural Rep proteins (Rep78, Rep68, Rep52, and Rep40, encoded by Rep genes) and one for three capsid or structural proteins (VP1, VP2, and VP3, encoded by capsid or Cap genes). The Rep proteins are used for replication and packaging, while the capsid proteins assemble to create the protein shell of AAV, or AAV capsid polypeptides, e.g., AAV capsid variants. Alternative splicing and alternative start codons and promoters result in the production of four different Rep proteins from one open reading frame and the production of three capsid proteins from one open reading frame. While variations vary depending on the AAV serotype, as a non-limiting example, for AAV9 / hu.14 (SEQ ID NO: 123 of US Pat. No. 7,906,111 , the contents of which are incorporated herein by reference in their entirety), VP1 refers to amino acids 1-736, VP2 refers to amino acids 138-736, and VP3 refers to amino acids 203-736. In some embodiments, for any one of the amino acid sequences set forth in SEQ ID NOs: 3636-3647, VP1 includes amino acids 1-743, VP2 includes amino acids 138-743, and VP3 includes amino acids 203-743. In other words, VP1 is the full-length capsid sequence, while VP2 and VP3 are shorter components of the whole. Consequently, while variations in the sequence of the VP3 region are also variations in VP1 and VP2, the percent difference compared to the parent sequence is greatest for VP3, as it is the shortest of the three sequences. While described herein with respect to amino acid sequences, the nucleic acid sequences encoding these proteins can be similarly described. Together, the three capsid proteins assemble to create the AAV capsid protein. Without wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of VP1:VP2:VP3 of 1:1:10.

[0242] The AAV vectors of the present disclosure may be recombinantly produced and may be based on the parent or reference sequence of an adeno-associated virus (AAV). In addition to single-stranded AAV viral genomes (e.g., ssAAV), the present disclosure also provides self-complementary AAV (scAAV) viral genomes. The scAAV vector genome contains DNA strands that anneal together to form double-stranded DNA. By omitting the synthesis of the second strand, scAAV allows for rapid expression in transduced cells. In some embodiments, the AAV particles of the present disclosure are scAAV. In some embodiments, the AAV particles of the present disclosure are ssAAV.

[0243] Methods for generating and / or modifying AAV particles have been disclosed in the art, such as in pseudotyped AAV vectors (PCT Patent Publication Nos. WO200028004; WO200123001; WO2004112727; WO2005005610; and WO2005072364, the contents of each of which are incorporated herein by reference in their entirety).

[0244] As described herein, AAV particles of the present disclosure comprising an AAV capsid polypeptide, e.g., an AAV capsid variant, and a viral genome have enhanced tropism for a cell type or tissue, e.g., a CNS cell type, region, or tissue, or a muscle cell type or tissue. In some embodiments, AAV particles of the present disclosure comprising a capsid with an inserted peptide, e.g., a targeting peptide, and a viral genome may have enhanced tropism for a cell type, region, or tissue of the human CNS or muscle.

[0245] Peptides, e.g., targeting peptides Disclosed herein are peptides, e.g., targeting peptides, for enhanced or improved transduction of target tissues (e.g., cells of the CNS or PNS), and related AAV particles comprising an AAV capsid polypeptide, e.g., an AAV capsid variant, with a peptide, e.g., a targeting peptide insert. In some embodiments, the peptide, e.g., the targeting peptide, is an isolated, e.g., recombinant, peptide, e.g., a targeting peptide. In some embodiments, the nucleic acid encoding the peptide, e.g., the targeting peptide, is an isolated, e.g., recombinant, nucleic acid.

[0246] In some embodiments, peptides, e.g., targeting peptides, may direct AAV particles to cells, regions, or tissues of the CNS. Cells of the CNS may be, but are not limited to, brain support cells such as neurons (e.g., excitatory, inhibitory, motor, sensory, autonomic, sympathetic, parasympathetic, Purkinje, Betz, etc.), glial cells (e.g., microglia, astrocytes, oligodendrocytes), and / or immune cells (e.g., T cells). Tissues of the CNS may be, but are not limited to, the cortex (e.g., frontal lobe, parietal lobe, occipital lobe, temporal lobe), thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, or deep cerebellar nuclei.

[0247] In some embodiments, a peptide, e.g., a targeting peptide, may direct the AAV particle to a cell, region, or tissue of the PNS, which may be, but is not limited to, the dorsal root ganglion (DRG).

[0248] In some embodiments, a peptide, e.g., a targeting peptide, may direct AAV particles to the CNS (e.g., the cortex) following intravenous administration. In some embodiments, a peptide, e.g., a targeting peptide, may direct AAV particles to the CNS (e.g., the cortex) following focused ultrasound (FUS), e.g., focused ultrasound in conjunction with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS in conjunction with intravenous administration.

[0249] In some embodiments, a peptide, e.g., a targeting peptide, may direct AAV particles to the PNS (e.g., DRG) following intravenous administration. In some embodiments, a peptide, e.g., a targeting peptide, may direct AAV particles to the PNS (e.g., DRG) following focused ultrasound (FUS), e.g., focused ultrasound in conjunction with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS in conjunction with intravenous administration.

[0250] In some embodiments, the peptide, e.g., a targeting peptide, may direct the AAV particle to a muscle cell, region, or tissue. In some embodiments, the muscle is cardiac muscle. In some embodiments, the peptide, e.g., a targeting peptide, may direct the AAV particle to a muscle cell, region, or tissue after intravenous administration.

[0251] Peptides, e.g., targeting peptides, can vary in length. In some embodiments, peptides, e.g., targeting peptides, are about 3 to about 20 amino acids in length. By way of non-limiting example, targeting peptides can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length. In some embodiments, peptides comprise about 6-12 amino acids in length, e.g., about 9 amino acids in length. In some embodiments, peptides comprise about 5-10 amino acids in length, e.g., about 7 amino acids in length.

[0252] Peptides, e.g., targeting peptides, may be contiguous (or consecutive) or non-contiguous (or not contiguous), or may be split, or may span two or more amino acid sequences by intervening amino acid sequences that may vary in length. Contiguous peptides, e.g., targeting peptides, may be of different lengths. As a non-limiting example, contiguous peptides, e.g., targeting peptides, may be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length. Non-contiguous or split peptides, e.g., targeting peptides, may be of different lengths. By way of non-limiting example, non-contiguous or split peptides, e.g., targeting peptides, can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length. Intervening amino acid sequences can vary in length. As non-limiting examples, an intervening peptide, e.g., a targeting peptide, can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 3-5, 3-8, 3-10, 3-12, 3-15, 3-18, 3-20, 5-10, 5-15, 5-20, 10-12, 10-15, 10-20, 12-20, or 15-20 amino acids in length.

[0253] In some embodiments, peptides of the present disclosure, e.g., targeting peptides, may be identified and / or designed by any sliding window algorithm known in the art.

[0254] In some embodiments, peptides, e.g., targeting peptides, and associated AAV particles (e.g., AAV particles comprising AAV capsid polypeptides, e.g., AAV capsid variants) may be identified from a library of AAV capsid polypeptides, e.g., AAV capsid variants. In some embodiments, the peptides, e.g., targeting peptides, may be 5-10 amino acid sequences, e.g., 6-10 amino acid sequences, 6-9 amino acid sequences, 7-10 amino acid sequences, 7-9 amino acid sequences, 8-10 amino acid sequences, 7 amino acid sequences, 8 amino acid sequences, or 9 amino acid sequences. In some embodiments, the peptides, e.g., targeting peptides, may be 5 amino acid sequences (5 amino acids in length). In some embodiments, the peptides, e.g., targeting peptides, may be 6 amino acid sequences (6 amino acids in length). In some embodiments, the peptides, e.g., targeting peptides, may be 7 amino acid sequences (7 amino acids in length). In some embodiments, the peptides, e.g., targeting peptides, may be 9 amino acid sequences (9 amino acids in length). In some embodiments, peptides, e.g., targeting peptides, may also differ in the way they are made or designed, non-limiting examples of which include random peptide selection, site-saturation mutagenesis, and / or optimization of specific regions of the peptide (e.g., flanking regions or the central core).

[0255] In some embodiments, a peptide library, eg, a targeting peptide library, comprises targeting peptides seven amino acids in length (7 amino acids in length) randomly generated by PCR.

[0256] In some embodiments, the library of peptides, e.g., targeting peptides, includes peptides, e.g., targeting peptides, with three mutated amino acids. In some embodiments, these three mutated amino acids are consecutive or adjacent amino acids. In other embodiments, these three mutated amino acids are non-consecutive, non-adjacent, or separated amino acids. In some embodiments, the peptides, e.g., targeting peptides, are 5 amino acids in length. In some embodiments, the peptides, e.g., targeting peptides, are 6 amino acids in length. In some embodiments, the parent peptide, e.g., targeting peptide, is 7 amino acids in length. In other embodiments, the parent peptide is 9 amino acids in length.

[0257] In some embodiments, the library of peptides, e.g., targeting peptides, includes peptides, e.g., targeting peptides, that are 7 amino acids in length, where amino acids in the peptides, e.g., targeting peptides, and / or flanking sequences are evolved via site-saturation mutagenesis of 3 consecutive amino acids. In some embodiments, NNK (N=any base; K=G or T) codons are used to generate the site-saturation mutant sequences.

[0258] AAV particles are generated containing capsid proteins with peptides, such as targeting peptides, inserted fragments (e.g., AAV capsid variants), and viral genomes encoding reporters (e.g., GFP) are packaged therein.These AAV particles (or AAV capsid libraries) containing AAV capsid variants are then administered to transgenic rodents (e.g., mice) via intravenous delivery to the tail vein.When these capsid libraries are administered to cre-expressing mice, the expression of Cre causes the reporter payload to be expressed in target tissues.

[0259] In some embodiments, expression of AAV capsid mRNA may be regulated under or driven by, for example, a cell-type specific promoter. Such capsids, which may contain a viral genome encoding a peptide, e.g., a targeting peptide, an insert, and a reporter encapsulated therein, may be administered to non-transgenic rodents (e.g., mice), such as, but not limited to, C57BL / 6 mice, BALB / C mice, and rats, for example, by intravenous delivery to the tail vein. Administration of such capsid libraries to non-transgenic rodents may result in expression of the reporter payload in target tissues by the cell-type specific promoter.

[0260] In some embodiments, expression of AAV capsid mRNA may be under the control of, or driven by, a cell-type specific promoter, for example. Such capsids, which may contain a viral genome encoding a peptide, e.g., a targeting peptide insert, and a reporter encapsulated therein, may be administered to non-human primates, such as, but not limited to, cynomolgus monkeys and rhesus monkeys, for example, by intravenous delivery to the saphenous vein. Administration of such capsid libraries to non-human primates may result in expression of the reporter payload in target tissues by the cell-type specific promoter.

[0261] In some embodiments, AAV particles comprising capsid proteins with peptide, e.g., targeting peptide, inserts may hereafter be referred to as peptide display capsid libraries.

[0262] AAV particles and / or viral genomes may be recovered from the target tissue for identification of peptides, e.g., targeting peptides, and associated AAV particles that are enriched exhibit enhanced transduction of the target tissue. Enrichment may be determined using standard methods in the art, such as, but not limited to, next-generation sequencing (NGS), viral genome quantification, biochemical assays, immunohistochemistry, and / or imaging of target tissue samples.

[0263] The target tissue may be any cell, tissue, or organ of interest. By way of non-limiting example, samples may be taken from the brain, spinal cord, dorsal root ganglion and associated roots, liver, heart, gastrocnemius muscle, soleus muscle, pancreas, kidney, spleen, lung, adrenal gland, stomach, sciatic nerve, saphenous nerve, thyroid gland, eye (with or without optic nerve), pituitary gland, skeletal muscle (rectus femoris), colon, duodenum, ileum, jejunum, leg skin, superior cervical ganglion, bladder, ovaries, uterus, prostate, testes, and / or any site identified as having a lesion or being of interest.

[0264] In some embodiments, the peptide, e.g., the targeting peptide, may comprise a sequence shown in Table 1. In some embodiments, the peptide, e.g., the targeting peptide, may comprise a sequence shown in Table 2. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of any one of peptides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, for example, as set forth in Table 2. In some embodiments, the peptide, e.g., the targeting peptide, is isolated, e.g., recombinant.

[0265] [Table 1-1]

[0266] [Table 1-2]

[0267] Table 1-3

[0268] Table 1-4

[0269] Table 1-5

[0270] Table 1-6

[0271] Table 1-7

[0272] Table 1-8

[0273] Table 1-9

[0274] Table 1-10

[0275] Table 1-11

[0276] Table 1-12

[0277] Table 2

[0278] In some embodiments, the peptide, e.g., the targeting peptide, comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622. In some embodiments, the peptide comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 3648-3659.

[0279] In some embodiments, three consecutive amino acids comprise PLN. In some embodiments, four consecutive amino acids comprise PLNG (SEQ ID NO: 3678). In some embodiments, five consecutive amino acids comprise PLNGA (SEQ ID NO: 3679). In some embodiments, six consecutive amino acids comprise PLNGAV (SEQ ID NO: 3680). In some embodiments, seven consecutive amino acids comprise PLNGAVH (SEQ ID NO: 3681). In some embodiments, eight consecutive amino acids comprise PLNGAVHL (SEQ ID NO: 3682). In some embodiments, nine consecutive amino acids comprise PLNGAVHLY (SEQ ID NO: 3648).

[0280] In some embodiments, the four consecutive amino acids comprise NGAV (SEQ ID NO: 3683). In some embodiments, the four consecutive amino acids comprise GAVH (SEQ ID NO: 3684). In some embodiments, the five consecutive amino acids comprise NGAVH (SEQ ID NO: 3685). In some embodiments, the five consecutive amino acids comprise GAVHL (SEQ ID NO: 3686). In some embodiments, the five consecutive amino acids comprise AVHLY (SEQ ID NO: 3687). In some embodiments, the six consecutive amino acids comprise NGAVHL (SEQ ID NO: 3688). In some embodiments, the seven consecutive amino acids comprise NGAVHLY (SEQ ID NO: 3689).

[0281] In some embodiments, three consecutive amino acids comprise YST. In some embodiments, four consecutive amino acids comprise YSTD (SEQ ID NO: 3690). In some embodiments, five consecutive amino acids comprise YSTDE (SEQ ID NO: 3691). In some embodiments, five consecutive amino acids comprise YSTDV (SEQ ID NO: 3700). In some embodiments, six consecutive amino acids comprise YSTDER (SEQ ID NO: 3692). In some embodiments, six consecutive amino acids comprise YSTDVR (SEQ ID NO: 3701). In some embodiments, seven consecutive amino acids comprise YSTDERM (SEQ ID NO: 3657). In some embodiments, seven consecutive amino acids comprise YSTDERK (SEQ ID NO: 3658). In some embodiments, seven consecutive amino acids comprise YSTDVRM (SEQ ID NO: 3650).

[0282] In some embodiments, three consecutive amino acids comprise IVM. In some embodiments, four consecutive amino acids comprise IVMN (SEQ ID NO: 3693). In some embodiments, five consecutive amino acids comprise IVMNS (SEQ ID NO: 3694). In some embodiments, six consecutive amino acids comprise IVMNS (SEQ ID NO: 3695). In some embodiments, seven consecutive amino acids comprise IVMNSL (SEQ ID NO: 3651).

[0283] In some embodiments, a peptide, e.g., a targeting peptide, comprises an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, comprises an amino acid sequence that includes at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 3648-3659.

[0284] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) or an amino acid sequence having at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), optionally with an H at position 7.

[0285] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of RDSPKGW (SEQ ID NO: 3649) or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RDSPKGW (SEQ ID NO: 3649).

[0286] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of IVMNSLK (SEQ ID NO: 3651).

[0287] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of YSTDVRM (SEQ ID NO: 3650) or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of YSTDVRM (SEQ ID NO: 3650).

[0288] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of RESPRGL (SEQ ID NO: 3652) or a sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RESPRGL (SEQ ID NO: 3652).

[0289] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of any of SEQ ID NOs: 1725-3622. In some embodiments, the peptide comprises the amino acid sequence of any of SEQ ID NOs: 3648-3659.

[0290] In some embodiments, a peptide, e.g., a targeting peptide, may comprise an amino acid sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the sequences shown in Table 1 or Table 2.

[0291] In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3648. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3649. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3650. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3651. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3652. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3653. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3654. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3655. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3656. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3657. In some embodiments, the peptide, e.g., the targeting peptide, comprises the amino acid sequence of SEQ ID NO: 3658. In some embodiments, the peptide, eg, the targeting peptide, comprises the amino acid sequence of SEQ ID NO:3659.

[0292] In some embodiments, the peptide, e.g., the targeting peptide, may comprise SEQ ID NO: 1725. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1726. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1729. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1760. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1769. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 3622. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1798. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1785. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1767. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1734. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1737. In some embodiments, the peptide, e.g., the targeting peptide may comprise SEQ ID NO: 1819.

[0293] In some embodiments, a peptide, e.g., a targeting peptide, may comprise four or more consecutive amino acids of any of the peptides, e.g., targeting peptides, disclosed herein. In some embodiments, a peptide, e.g., a targeting peptide, may comprise four consecutive amino acids of any of the sequences shown in Table 1 or Table 2. In some embodiments, a peptide, e.g., a targeting peptide, may comprise five consecutive amino acids of any of the sequences shown in Table 1 or Table 2. In some embodiments, a peptide, e.g., a targeting peptide, may comprise six consecutive amino acids of any of the sequences shown in Table 1 or 2.

[0294] In some embodiments, the peptide, e.g., a targeting peptide, comprises an amino acid sequence encoded by a nucleotide sequence described herein, e.g., a nucleotide sequence in Table 2. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence that includes at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of the nucleotide sequence of any one of SEQ ID NOs: 3660-3671. In some embodiments, the peptide comprises an amino acid sequence encoded by a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0295] In some embodiments, a peptide, e.g., a targeting peptide, comprises an amino acid sequence encoded by a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3660. In some embodiments, a peptide comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0296] In some embodiments, a peptide, e.g., a targeting peptide, comprises an amino acid sequence encoded by a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3663. In some embodiments, a peptide comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0297] In some embodiments, a nucleotide sequence encoding a peptide described herein, e.g., a targeting peptide, e.g., peptides 1-12, comprises a nucleotide sequence described herein, e.g., as set forth in Table 2. In some embodiments, a nucleic acid sequence encoding a peptide described herein comprises the nucleotide sequence of any of SEQ ID NOs: 3660-3671, or a nucleotide sequence substantially identical thereto (e.g., at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, a nucleic acid sequence encoding a peptide described herein comprises a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660-3671. In some embodiments, a nucleotide sequence encoding a peptide described herein, e.g., a targeting peptide, is isolated, e.g., recombinant.

[0298] In some embodiments, a nucleotide sequence encoding a peptide described herein, e.g., a targeting peptide, comprises a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3660. In some embodiments, a nucleic acid sequence encoding a peptide described herein comprises a nucleotide sequence that includes the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0299] In some embodiments, nucleic acids encoding peptides described herein comprise a nucleotide sequence that includes at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3663. In some embodiments, nucleic acids encoding peptides described herein comprise a nucleotide sequence that includes the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0300] In some embodiments, the AAV particles of the present disclosure comprise a peptide, e.g., a targeting peptide, an AAV capsid, e.g., and an AAV capsid variant, with an insert (e.g., an AAV capsid variant), wherein the peptide, e.g., the targeting peptide, has an amino acid sequence as set forth in either Table 1 or Table 2.

[0301] In some embodiments, the AAV particles of the present disclosure comprise a peptide, e.g., a targeting peptide, an AAV capsid polypeptide, e.g., an AAV capsid variant, together with an insert (e.g., an AAV capsid variant), wherein the peptide has amino acids with at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of either Table 1 or Table 2.

[0302] In some embodiments, the AAV particles of the present disclosure comprise a peptide, e.g., a targeting peptide, an AAV capsid polypeptide, e.g., with an insert (e.g., an AAV capsid variant), and an AAV capsid variant, wherein the peptide, e.g., the targeting peptide, has an amino acid sequence comprising at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids of any of the sequences set forth in either Table 1 or 2.

[0303] In some embodiments, the AAV particles of the present disclosure comprise a peptide, e.g., a targeting peptide, an AAV capsid, e.g., and AAV capsid variant, with an insert (e.g., an AAV capsid variant), wherein the peptide, e.g., the targeting peptide, has an amino acid sequence substantially identical (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity) to any of the sequences shown in either Table 1 or Table 2.

[0304] In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 2 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 3 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 2 through amino acid 3 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 4 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 2 through amino acid 4 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 3 through amino acid 4 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 5 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 2 through amino acid 5 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 3 through amino acid 5 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 4 through amino acid 5 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, can include amino acids 1 through 6 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, can include amino acids 2 through 6 of SEQ ID NOs: 1725-3622 or 3648-3659.In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 3 through amino acid 6 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 4 through amino acid 6 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 5 through amino acid 6 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 7 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 2 through amino acid 7 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 3 through amino acid 7 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 4 through amino acid 7 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 5 through amino acid 7 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 6 through amino acid 7 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 8 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, can include amino acids 2 through 8 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, can include amino acids 3 through 8 of SEQ ID NOs: 1725-3622 or 3648-3659.In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 4 through amino acid 8 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 5 through amino acid 8 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 6 through amino acid 8 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 7 through amino acid 8 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 2 through amino acid 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 3 through amino acid 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 4 through amino acid 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 5 through amino acid 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 6 through amino acid 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, can include amino acids 7 through 9 of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, a peptide, e.g., a targeting peptide, can include amino acids 8 through 9 of SEQ ID NOs: 1725-3622 or 3648-3659.In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 1 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 2 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 3 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 4 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 5 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 6 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 7 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 8 through amino acid 10 of SEQ ID NOs: 1725-3622. In some embodiments, a peptide, e.g., a targeting peptide, may comprise amino acid 9 through amino acid 10 of SEQ ID NOs: 1725-3622.

[0305] The present disclosure also provides nucleic acids or polynucleotides encoding any of the above-described peptides, e.g., targeting peptides, and AAV capsid polypeptides, e.g., AAV capsid variants, and AAV particles, vectors, and cells containing them.

[0306] In some embodiments, the insertion of a peptide, e.g., a targeting peptide, into a parent AAV capsid, e.g., a parent sequence, results in a combination insertion / substitution compared to the parent amino acid sequence. In some embodiments, all of the amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, one amino acid of the peptide, e.g., a targeting peptide, replaces one amino acid in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, two amino acids of the peptide, e.g., a targeting peptide, replace two amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, three amino acids of the peptide, e.g., a targeting peptide, replace three amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, four amino acids of the peptide, e.g., a targeting peptide, replace four amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, five amino acids of the peptide, e.g., a targeting peptide, replace five amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, six amino acids of the peptide, e.g., a targeting peptide, replace six amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, seven amino acids of the peptide, e.g., a targeting peptide, replace seven amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence.In some embodiments, eight amino acids of the peptide, e.g., a targeting peptide, replace eight amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence. In some embodiments, nine amino acids of the peptide, e.g., a targeting peptide, replace nine amino acids in the parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, are inserted into the parent AAV capsid sequence.

[0307] In some embodiments, one amino acid of a peptide, e.g., a targeting peptide, is inserted into a parent AAV capsid, e.g., a parent sequence, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, two amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, three amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, four amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, five amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, six amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, seven amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, eight amino acids of a peptide, e.g., a targeting peptide, are inserted into a parent AAV capsid, while the remaining amino acids of the peptide, e.g., a targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence. In some embodiments, nine amino acids of the peptide, e.g., the targeting peptide, are inserted into the parent AAV capsid, while the remaining amino acids of the peptide, e.g., the targeting peptide, replace the corresponding amino acids in the parent AAV capsid sequence.

[0308] In some embodiments, certain amino acids of a peptide, e.g., a targeting peptide, may be anchored and / or retained as in the original parent AAV capsid sequence, e.g., the parent sequence. In certain embodiments, these anchored amino acids are centrally located in the peptide, e.g., the targeting peptide, resulting in a split-insertion-anchor-insertion design. Similarly, by way of non-limiting example, insertion of a peptide, e.g., a targeting peptide, may result in a split-insertion-replacement-insertion design. By way of non-limiting example, insertion of a peptide, e.g., a targeting peptide, into the parent AAV capsid sequence may result in a split-replacement-insertion-replacement design. By way of non-limiting example, a peptide, e.g., a targeting peptide, of a split design may be as shown in Figure 4 (e.g., TNHQSXXXXAVXXXAQAQT (SEQ ID NO: 3699)).

[0309] In some embodiments, insertion of a peptide, e.g., a targeting peptide, into a parent AAV capsid sequence, e.g., a parent sequence, may result in the substitution or mutation of at least one amino acid in the parent AAV capsid. In certain embodiments, the first amino acid (N-terminus) of the peptide, e.g., a targeting peptide, replaces an amino acid in the parent AAV capsid sequence. In certain embodiments, the first two amino acids (N-terminus) of the peptide, e.g., a targeting peptide, replace two amino acids in the parent AAV capsid sequence. In certain embodiments, the first three amino acids (N-terminus) of the peptide, e.g., a targeting peptide, replace three amino acids in the parent AAV capsid sequence. In certain embodiments, the first four amino acids (N-terminus) of the peptide, e.g., a targeting peptide, replace four amino acids in the parent AAV capsid sequence. In certain embodiments, the first five amino acids (N-terminus) of the peptide, e.g., a targeting peptide, replace five amino acids in the parent AAV capsid sequence. In certain embodiments, the first six amino acids (N-terminus) of a peptide, e.g., a targeting peptide, replace six amino acids in the parent AAV capsid sequence. In certain embodiments, the first seven amino acids (N-terminus) of a peptide, e.g., a targeting peptide, replace seven amino acids in the parent AAV capsid sequence. In certain embodiments, the first eight amino acids (N-terminus) of a peptide, e.g., a targeting peptide, replace eight amino acids in the parent AAV capsid sequence. In certain embodiments, the first nine amino acids (N-terminus) of a peptide, e.g., a targeting peptide, replace nine amino acids in the parent AAV capsid sequence. In certain embodiments, the first ten amino acids (N-terminus) of a peptide, e.g., a targeting peptide, replace ten amino acids in the parent AAV capsid sequence.

[0310] In certain embodiments, the last amino acid (C-terminus) of a peptide, e.g., a targeting peptide, replaces an amino acid in the parent AAV capsid sequence. In certain embodiments, the last two amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace two amino acids in the parent AAV capsid sequence. In certain embodiments, the last three amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace three amino acids in the parent AAV capsid sequence. In certain embodiments, the last four amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace four amino acids in the parent AAV capsid sequence. In certain embodiments, the last five amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace five amino acids in the parent AAV capsid sequence. In certain embodiments, the last six amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace six amino acids in the parent AAV capsid sequence. In certain embodiments, the last seven amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace seven amino acids in the parent AAV capsid sequence. In certain embodiments, the last eight amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace eight amino acids in the parent AAV capsid sequence. In certain embodiments, the last nine amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace nine amino acids in the parent AAV capsid sequence. In certain embodiments, the last ten amino acids (C-terminus) of a peptide, e.g., a targeting peptide, replace ten amino acids in the parent AAV capsid sequence.

[0311] In certain embodiments, the first (N-terminal) and last (C-terminal) amino acids of a peptide, e.g., a targeting peptide, may substitute amino acids in a parent AAV capsid sequence, e.g., a parent sequence. In certain embodiments, the first (N-terminal) two and last (C-terminal) two amino acids of a peptide, e.g., a targeting peptide, may substitute amino acids in a parent AAV capsid sequence. In certain embodiments, the first (N-terminal) three and last (C-terminal) three amino acids of a peptide, e.g., a targeting peptide, may substitute amino acids in a parent AAV capsid sequence. In certain embodiments, the substitutions are asymmetric with respect to the N-terminal substitutions and C-terminal substitutions, and may be any combination of any of the above.

[0312] In certain embodiments, one amino acid in a peptide, e.g., a targeting peptide, replaces one amino acid in a parent AAV capsid sequence, e.g., a parent sequence. In certain embodiments, two amino acids in a peptide, e.g., a targeting peptide, replace two amino acids in a parent AAV capsid sequence. In certain embodiments, three amino acids in a peptide, e.g., a targeting peptide, replace three amino acids in a parent AAV capsid sequence. In certain embodiments, four amino acids in a peptide, e.g., a targeting peptide, replace four amino acids in a parent AAV capsid sequence. In certain embodiments, five amino acids in a peptide, e.g., a targeting peptide, replace five amino acids in a parent AAV capsid sequence. In certain embodiments, six amino acids in a peptide, e.g., a targeting peptide, replace six amino acids in a parent AAV capsid sequence. In certain embodiments, seven amino acids in a peptide, e.g., a targeting peptide, replace seven amino acids in a parent AAV capsid sequence. In certain embodiments, eight amino acids of a peptide, e.g., a targeting peptide, replace eight amino acids in the parent AAV capsid sequence. In certain embodiments, nine amino acids of a peptide, e.g., a targeting peptide, replace nine amino acids in the parent AAV capsid sequence. In certain embodiments, ten amino acids of a peptide, e.g., a targeting peptide, replace ten amino acids in the parent AAV capsid sequence. In certain embodiments, all of the amino acids of a peptide, e.g., a targeting peptide, replace the same number of amino acids in the parent AAV capsid sequence.

[0313] In some embodiments, the AAV particles of the present disclosure may comprise a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, wherein the polynucleotide further comprises a nucleic acid insert, e.g., a targeting nucleic acid insert, having a nucleotide sequence substantially comprising any of those described in Hanlon et al., 2019 (Hanlon et al., Mol. Ther. Methods Clin. Dev. 2019, Oct. 23;15:320-332, the contents of which are incorporated herein by reference in their entirety). Non-limiting examples of nucleic acid inserts, e.g., targeting nucleic acid inserts, include those having a nucleotide sequence substantially comprising AAV-S. Non-limiting examples of targeting nucleic acid inserts include those having a nucleotide sequence substantially comprising AAV-F.

[0314] AAV particles of the present disclosure comprising a nucleic acid insert, e.g., a targeting nucleic acid insert, may have a polynucleotide sequence encoding a capsid polypeptide that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the parent capsid sequence.

[0315] AAV particles of the present disclosure that include a peptide, e.g., a targeting peptide, insert may have an amino acid sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the parent capsid sequence.

[0316] AAV capsid polypeptides, e.g., AAV capsid variants In some embodiments, a peptide, e.g., a targeting peptide, is inserted, included, or otherwise incorporated into a polypeptide. In some embodiments, such a polypeptide may be referred to as a "parent polypeptide" or "starting polypeptide" or "parent amino acid sequence," and such an insert may be referred to as a "targeting peptide insert," "peptide insert," or "amino acid sequence insert." In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, described herein may comprise a peptide, e.g., a targeting peptide, an insert, and a polypeptide, e.g., a larger polypeptide, described herein. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, has a longer amino acid sequence than the parent AAV capsid. In other embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, has an amino acid sequence of the same length as the parent AAV capsid. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, has a shorter amino acid sequence than the parent AAV capsid.

[0317] When a peptide comprising a sequence of 5 to 50 contiguous amino acids, e.g., a targeting peptide, is inserted into a parent polypeptide, the insertion can be between two amino acids of the parent polypeptide. The insertion can be a split insertion, whereby one contiguous portion of the peptide insert is inserted between a first set of two amino acids of the parent polypeptide and a second portion of the peptide is inserted between a second set of two amino acids of the parent polypeptide, e.g., at a different site. Any number of amino acids of the parent polypeptide can be retained between the first and second sites. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of the parent polypeptide can be retained between the first and second peptide inserts.

[0318] In some embodiments, a peptide, e.g., a targeting peptide, insert may wholly or partially replace one or more amino acids in a parent polypeptide. For example, a four-amino acid peptide insert may be inserted immediately after position 586 of the parent polypeptide, with the last two amino acids of the peptide insert replacing amino acids 587 and 588 of the parent polypeptide. As a result, the newly formed polypeptide is increased in length by two amino acids, e.g., two amino acids are inserted and two amino acids are replaced. In some embodiments, combinatorial insertion / substitution (i / s) variants may include one or more amino acid insertions and one or more amino acid substitutions, e.g., 1-15 amino acids in length and 1-15 amino acids in number, respectively.

[0319] The peptide, e.g., a targeting peptide, may be a free-standing peptide or may be inserted into or attached to a parent sequence, hi some embodiments, the peptide, e.g., a targeting peptide, is inserted into the capsid protein of an AAV particle.

[0320] One or more peptides, for example, targeting peptides, may be inserted into the parent AAV capsid sequence to generate the AAV particles of the present disclosure. A peptide, e.g., a targeting peptide, may be inserted into the parent AAV capsid sequence at any position that results in a fully functional AAV particle, e.g., an AAV particle comprising an AAV capsid polypeptide, e.g., an AAV capsid variant, described herein. A peptide, e.g., a targeting peptide, may be inserted into VP1, VP2, and / or VP3. The numbering of amino acid residues may vary depending on the AAV serotype. As used herein, the amino acid positions of the parent AAV capsid sequence are described using AAV9 (SEQ ID NO: 138) as a reference.

[0321] In some embodiments, peptides, e.g., targeting peptides, are inserted into hypervariable regions of the AAV capsid sequence. Non-limiting examples of such hypervariable regions include loop I, loop II, loop IV, loop VI, and loop VIII of the parent AAV capsid. Without wishing to be bound by theory, in some embodiments, these surface-exposed loops, hereafter also referred to as surface loops, are typically unstructured and less conserved, making them suitable regions for inserting peptides, e.g., targeting peptides.

[0322] In some embodiments, a peptide, e.g., a targeting peptide, is inserted into Loop I. In other embodiments, a peptide, e.g., a targeting peptide, is used to replace part or all of Loop I. As a non-limiting example, the addition of a peptide, e.g., a targeting peptide, to a parent AAV capsid sequence may result in the substitution or mutation of at least one amino acid in the parent AAV capsid.

[0323] In some embodiments, the peptide, e.g., a targeting peptide, is inserted into Loop II. In other embodiments, the targeting peptide is used to replace part or all of Loop II. As a non-limiting example, the addition of a targeting peptide to the parent AAV capsid sequence may result in the substitution or mutation of at least one amino acid in the parent AAV capsid.

[0324] In some embodiments, the peptide, e.g., a targeting peptide, is inserted into loop IV. In other embodiments, the peptide, e.g., a targeting peptide, is used to replace part or all of loop IV. As a non-limiting example, the addition of a peptide, e.g., a targeting peptide, to the parent AAV capsid sequence may result in the substitution or mutation of at least one amino acid in the parent AAV capsid.

[0325] In some embodiments, the peptide, e.g., a targeting peptide, is inserted into loop VI. In other embodiments, the peptide, e.g., a targeting peptide, is used to replace part or all of loop VI. As a non-limiting example, the addition of a peptide, e.g., a targeting peptide, to the parent AAV capsid sequence may result in the substitution or mutation of at least one amino acid in the parent AAV capsid.

[0326] In some embodiments, the peptide, e.g., a targeting peptide, is inserted into loop VIII. In other embodiments, the peptide, e.g., a targeting peptide, is used to replace part or all of loop VIII. As a non-limiting example, the addition of a peptide, e.g., a targeting peptide, to the parent AAV capsid sequence may result in the substitution or mutation of at least one amino acid in the parent AAV capsid.

[0327] In some embodiments, more than one peptide, e.g., a targeting peptide, is inserted into the parent AAV capsid sequence. As a non-limiting example, a peptide, e.g., a targeting peptide, may be inserted into both loop IV and loop VIII of the same parent AAV capsid sequence.

[0328] Peptides, e.g., targeting peptides, may be inserted at any amino acid position in the parent AAV capsid sequence, for example, between amino acids such as, but not limited to, positions 586-592, 588-589, 586-589, 452-458, 262-269, 464-473, 491-495, 546-557, and / or 659-668.

[0329] In some embodiments, a peptide, e.g., a targeting peptide, is inserted into the parent AAV capsid sequence between amino acids 588 and 589 (loop VIII). In some embodiments, the parent AAV capsid is AAV9 (SEQ ID NO: 138). In some embodiments, the parent AAV capsid is K449R AAV9 (SEQ ID NO: 11).

[0330] In some embodiments, a peptide, e.g., a targeting peptide, is inserted into the parent AAV capsid sequence between amino acids 454, 455, 457, 458, 459, 460, and / or 461 (loop IV).

[0331] In some embodiments, a peptide, e.g., a targeting peptide, is inserted into the parent AAV capsid sequence between amino acids 586, 587, 589, and / or 590 (loop VIII).

[0332] In some embodiments, a peptide, e.g., a targeting peptide, is inserted into loop IV of the parent AAV capsid sequence. As a non-limiting example, the parent AAV capsid may be AAV5. As a non-limiting example, the parent AAV capsid may be AAV9. As a non-limiting example, the parent AAV capsid may be AAV9hu.14 (SEQ ID NO: 137 or 138). As a non-limiting example, the parent AAV capsid may be AAV9 K449R (SEQ ID NO: 11). As a non-limiting example, the parent AAV capsid may be PHP.B (SEQ ID NO: 5 or 6). As a non-limiting example, the parent AAV capsid may be PHP.N (SEQ ID NO: 4). As a non-limiting example, the parent AAV capsid may be VOY101 (SEQ ID NO: 1 or 2). As a non-limiting example, the parent AAV capsid may be VOY201 (SEQ ID NO: 3 or 1724).

[0333] In some embodiments, a peptide, e.g., a targeting peptide, is inserted into loop VIII of the parent AAV capsid sequence. As a non-limiting example, the parent AAV capsid may be AAV5. As a non-limiting example, the parent AAV capsid may be AAV9. As a non-limiting example, the parent AAV capsid may be AAV9hu.14 (SEQ ID NO: 137 or 138). As a non-limiting example, the parent AAV capsid may be AAV9 K449R (SEQ ID NO: 11). As a non-limiting example, the parent AAV capsid may be PHP.B (SEQ ID NO: 5 or 6). As a non-limiting example, the parent AAV capsid may be PHP.N (SEQ ID NO: 4). As a non-limiting example, the parent AAV capsid may be VOY101 (SEQ ID NO: 1 or 2). As a non-limiting example, the parent AAV capsid may be VOY201 (SEQ ID NO: 3 or 1724).

[0334] In some embodiments, the peptide, e.g., a targeting peptide, is present in an AAV capsid polypeptide, e.g., in loop VIII of an AAV capsid variant. In some embodiments, the peptide of an AAV capsid variant described herein is present immediately after position 586 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the peptide of an AAV capsid variant described herein is present immediately after position 588 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the peptide of an AAV capsid variant described herein is present immediately after position 589 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0335] In some embodiments, an AAV capsid polypeptide described herein, e.g., an AAV capsid variant peptide, e.g., a targeting peptide, comprises the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), wherein the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) occurs immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0336] In some embodiments, an AAV capsid polypeptide described herein, e.g., an AAV capsid variant peptide, e.g., a targeting peptide, comprises the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654), wherein the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654) occurs immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0337] In some embodiments, an AAV capsid polypeptide described herein, e.g., an AAV capsid variant peptide, e.g., a targeting peptide, comprises the amino acid sequence of IVMNSLK (SEQ ID NO: 3651), wherein the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) occurs immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0338] In some embodiments, an AAV capsid polypeptide described herein, e.g., an AAV capsid variant peptide, e.g., a targeting peptide, comprises the amino acid sequence of any of SEQ ID NOs: 3649, 3650, 3652, 3653, or 3655-3659, wherein the amino acid sequence of any of said sequences occurs immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0339] A peptide, e.g., a targeting peptide, described herein may increase transduction of an AAV particle of the present disclosure (e.g., an AAV particle comprising an AAV capsid polypeptide, e.g., an AAV capsid variant) into a target tissue compared to a parent AAV particle lacking the peptide, e.g., targeting peptide, insert. In some embodiments, the peptide, e.g., a targeting peptide, increases transduction of the AAV particle into a target tissue by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more compared to a parent AAV particle lacking the peptide, e.g., targeting peptide, insert.

[0340] In some embodiments, the peptide, e.g., a targeting peptide, increases transduction of AAV particles (e.g., AAV particles comprising an AAV capsid polypeptide, e.g., an AAV capsid variant) into cells, regions, or tissues of the CNS (e.g., brain cells, brain tissue, brain regions, spinal cord cells, spinal cord regions, or spinal cord tissue) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more, compared to a parent AAV particle lacking the peptide, e.g., targeting peptide, insert. In some embodiments, the brain region comprises the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus. In some embodiments, the spinal cord region comprises the cervical, thoracic, and / or lumbar regions.

[0341] In some embodiments, the peptide, e.g., a targeting peptide, increases transduction of AAV particles (e.g., AAV particles comprising an AAV capsid polypeptide, e.g., an AAV capsid variant) into cells or tissues of the PNS by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more, compared to a parent AAV particle lacking the peptide, e.g., targeting peptide, insert.

[0342] In some embodiments, the peptide, e.g., a targeting peptide, increases transduction of AAV particles (e.g., AAV particles comprising an AAV capsid polypeptide, e.g., an AAV capsid variant) into DRG cells or tissues by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more, compared to a parent AAV particle lacking the peptide, e.g., targeting peptide, insert.

[0343] In some embodiments, the peptide, e.g., a targeting peptide, increases transduction of AAV particles (e.g., AAV particles comprising an AAV capsid polypeptide, e.g., an AAV capsid variant) into muscle cells, regions, or tissues by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 200%, 300%, 400%, 500%, or more, compared to parent AAV particles lacking the targeting peptide insert. In some embodiments, the muscle region may include myocardial, quadriceps, and / or diaphragm muscle regions. In some embodiments, the muscle region includes myocardial regions, e.g., atrial or ventricular muscle regions.

[0344] In some embodiments, the AAV particles described herein comprise an AAV capsid polypeptide, e.g., an AAV capsid polypeptide, e.g., an AAV capsid variant. In some embodiments, the AAV capsid variant comprises a peptide, e.g., a targeting peptide, sequence listed in Table 1 or 2.

[0345] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622. In some embodiments, the AAV capsid variant comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 3648-3659. In some embodiments, the amino acid sequence is located in Loop VIII. In some embodiments, the amino acid sequence is located immediately after position 586, 588, or 589 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0346] In some embodiments, three consecutive amino acids comprise PLN. In some embodiments, four consecutive amino acids comprise PLNG (SEQ ID NO: 3678). In some embodiments, five consecutive amino acids comprise PLNGA (SEQ ID NO: 3679). In some embodiments, six consecutive amino acids comprise PLNGAV (SEQ ID NO: 3680). In some embodiments, seven consecutive amino acids comprise PLNGAVH (SEQ ID NO: 3681). In some embodiments, eight consecutive amino acids comprise PLNGAVHL (SEQ ID NO: 3682). In some embodiments, nine consecutive amino acids comprise PLNGAVHLY (SEQ ID NO: 3648).

[0347] In some embodiments, three consecutive amino acids comprise YST. In some embodiments, four consecutive amino acids comprise YSTD (SEQ ID NO: 3690). In some embodiments, five consecutive amino acids comprise YSTDE (SEQ ID NO: 3691). In some embodiments, five consecutive amino acids comprise YSTDV (SEQ ID NO: 3700). In some embodiments, six consecutive amino acids comprise YSTDER (SEQ ID NO: 3692). In some embodiments, six consecutive amino acids comprise YSTDVR (SEQ ID NO: 3701). In some embodiments, seven consecutive amino acids comprise YSTDERM (SEQ ID NO: 3657). In some embodiments, seven consecutive amino acids comprise YSTDERK (SEQ ID NO: 3658). In some embodiments, seven consecutive amino acids comprise YSTDVRM (SEQ ID NO: 3650).

[0348] In some embodiments, three consecutive amino acids comprise IVM. In some embodiments, four consecutive amino acids comprise IVMN (SEQ ID NO: 3693). In some embodiments, five consecutive amino acids comprise IVMNS (SEQ ID NO: 3694). In some embodiments, six consecutive amino acids comprise IVMNS (SEQ ID NO: 3695). In some embodiments, seven consecutive amino acids comprise IVMNSL (SEQ ID NO: 3651).

[0349] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622. In some embodiments, the AAV capsid variant comprises an amino acid sequence that contains at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 3648-3659. In some embodiments, the amino acid sequence is located in Loop VIII. In some embodiments, the amino acid sequence is located immediately after position 586, 588, or 589 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0350] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), or an amino acid sequence having at least one, two, or three, but not more than four, modifications, e.g., substitutions, relative to the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), optionally with an H at position 7.

[0351] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of RDSPKGW (SEQ ID NO: 3649), or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RDSPKGW (SEQ ID NO: 3649).

[0352] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of IVMNSLK (SEQ ID NO: 3651).

[0353] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of YSTDVRM (SEQ ID NO: 3650), or an amino acid sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of YSTDVRM (SEQ ID NO: 3650).

[0354] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of RESPRGL (SEQ ID NO: 3652) or a sequence having at least one, two, or three modifications, but no more than four modifications, e.g., substitutions, relative to the amino acid sequence of RESPRGL (SEQ ID NO: 3652).

[0355] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of any of SEQ ID NOs: 1725-3622. In some embodiments, the AAV capsid variant comprises the amino acid sequence of any of SEQ ID NOs: 3648-3659. In some embodiments, the amino acid sequence is present in loop VIII of an AAV capsid variant described herein. In some embodiments, the amino acid sequence is present immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence is present immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0356] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., an AAV capsid variant described herein), comprises an amino acid sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, an AAV capsid described herein, e.g., an AAV capsid variant, comprises an amino acid sequence encoded by a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but not more than 10 modifications, of the nucleotide sequence of any one of SEQ ID NOs: 3660-3671.

[0357] In some embodiments, a nucleotide sequence encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, a nucleic acid sequence encoding an AAV capsid variant, e.g., an AAV capsid variant described herein, comprises a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660-3671.

[0358] In some embodiments, a nucleotide sequence encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of SEQ ID NO: 3660, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the nucleic acid sequence encoding an AAV capsid variant comprises a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3660.

[0359] In some embodiments, a nucleotide sequence encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., an AAV capsid variant described herein), comprises the nucleotide sequence of SEQ ID NO: 3663, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the nucleic acid sequence encoding an AAV capsid variant comprises a nucleotide sequence containing at least 1, 2, 3, 4, 5, 6, or 7 modifications, but no more than 10 modifications, of the nucleotide sequence of SEQ ID NO: 3663.

[0360] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid residue other than "A" at position 587 and / or an amino acid residue other than "Q" at position 588, numbered according to SEQ ID NO:138.

[0361] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), wherein the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) is located immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0362] In some embodiments, the polypeptide, e.g., the AAV capsid variant, comprises the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654), wherein the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654) is located immediately after position 586 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0363] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of IVMNSLK (SEQ ID NO: 3651), wherein the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) is located immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0364] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises the amino acid sequence of any of SEQ ID NOs: 3649, 3650, 3652, 3653, or 3655-3659, wherein the amino acid sequence of any of said sequences occurs immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138.

[0365] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, further comprises a substitution at position K449, e.g., a K449R substitution, numbered according to SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises a modification, e.g., an insertion, substitution, and / or deletion, in loops I, II, IV, and / or VI.

[0366] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, further comprises an amino acid sequence having at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, of the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variant further comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence that has at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid variant further comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence that has at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0367] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, of the present disclosure comprises a parent amino acid sequence with an insert, e.g., a peptide, e.g., a targeting peptide, wherein the insert comprises the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, the insert comprises at least 3, 4, 5, 6, 7, 8, or 9 contiguous amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659. In some embodiments, the insert comprises an amino acid sequence that includes at least 1, 2, or 3 modifications, but no more than 4 modifications, e.g., substitutions, relative to the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659.

[0368] In some embodiments, a parent sequence of an AAV capsid polypeptide described herein, e.g., an AAV capsid variant, comprises an amino acid sequence that contains at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the parent sequence comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the parent sequence further comprises a substitution at position K449, e.g., a K449R substitution. In some embodiments, the parent sequence comprises the nucleotide sequence of SEQ ID NO: 137, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the polynucleotide encoding the parent sequence described herein comprises the nucleotide sequence of SEQ ID NO: 137, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0369] In some embodiments, the parent sequence of an AAV capsid polypeptide, e.g., an AAV capsid variant described herein, comprises an amino acid sequence that includes at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 11, with the proviso that, e.g., position 449 of SEQ ID NO: 11 is not K, e.g., R. In some embodiments, the parent sequence of an AAV capsid variant described herein comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity), with the proviso that, e.g., position 449 of SEQ ID NO: 11 is not K, e.g., R.

[0370] In some embodiments, the insert of the AAV capsid variant is inserted into loop VIII of the parent amino acid sequences described herein, hi some embodiments, the insert is inserted immediately after position 586, 588, or 589 of the parent amino acid sequences described herein.

[0371] In some embodiments, the AAV capsid variant further comprises an amino acid other than "A" at position 587 and / or an amino acid other than "Q" at position 588 of the parental amino acid sequences described herein. In some embodiments, the AAV capsid variant further comprises a deletion at position 587 and / or a deletion at position 588 of the parental amino acid sequences described herein. In some embodiments, the AAV capsid variant comprises a deletion of amino acids "AQ" at positions 587-588 of the parental amino acid sequences described herein.

[0372] In some embodiments, the insert of an AAV capsid variant described herein comprises the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648). In some embodiments, the insert sequence of PLNGAVHLY (SEQ ID NO: 3648) is inserted immediately after position 586 of the parent amino acid sequence. In some embodiments, the AAV capsid variant further comprises a deletion of amino acids "AQ" at positions 587-588 of the parent amino acid sequence. In some embodiments, the AAV capsid variant comprises an insert comprising the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) inserted immediately after position 586 of the parent amino acid sequence, and a deletion of amino acids "AQ" at positions 587-588 of the parent amino acid sequence.

[0373] In some embodiments, an insert of an AAV capsid polypeptide described herein, e.g., an AAV capsid variant, comprises the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654). In some embodiments, the insert sequence of GGTLAVVSL (SEQ ID NO: 3654) is inserted immediately after position 586 of the parent amino acid sequence. In some embodiments, the AAV capsid variant further comprises a deletion of amino acids "AQ" at positions 587-588 of the parent amino acid sequence. In some embodiments, the AAV capsid variant comprises an insert comprising the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654) inserted immediately after position 586 of the parent amino acid sequence, and a deletion of amino acids "AQ" at positions 587-588 of the parent amino acid sequence.

[0374] In some embodiments, the insert of an AAV capsid polypeptide described herein, e.g., an AAV capsid variant, comprises the amino acid sequence of IVMNSLK (SEQ ID NO: 3651). In some embodiments, the insert sequence of IVMNSLK (SEQ ID NO: 3651) is inserted immediately after position 588 of the parent amino acid sequence. In some embodiments, the AAV capsid variant comprises an insert comprising the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) inserted immediately after position 589 of the parent amino acid sequence.

[0375] In some embodiments, the insert of an AAV capsid polypeptide described herein, e.g., an AAV capsid variant, comprises the amino acid sequence of RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656). In some embodiments, the RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656) insert sequence is inserted immediately after position 589 of the parent amino acid sequence. In some embodiments, the AAV capsid variant comprises an insert comprising the amino acid sequence of RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656) inserted immediately after position 589 of the parent amino acid sequence.

[0376] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, of the present disclosure comprises an amino acid sequence described herein, e.g., the amino acid sequence of an AAV capsid variant selected from TTD-001, TTD-002, TTD-003, TTD-004, TTD-005, TTD-006, TTD-007, TTD-008, TTD-009, TTD-010, TTD-011, or TTD-012, as described in Tables 3 and 4.

[0377] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid sequence described herein, e.g., a VP1, VP2, and / or VP3 protein, comprising the amino acid sequence of an AAV capsid variant selected from TTD-001, TTD-002, TTD-003, TTD-004, TTD-005, TTD-006, TTD-007, TTD-008, TTD-009, TTD-010, TTD-011, or TTD-012, as described in Tables 3 and 4.

[0378] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid sequence encoded by a nucleotide sequence as described herein, e.g., the nucleotide sequence of an AAV capsid variant selected from TTD-001, TTD-002, TTD-003, TTD-004, TTD-005, TTD-006, TTD-007, TTD-008, TTD-009, TTD-010, TTD-011, or TTD-012, as described in Tables 3 and 5.

[0379] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, of the present disclosure comprises a nucleotide sequence described herein, e.g., the nucleotide sequence of an AAV capsid variant selected from TTD-001, TTD-002, TTD-003, TTD-004, TTD-005, TTD-006, TTD-007, TTD-008, TTD-009, TTD-010, TTD-011, or TTD-012, as described in Tables 3 and 5.

[0380] In some embodiments, insertion of a nucleic acid sequence, targeting nucleic acid sequence, or peptide, e.g., a targeting peptide, into a parent AAV sequence generates non-limiting exemplary full-length capsid sequences, e.g., AAV capsid polypeptides, e.g., AAV capsid variants, such as those set forth in Tables 3, 4, and 5.

[0381] Table 3

[0382] Table 4-1

[0383] Table 4-2

[0384] Table 4-3

[0385] Table 4-4

[0386] Table 4-5

[0387] Table 5-1

[0388] Table 5-2

[0389] Table 5-3

[0390] Table 5-4

[0391] Table 5-5

[0392] Table 5-6

[0393] Table 5-7

[0394] Table 5-8

[0395] Table 5-9

[0396] Table 5-10

[0397] Table 5-11

[0398] Table 5-12

[0399] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide described herein, e.g., an AAV capsid variant, comprises the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence that has at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, a polynucleotide encoding an AAV capsid variant described herein comprises the nucleotide sequence of SEQ ID NO: 3623, or a nucleotide sequence that has at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, a polynucleotide encoding an AAV capsid variant described herein comprises the nucleotide sequence of SEQ ID NO: 3627, or a nucleotide sequence that has at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, nucleic acid sequences encoding the AAV capsid polypeptides described herein, eg, AAV capsid variants, are codon-optimized.

[0400] In some embodiments, the polynucleotide encoding the AAV capsid polypeptide of the AAV particle, e.g., an AAV capsid variant (e.g., VP1), is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 1 , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of the present invention.

[0401] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 80% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 85% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), is encoded by a nucleic acid sequence having at least 90% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 95% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 96% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 97% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 98% identity to any of SEQ ID NOs: 3623-3635. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise a nucleic acid sequence having at least 99% identity to any of SEQ ID NOs: 3623-3635.In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant (eg, VP1), may comprise a nucleic acid sequence provided by any of SEQ ID NOs: 3623-3635.

[0402] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3623. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 95% identity to SEQ ID NO: 3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 96% identity to SEQ ID NO: 3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 97% identity to SEQ ID NO: 3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 98% identity to SEQ ID NO: 3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 99% identity to SEQ ID NO: 3623.

[0403] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence comprising SEQ ID NO:3623. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence consisting of SEQ ID NO:3623.

[0404] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 95% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 96% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 97% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 98% identity to SEQ ID NO: 3624 or 3625. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 99% identity to SEQ ID NO: 3624 or 3625.

[0405] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence comprising SEQ ID NO: 3624 or 3625.

[0406] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence consisting of SEQ ID NO: 3624 or 3625.

[0407] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 95% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 96% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 97% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 98% identity to SEQ ID NO: 3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 99% identity to SEQ ID NO: 3626.

[0408] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence comprising SEQ ID NO:3626. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence consisting of SEQ ID NO:3626.

[0409] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 95% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 96% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 97% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 98% identity to SEQ ID NO: 3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 99% identity to SEQ ID NO: 3627.

[0410] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence comprising SEQ ID NO:3627. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence consisting of SEQ ID NO:3627.

[0411] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 85% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 90% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 95% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 96% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 97% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 98% identity to SEQ ID NO: 3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise a nucleic acid sequence having at least 99% identity to SEQ ID NO: 3628.

[0412] In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence comprising SEQ ID NO:3628. In some embodiments, a polynucleotide encoding an AAV capsid polypeptide, eg, an AAV capsid variant, may have a nucleic acid sequence consisting of SEQ ID NO:3628.

[0413] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises a VP2 protein comprising an amino acid sequence corresponding to positions 138-743 of any one of SEQ ID NOs: 3636-3647, or a sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, the AAV capsid comprises a VP3 protein comprising an amino acid sequence corresponding to positions 203-743 of any one of SEQ ID NOs: 3636-3647, or a sequence having at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto.

[0414] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, e.g., an AAV capsid variant described herein, comprises the amino acid sequence of any one of SEQ ID NOs: 3636-3647, or an amino acid sequence that has at least 80% (e.g., at least about 85, 90, 95, 96, 97, 98, or 99%) sequence identity thereto. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, e.g., an AAV capsid variant described herein, comprises an amino acid sequence having at least one, two, or three modifications, but not more than 30, 20, or 10 modifications, of the amino acid sequence of any one of SEQ ID NOs: 3636-3647.

[0415] In some embodiments, the AAV capsid polypeptide of the AAV particle, e.g., an AAV capsid variant (e.g., VP1), is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153 It may comprise an amino acid sequence that is 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence.

[0416] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 80% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid (e.g., VP1) may comprise an amino acid sequence having at least 85% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 90% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 95% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 96% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 97% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 98% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may comprise an amino acid sequence having at least 99% identity to any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may have an amino acid sequence comprising any of SEQ ID NOs: 3636-3647. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant (e.g., VP1), may have an amino acid sequence consisting of any of SEQ ID NOs: 3636-3647.

[0417] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 96% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 97% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 3636.

[0418] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, may have an amino acid sequence that includes SEQ ID NO: 3636. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3636.

[0419] In some embodiments, an AAV capsid polypeptide, eg, an AAV capsid variant, may have an amino acid sequence consisting of SEQ ID NO:3636. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence provided as SEQ ID NO: 3636 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3623. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence provided as SEQ ID NO: 3636 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3623, wherein the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 13 Encoded by a codon-optimized nucleic acid sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0420] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 3637. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 3637. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 3637. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 3637. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 96% identity to SEQ ID NO: 3637. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 97% identity to SEQ ID NO: 3637. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 3637. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 3637.

[0421] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, may have an amino acid sequence that includes SEQ ID NO: 3637. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3637.

[0422] In some embodiments, an AAV capsid polypeptide, eg, an AAV capsid variant, may have an amino acid sequence consisting of SEQ ID NO:3637. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence given as SEQ ID NO: 3637 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3624 or 3625. ... 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, Encoded by a codon-optimized nucleic acid sequence having 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0423] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 96% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 97% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 3638.

[0424] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, may have an amino acid sequence that includes SEQ ID NO: 3638. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3638.

[0425] In some embodiments, an AAV capsid polypeptide, eg, an AAV capsid variant, may have an amino acid sequence consisting of SEQ ID NO:3638. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence provided as SEQ ID NO: 3638 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3626. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence provided as SEQ ID NO: 3638 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3626, wherein the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 13 Encoded by a codon-optimized nucleic acid sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0426] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 96% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 97% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 3639.

[0427] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, may have an amino acid sequence that includes SEQ ID NO: 3639. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3639.

[0428] In some embodiments, an AAV capsid polypeptide, eg, an AAV capsid variant, may have an amino acid sequence consisting of SEQ ID NO:3639. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence given as SEQ ID NO: 3639 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3627. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence given as SEQ ID NO: 3639 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3627, wherein the amino acid sequence is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, Encoded by a codon-optimized nucleic acid sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0429] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 80% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 85% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 90% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 95% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 96% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 97% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 98% identity to SEQ ID NO: 3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, may comprise an amino acid sequence having at least 99% identity to SEQ ID NO: 3640.

[0430] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, may have an amino acid sequence that includes SEQ ID NO: 3640. In some embodiments, the AAV capsid variant comprises the amino acid sequence of SEQ ID NO: 3640.

[0431] In some embodiments, an AAV capsid polypeptide, eg, an AAV capsid variant, may have an amino acid sequence consisting of SEQ ID NO:3640. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence given as SEQ ID NO: 3640 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3628. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, comprising the amino acid sequence given as SEQ ID NO: 3640 is encoded by a nucleic acid sequence comprising SEQ ID NO: 3628, wherein the AAV capsid polypeptide, e.g., an AAV capsid variant, comprises an amino acid sequence that is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 13 Encoded by a codon-optimized nucleic acid sequence having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0432] In some embodiments, the AAV capsid polypeptides described herein, e.g., AAV capsid variants, have increased tropism for cells or tissues of the CNS, e.g., brain cells, brain tissue, spinal cord cells, or spinal cord tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138.

[0433] In some embodiments, the AAV capsid polypeptides described herein, e.g., AAV capsid variants, transduce a brain region selected from, e.g., the dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, and putamen, and in some embodiments, the level of transduction of the brain region is at least 5, 10, 50, 100, 200, 500, 1,000, 2,000, 5,000, or 10,000 times greater than the reference sequence of SEQ ID NO: 138.

[0434] In some embodiments, the AAV capsid polypeptides described herein, e.g., AAV capsid variants, are enriched in the brain by at least about 5, 6, 7, 8, 9, or 10-fold compared to the reference sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variants described herein are enriched in the brain by at least about 20, 30, 40, or 50-fold compared to the reference sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variants described herein are enriched in the brain by at least about 100, 200, 300, or 400-fold compared to the reference sequence of SEQ ID NO: 138.

[0435] In some embodiments, the AAV capsid polypeptides, e.g., AAV capsid variants described herein, deliver high levels of viral genomes to brain regions. In some embodiments, the level of viral genomes is increased by at least 5, 10, 20, 30, 40, or 50-fold compared to the reference sequence of SEQ ID NO: 138. In some embodiments, the brain region includes the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0436] In some embodiments, the AAV capsid polypeptides, e.g., AAV capsid variants described herein, deliver high levels of payload to brain regions. In some embodiments, the levels of payload are at least 5, 10, 50, 100, 200, 500, 1,000, 2,000, 5,000, or 10,000 times higher than the reference sequence of SEQ ID NO: 138. In some embodiments, the brain region includes the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

[0437] In some embodiments, the AAV capsid polypeptides described herein, e.g., AAV capsid variants, deliver high levels of payload to the spinal cord region. In some embodiments, the level of payload is at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 times higher compared to the reference sequence of SEQ ID NO: 138. In some embodiments, the spinal cord region includes the cervical, thoracic, and / or lumbar regions.

[0438] In some embodiments, the AAV capsid polypeptides described herein, eg, AAV capsid variants, exhibit preferential transduction in brain regions compared to transduction in the dorsal root ganglia (DRG).

[0439] In some embodiments, the AAV capsid polypeptides described herein, e.g., AAV capsid variants, have increased tropism for muscle cells or tissues, e.g., cardiac cells or tissues, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138. In some embodiments, the AAV capsid variants deliver increased levels of payload to muscle regions. In some embodiments, the payload is increased by at least 10, 15, 20, 30, or 40-fold relative to the reference sequence of SEQ ID NO: 138. In some embodiments, the muscle region comprises a region of myocardium, quadriceps, and / or diaphragm muscle. In some embodiments, the muscle region comprises a myocardial region, e.g., an atrial muscle region or a ventricular muscle region.

[0440] In some embodiments, the AAV capsid polypeptides, e.g., AAV capsid variants, of the present disclosure are isolated, e.g., recombinant. In some embodiments, the polynucleotides encoding the AAV capsid polypeptides, e.g., AAV capsid variants, of the present disclosure are isolated, e.g., recombinant.

[0441] In any of the DNA and RNA sequences referenced and / or described herein, the single letter symbols have the following explanations: A for adenine; C for cytosine; G for guanine; T for thymine; U for uracil; W for a weak base such as adenine or thymine; S for a strong nucleotide such as cytosine and guanine; M for an amino nucleotide such as adenine and cytosine; K for a keto nucleotide such as guanine and thymine; R for the purines adenine and guanine; Y for the pyrimidines cytosine and thymine; B for any base that is not A (e.g., cytosine, guanine, thymine); D for any base that is not C (e.g., adenine, guanine, thymine); H for any base that is not G (e.g., adenine, cytosine, thymine); V for any base that is not T (e.g., adenine, cytosine, guanine); N for any nucleotide (not a gap); and Z is zero.

[0442] In any of the amino acid sequences referred to and / or described herein, the single letter codes have the following explanation: G for glycine (Gly); A for alanine (Ala); L for leucine (Leu); M for methionine (Met); F for phenylalanine (Phe); W for tryptophan (Trp); K for lysine (Lys); Q for glutamine (Gln); E for glutamic acid (Glu); S for serine (Ser); P for proline (Pro); V for valine (Val); I for isoleucine (Ile); Ile); C for cysteine ​​(Cys); Y for tyrosine (Tyr); H for histidine (His); R for arginine (Arg); N for asparagine (Asn); D for aspartic acid (Asp); T for threonine (Thr); B for aspartic acid or asparagine (Asx); J for leucine or isoleucine (Xle); O for pyrrolysine (Pyl); U for selenocysteine ​​(Sec); X for any amino acid (Xaa); and Z for glutamine or glutamic acid (Glx).

[0443] Also provided herein are polynucleotide sequences encoding any of the above AAV capsid variants, as well as AAV particles, vectors, and cells containing same.

[0444] AAV serotypes and capsids In some embodiments, the AAV particles of the present disclosure may comprise or be derived from any natural or recombinant AAV serotype. AAV serotypes may differ in properties such as, but not limited to, packaging, tropism, transduction, and immunogenicity profiles. Without wishing to be bound by theory, it is believed that in some embodiments, AAV capsid proteins, e.g., AAV capsid variants, can modulate the tropism of AAV particles, e.g., direct the tropism of AAV particles to specific tissues.

[0445] In some embodiments, AAV particles may have capsid proteins, e.g., AAV capsid variants, and ITR sequences derived from the same parent serotype (e.g., AAV2 capsid and AAV2 ITRs). In other embodiments, AAV particles may be pseudotyped AAV particles derived from parent serotypes with different capsid proteins and ITR sequences (e.g., AAV9 capsid and AAV2 ITRs; AAV2 / 9).

[0446] The AAV particles of the present disclosure may include an AAV capsid protein, e.g., an AAV capsid mutant, with a peptide, e.g., a targeting peptide, inserted into the parent sequence. The parent capsid or parent serotype may include or be derived from any natural or recombinant AAV serotype. As used herein, a "parent" sequence is a nucleotide sequence or amino acid sequence into which a targeting sequence is inserted (e.g., a nucleotide insertion into a nucleic acid sequence or an amino acid sequence insertion into an amino acid sequence).

[0447] In certain embodiments, the parent AAV capsid nucleotide sequence, e.g., the parent nucleic acid sequence, is as set forth in SEQ ID NO: 137. In some embodiments, the nucleotide sequence of the parent AAV capsid comprises the nucleic acid sequence of SEQ ID NO: 137, or a nucleic acid sequence at least substantially identical thereto (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the amino acid sequence of the parent AAV capsid, e.g., the parent sequence, is encoded by the nucleic acid sequence of SEQ ID NO: 137, or a nucleic acid sequence at least substantially identical thereto (e.g., having at least about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0448] In some embodiments, the nucleotide sequence of the parent AAV capsid is the K449R variant of SEQ ID NO: 137, in which the codon encoding lysine at position 449 in the amino acid sequence (nucleotides 1345-1347) (e.g., AAA or AAG) is replaced with a codon encoding arginine (CGT, CGC, CGA, CGG, AGA, AGG). In some embodiments, the K449R variant has the same functionality as wild-type AAV9.

[0449] In some embodiments, the amino acid sequence of the parent AAV capsid, e.g., the parent amino acid sequence, is as set forth in SEQ ID NO:138. (SEQ ID NO: 138) In some embodiments, the parent sequence comprises an amino acid sequence that includes at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 138. In some embodiments, the parent sequence comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity). In some embodiments, the parent sequence includes a substitution at position K449, e.g., a K449R substitution.

[0450] In some embodiments, the amino acid sequence of the parent AAV capsid, e.g., the parent amino acid sequence, is as set forth in SEQ ID NO: 11. In some embodiments, the parent sequence comprises an amino acid sequence that includes at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the parent sequence comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity).

[0451] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be as described in Jackson et al. (Frontiers in Molecular Neuroscience, 9:154 (2016)), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the AAV serotype of the parent AAV capsid described herein is PHP.B or AAV9. In some embodiments, the AAV serotype of the parent AAV capsid or AAV capsid variant is paired with a synapsin promoter to enhance neuronal transduction compared to when a more ubiquitous promoter (e.g., CBA or CMV) is used.

[0452] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, is AAV9 or a variant thereof. In some embodiments, the AAV9 capsid comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the amino acid sequence of AAV9 is encoded by a nucleotide sequence comprising SEQ ID NO: 137. In some embodiments, the AAV9 capsid comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 138, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical. In some embodiments, the AAV9 capsid comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 137, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical.

[0453] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant or parent AAV capsid, is AAV9 K449R or a variant thereof. In some embodiments, the AAV9 K449R capsid comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the AAV9 K449R capsid comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 11, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% identical.

[0454] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, comprises an AAVDJ sequence. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, comprises an AAVDJ8 sequence. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, comprises an AAVrhlO sequence. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, comprises an AAVl sequence. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, comprises AAVF7 / HSC7 (SEQ ID NOs: 8 and 27 of WO2016049230). In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant or parent AAV capsid, comprises AAVF15 / HSC15 (SEQ ID NOs: 16 and 33 in WO2016049230). In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant or parent AAV capsid, comprises AAVF17 / HSC17 (SEQ ID NOs: 13 and 35 in WO2016049230). In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant or parent AAV capsid, comprises an AAV5 sequence. As a non-limiting example, the AAV5 sequence is SEQ ID NO: 4 in U.S. Patent No. 6,984,517, the contents of which are incorporated herein by reference in their entirety.

[0455] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid mutant, is capable of penetrating the blood-brain barrier after intravenous administration. In some embodiments, the AAV capsid, e.g., an AAV capsid mutant, is capable of penetrating the blood-brain barrier following focused ultrasound (FUS), e.g., focused ultrasound combined with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS combined with intravenous administration. In some embodiments, the AAV capsid, e.g., an AAV capsid mutant, is capable of increasing distribution to brain regions. In some embodiments, the brain regions include the frontal cortex, sensory cortex, motor cortex, caudate nucleus, dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, putamen, or a combination thereof. In some embodiments, the AAV capsid, e.g., an AAV capsid mutant, is capable of preferential transduction in brain regions compared to transduction in the dorsal root ganglion (DRG).

[0456] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, allows for increased distribution to spinal cord regions, hi some embodiments, the spinal cord regions include the cervical spinal cord region, the thoracic spinal cord region, and / or the lumbar spinal cord region.

[0457] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, is suitable for intramuscular administration and / or transduction of muscle fibers. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, allows for increased distribution to muscle regions. In some embodiments, the muscle region includes myocardium, quadriceps, diaphragm muscle regions, or a combination thereof. In some embodiments, the muscle region includes myocardium regions, e.g., atrial muscle regions or ventricular muscle regions.

[0458] In some embodiments, one or more targeting sequences are inserted into the parent AAV capsid sequence or AAV capsid polypeptide, for example, the sequence of an AAV capsid mutant.For example, one targeting sequence may be inserted into the parent AAV capsid sequence or the AAV capsid mutant sequence.For example, two targeting sequences may be inserted into the parent AAV capsid sequence or the AAV capsid mutant sequence.For example, three targeting sequences may be inserted into the parent AAV capsid sequence or the AAV capsid mutant sequence.For example, more than three targeting sequences may be inserted into the parent AAV capsid sequence or the AAV capsid mutant sequence.

[0459] In some embodiments, the AAV capsid polypeptides described herein, e.g., AAV capsid variants, comprise an insert, e.g., a parent AAV capsid sequence having an amino acid sequence as set forth in Table 1 or 2. In some embodiments, the parent sequence may comprise at least one, two, three, or more insert sequences.

[0460] Without wishing to be bound by theory, it is understood that the parent AAV capsid sequence or AAV capsid polypeptide, e.g., an AAV capsid variant, comprises a VP1 region. In some embodiments, the parent AAV capsid sequence or AAV capsid variant comprises a VP1 region, a VP2 region, and / or a VP3 region, or any combination thereof.

[0461] In some embodiments, the start codon for translation of the VP1 capsid protein of AAV, e.g., a capsid variant, may be CTG, TTG, or GTG, as described in U.S. Pat. No. 8,163,543, the contents of which are incorporated herein by reference in their entirety.

[0462] This disclosure refers to structural capsid proteins (including VP1, VP2, and VP3) encoded by capsid (Cap) genes. These capsid proteins form the outer protein shell (e.g., capsid) of viral vectors such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally contain a methionine (Met1) as the first amino acid in the peptide sequence relative to the initiation codon (AUG or ATG) of the corresponding Cap nucleotide sequence. However, cleavage of the first methionine (Met1) residue, or generally any first amino acid (AA1), after or during polypeptide synthesis by protein-processing enzymes such as Met-aminopeptidases is common. This "Met / AA-clipping" process often correlates with the corresponding acetylation of a second amino acid (e.g., alanine, valine, serine, threonine, etc.) in the polypeptide sequence. Met-clipping typically occurs in VP1 and VP3 capsid proteins, but can also occur in VP2 capsid proteins.

[0463] Incomplete Met / AA-clipping may result in a mixture of one or more (1, 2, or 3) VP capsid proteins that make up the viral capsid, some of which may contain the Met1 / AA1 amino acid (Met+ / AA+) and some of which may lack the Met1 / AA1 amino acid as a result of Met / AA-clipping (Met- / AA-). For further discussion regarding Met / AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017, Oct. 28(5):255-267; Hwang, et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010, February 19. 327(5968):973-977, the contents of each of which are incorporated herein by reference in their entireties.

[0464] According to the present disclosure, reference to a capsid protein, e.g., an AAV capsid variant, is not limited to either clipped (Met- / AA-) or unclipped (Met+ / AA+) and, in context, may refer to an individual capsid protein, a viral capsid composed of a mixture of capsid proteins, and / or a polynucleotide sequence (or fragment thereof) that encodes, describes, produces, or results in a capsid protein of the present disclosure. Direct reference to a capsid protein or capsid polypeptide (e.g., VP1, VP2, or VP2) may also include a VP capsid protein that includes the Met1 / AA1 amino acids (Met+ / AA+) and the corresponding VP capsid protein that lacks the Met1 / AA1 amino acids as a result of Met / AA- clipping (Met- / AA-).

[0465] Further, according to the present disclosure, reference to a particular SEQ ID NO: (whether protein or nucleic acid) that each comprises or encodes one or more (Met+ / AA+) capsid proteins that comprise the Met1 / AA1 amino acids should be understood to teach VP capsid proteins that lack the Met1 / AA1 amino acids, as any sequence (whether Met1 / AA1 or otherwise) that simply lacks the originally listed amino acids is readily apparent upon inspection of the sequence.

[0466] As a non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and that includes a "Met1" amino acid (Met+) encoded by an AUG / ATG start codon may also be understood to teach a VP1 polypeptide sequence that is 735 amino acids in length and that does not include the "Met1" amino acid of the 736 amino acid Met+ sequence (Met-). As a second non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and that includes an "AA1" amino acid (AA1+) encoded by an NNN start codon may also be understood to teach a VP1 polypeptide sequence that is 735 amino acids in length and that does not include the "AA1" amino acid of the 736 amino acid AA1+ sequence (AA1-).

[0467] Reference to a viral capsid formed from VP capsid proteins (e.g., reference to a particular AAV capsid serotype) can incorporate VP capsid proteins containing Met1 / AA1 amino acids (Met+ / AA1+), corresponding VP capsid proteins lacking Met1 / AA1 amino acids as a result of Met / AA1-clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-).

[0468] As non-limiting examples, AAV capsid serotypes can include VP1(Met+ / AA1+), VP1(Met- / AA1-), or a combination of VP1(Met+ / AA1+) and VP1(Met- / AA1-). AAV capsid serotypes can also include VP3(Met+ / AA1+), VP3(Met- / AA1-), or a combination of VP3(Met+ / AA1+) and VP3(Met- / AA1-), as well as any similar combination of VP2(Met+ / AA1) and VP2(Met- / AA1-).

[0469] Additional AAV sequences In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or a parent AAV capsid, is constructed or modified such that immediately following position 586, 588, or 589, numbered relative to SEQ ID NO: 138, the AAV capsid variant, or parent AAV capsid polypeptide, comprises at least 3, 4, 5, 6, 7, 8, or 9 consecutive amino acids of any of SEQ ID NOs: 1725-3622 or 3648-3659.

[0470] In some embodiments, an AAV capsid polypeptide described herein, e.g., an AAV capsid variant, or a parent AAV capsid, does not include an insert sequence having at least five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, which is located immediately after positions 586, 588, or 589 numbered relative to SEQ ID NO: 138 of any of SEQ ID NOs: 1-1724, as set forth in Table 6.

[0471] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, or parent AAV capsid, has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103 It may comprise an amino acid sequence that is 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of the present invention.

[0472] In any of the embodiments described herein, positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138 can be identified by providing a sequence comparison of a reference sequence and a query sequence, where the reference sequence is SEQ ID NO: 138, and identifying residues corresponding to positions in the query sequence that correspond to positions 586-594 in the reference sequence.

[0473] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, or parent AAV capsid, does not share any of the sequences described herein at positions other than the five consecutive amino acids corresponding to positions 586-594, numbered relative to SEQ ID NO: 138, with a sequence similar to any of the sequences described herein at 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 %, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence.

[0474] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be AAV9 at a position other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be AAV9hu.14 (SEQ ID NO: 137 or 138) at a position other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be AAV9K449R (SEQ ID NO: 11) at a position other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be PHP.B (SEQ ID NO: 5 or 6) at a position other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be PHP.N (SEQ ID NO: 4) at a position other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138. In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be VOY101 (SEQ ID NO: 1 or 2) at a position other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138. In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, or the parent AAV capsid, may be VOY201 (SEQ ID NO: 3 or 1724) at positions other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138.

[0475] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant or parent AAV capsid, may be AAV5 at positions other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138.

[0476] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has one or more of the following at positions other than the five consecutive amino acids corresponding to positions 586 to 594, numbered relative to SEQ ID NO: 138: VOY101, VOY201, AAVPHP.B (PHP.B), AAVPHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVT H1.1-35, AAVPHP.B2(PHP.B2), AAVPHP.B3(PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPH P.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AA VPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B-EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AA VPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPH P.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3(G2A3), AAVG2B4(G2B4), AAVG2B5(G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9 K449R, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-1 5、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AA V223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / rh.6、AAV3.1 / rh.61 .9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10 AV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、A AV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAV-DJ、AAV-DJ8、AAV3、AAV5、AAV2 rh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、AAVrh.65、AAVrh.55、AAVrh.47、AAVrh.69、AAVrh.45、AAVrh.59、AAVhu.12、AAVH6、AAVLK03、AAVH-1 / hu.1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh. R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5、AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AA Vhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23、AAVhu.2 AVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41 、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu. .48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu.57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AA Vhu.67、AAVhu.14 / 9、AAVhu.t19、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.1 8、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh.46、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.51、AAVrh.52、AAVrh. rh.53、AAVrh.54、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74、AAVrh.8R、AAVrh.8R. A586R mutation、AAVrh8R R533A mutation type、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhER1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAV hEr2.29、AAVhEr2.4、AAVhEr2.16、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhER1.23、AAVhEr3.1、AAV2.5T、AAV-PAEC、AAV-LK01、AAV-LK02、AAV-LK03、AAV-LK03 LK04、AAV-LK05、AAV-LK06、AAV-LK07、AAV-LK08、AAV-LK09、AAV-LK10、AAV-LK11、AAV-LK12、AAV-LK13、AAV-LK14、AAV-LK15、AAV-LK16、AAV-LK17、AAV-LK18 、AAV-LK19、AAV-PAEC2、AAV-PAEC4、AAV-PAEC6、AAV-PAEC7、AAV-PAEC8、AAV-PAEC11、AAV-PAEC12、AAV-2-pre-miRNA-101、AAV-8h、AAV-8b、AAV-h、AAV-b、AAV SM10-2、AAVシャッフル100-1、AAVシャッフル100-3、AAVシャッフル100-7、AAVシャッフル10-6、AAVシャッフル10-8、AAV SM10-1、AAV SM10-8、AAV SM100-3 SM100-10、BNP61AAV、BNP62AAV、BNP63AAV、AAVrh.50、AAVrh.43、AAVrh.62、AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, authentic AAV (ttAAV), UPENN AAV10, Japanese AAV10 serotype, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-E1, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-​CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV Clv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AA V CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAV CLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-M9、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV It may be selected from any one of CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and any variant thereof.

[0477] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has a sequence similar to that of AAV1 (SEQ ID NOs: 6 and 64 in US20030138772), AAV2 (SEQ ID NOs: 7 and 70 in US20030138772), AAV3 (SEQ ID NOs: 138), or AAV4 (SEQ ID NOs: 138), e.g., AAV5 (SEQ ID NOs: 138), AAV6 (SEQ ID NOs: 138), AAV7 (SEQ ID NOs: 138), AAV8 (SEQ ID NOs: 138), AAV9 (SEQ ID NOs: 138), AAV10 (SEQ ID NOs: 138), AAV11 (SEQ ID NOs: 138), AAV12 (SEQ ID NOs: 138), AAV13 (SEQ ID NOs: 138), AAV14 (SEQ ID NOs: 138), AAV15 (SEQ ID NOs: 138), AAV16 (SEQ ID NOs: 138), AAV17 (SEQ ID NOs: 138), AAV18 (SEQ ID NOs: 138), AAV19 (SEQ ID NOs: 138), AAV20 (SEQ ID NOs: 138), AAV21 (SEQ ID NOs: 138), AAV22 (SEQ ID NOs: 138), AAV23 (SEQ ID NOs: 138), AAV24 (SEQ ID NOs: 138), AAV25 (SEQ ID NOs: 138), AAV26 (SEQ ID NOs: 138), AAV27 (SEQ ID NOs: 138), AAV28 (SEQ ID NOs: AAV4 (SEQ ID NO: 63 in US20030138772), AAV5 (SEQ ID NO: 114 in US20030138772), AAV6 (SEQ ID NO: 65 in US20030138772), AAV7 (SEQ ID NOs: 1 to 3 in US20030138772), AAV8 (SEQ ID NOs: 4 and 95 in US20030138772), AAV9 (SEQ ID NOs: 5 and 100 in US20030138772), AAV10 (SEQ ID NO: 117 in US20030138772), AAV11 (SEQ ID NO: 118 in US20030138772), AAV12 (SEQ ID NO: 119 in US20030138772), AAVrh10 (amino acids 1 to 738 of SEQ ID NO: 81 in US20030138772), AAV16.3 (SEQ ID NO: 10 in US20030138772), AAV29.3 / bb.1 (SEQ ID NO: 11 in US20030138772), AAV AV29.4 (SEQ ID NO: 12 in US20030138772), AAV29.5 / bb.2 (SEQ ID NO: 13 in US20030138772), AAV1.3 (SEQ ID NO: 14 in US20030138772), AAV13.3 (SEQ ID NO: 15 in US20030138772), AAV24.1 (SEQ ID NO: 16 in US20030138772), AAV27.3 (SEQ ID NO: 17 in US20030138772), AAV7.2 (SEQ ID NO: 18 of US20030138772), AAVC1 (SEQ ID NO: 19 of US20030138772), AAVC3 (SEQ ID NO: 20 of US20030138772), AAVC5 (SEQ ID NO: 21 of US20030138772), AAVF1 (SEQ ID NO: 22 of US20030138772), AAVF3 (SEQ ID NO: 23 of US20030138772), AAVF5 (SEQ ID NO: 24 of US20030138772), AAVH6 (SEQ ID NO: 25 of US20030138772), AAVH2 (SEQ ID NO: 26 of US20030138772), AAV42-8 (SEQ ID NO: 27 in US20030138772), AAV42-15 (SEQ ID NO: 28 in US20030138772), AAV42-5b (SEQ ID NO: 29 in US20030138772), AAV42-1b (SEQ ID NO: 30 in US20030138772), AAV42-13 (SEQ ID NO: 31 in US20030138772), AAV42-3a (SEQ ID NO: 32 in US20030138772), AAV42-4 (SEQ ID NO: 33 in US20030138772), AAV42-5a (SEQ ID NO: 34 in US20030138772), AA AAV42-10 (SEQ ID NO: 35 in US20030138772), AAV42-3b (SEQ ID NO: 36 in US20030138772), AAV42-11 (SEQ ID NO: 37 in US20030138772), AAV42-6b (SEQ ID NO: 38 in US20030138772), AAV43-1 (SEQ ID NO: 39 in US20030138772), AAV43-5 (SEQ ID NO: 40 in US20030138772), AAV43-12 (SEQ ID NO: 41 in US20030138772), AAV43-20 (SEQ ID NO: 42 in US20030138772), AAV4 3-21 (SEQ ID NO: 43 in US20030138772), AAV43-23 (SEQ ID NO: 44 in US20030138772), AAV43-25 (SEQ ID NO: 45 in US20030138772), AAV44.1 (SEQ ID NO: 46 in US20030138772), AAV44.5 (SEQ ID NO: 47 in US20030138772), AAV223.1 (SEQ ID NO: 48 in US20030138772), AAV223.2 (SEQ ID NO: 49 in US20030138772), AAV223.4 (SEQ ID NO: 50 in US20030138772), AAV223.AAV223.5 (SEQ ID NO: 51 in US20030138772), AAV223.6 (SEQ ID NO: 52 in US20030138772), AAV223.7 (SEQ ID NO: 53 in US20030138772), AAVA3.4 (SEQ ID NO: 54 in US20030138772), AAVA3.5 (SEQ ID NO: 55 in US20030138772), AAVA3.7 (SEQ ID NO: 56 in US20030138772), AAVA3.3 (SEQ ID NO: 57 in US20030138772) The AAV vector may be or may have a sequence as described in U.S. Publication No. US20030138772, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV42.12 (SEQ ID NO: 57), AAV42.12 (SEQ ID NO: 58 of US20030138772), AAV44.2 (SEQ ID NO: 59 of US20030138772), AAV42-2 (SEQ ID NO: 9 of US20030138772), or variants thereof.

[0478] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, contains any of Cy5R1, Cy5R2, Cy5R3, Cy5R4, rh.13R, rh.37R2, rh.2R, rh.8R, rh.48.1, rh.48.2, rh.48.1.2, hu.44R1, hu.44R2, hu.44R3, hu.29R, ch.5R1, rh64R1, rh64R2, AAV6.2, AA Examples of AAV vectors include, but are not limited to, AAV2 (SEQ ID NOs: 7 and 23 in US20150159173), rh20 (SEQ ID NO: 1 in US20150159173), rh32 / 33 (SEQ ID NO: 2 in US20150159173), rh39 (SEQ ID NOs: 3, 20, and 36 in US20150159173), rh46 (SEQ ID NOs: 4 and 22 in US20150159173), rh73 (SEQ ID NO: 5 in US20150159173), rh74 (SEQ ID NO: 6 in US20150159173), and AAV vectors. US20150159173 SEQ ID NO: 6), AAV6.1 (US20150159173 SEQ ID NO: 29), rh.8 (US20150159173 SEQ ID NO: 41), rh.48.1 (US20150159173 SEQ ID NO: 44), hu.44 (US20150159173 SEQ ID NO: 45), hu.29 (US20150159173 SEQ ID NO: 42), hu.48 (US20150159173 SEQ ID NO: 38), rh54 (US20150159173 SEQ ID NO: 49), AAV2 (US20150159173 SEQ ID NO: 7), cy.5 (US20150159173 SEQ ID NO: 10), US20150159173 SEQ ID NOs: 8 and 24), rh.10 (US20150159173 SEQ ID NOs: 9 and 25), rh.13 (US20150159173 SEQ ID NOs: 10 and 26), AAV1 (US20150159173 SEQ ID NOs: 11 and 27), AAV3 (US20150159173 SEQ ID NOs: 12 and 28), AAV6 (US20150159173 SEQ ID NOs: 13 and 29), AAV7 (US20150159173 SEQ ID NOs: 14 and 30), AAV8 (US20150159173 SEQ ID NOs: 15 and 31), hu.13 (SEQ ID NOs: 16 and 32 in US20150159173), hu.26 (SEQ ID NOs: 17 and 33 in US20150159173), hu.37 (SEQ ID NOs: 18 and 34 in US20150159173), hu.53 (SEQ ID NOs: 19 and 35 in US20150159173), rh.43 (SEQ ID NOs: 21 and 37 in US20150159173), rh2 (SEQ ID NO: 39 in US20150159173), rh.37 (SEQ ID NO: 40 in US20150159173), rh.64 (SEQ ID NO: 41 in US20150159173), The rh.58 (SEQ ID NO: 48 of US20150159173) or rh.67 (SEQ ID NO: 49 of US20150159173), or may have a sequence as described in U.S. Publication No. US20150159173, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, ch.3 (SEQ ID NO: 43 of US20150159173), rh.48 (SEQ ID NO: 44 of US20150159173), ch.5 (SEQ ID NO: 46 of US20150159173), rh.67 (SEQ ID NO: 47 of US20150159173), rh.58 (SEQ ID NO: 48 of US20150159173), or variants thereof.

[0479] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Pat. No. 7,198,951, the contents of which are incorporated by reference in their entirety, such as, but not limited to, AAV9 (SEQ ID NOs: 1-3 of U.S. Pat. No. 7,198,951), AAV2 (SEQ ID NO: 4 of U.S. Pat. No. 7,198,951), AAV1 (SEQ ID NO: 5 of U.S. Pat. No. 7,198,951), AAV3 (SEQ ID NO: 6 of U.S. Pat. No. 7,198,951).

[0480] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may be or may have mutations in an AAV9 sequence described by N. Pulicherla et al. (Molecular Therapy 19(6):1070-1078 (2011), incorporated herein by reference in its entirety), such as, but not limited to, AAV9.9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, and AAV9.84, at positions other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138.

[0481] In some embodiments, an AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Pat. No. 6,156,303, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV3B (SEQ ID NOs: 1 and 10 of U.S. Pat. No. 6,156,303), AAV6 (SEQ ID NOs: 2, 7, and 11 of U.S. Pat. No. 6,156,303), AAV2 (SEQ ID NOs: 3 and 8 of U.S. Pat. No. 6,156,303), AAV3A (SEQ ID NOs: 4 and 9 of U.S. Pat. No. 6,156,303), or derivatives thereof.

[0482] In some embodiments, the AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Publication No. US20140359799, the contents of which are incorporated by reference in their entirety, such as, but not limited to, AAV8 (SEQ ID NO: 1 of US20140359799), AAVDJ (SEQ ID NOs: 2 and 3 of US20140359799), or variants thereof.

[0483] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may be AAVDJ or a variant thereof, e.g., AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology, 82(12):5887-5911 (2008), incorporated herein by reference in its entirety), at positions other than the five consecutive amino acids corresponding to positions 586-594, numbered relative to SEQ ID NO: 138. In some embodiments, the amino acid sequence of AAVDJ8 may include two or more mutations to remove the heparin-binding domain (HBD). In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may comprise the AAV-DJ sequence set forth as SEQ ID NO: 1 in U.S. Patent No. 7,588,722, the contents of which are incorporated by reference herein in their entirety, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138.

[0484] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may be or have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, the sequence of AAV4 as described in International Publication No. WO1998011244, the contents of which are incorporated by reference in their entirety, such as, but not limited to, AAV4 (SEQ ID NOs: 1-20 of WO1998011244).

[0485] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may be or may have mutations in the AAV2 sequence at positions other than five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138 to generate AAV2G9, as described in International Publication No. WO2014144229, and incorporated herein by reference in its entirety.

[0486] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has one or more of the following sequences at positions other than the five consecutive amino acids corresponding to positions 586 to 594, numbered relative to SEQ ID NO: 138: AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.6, AAVrh.12, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.25 / 42 AAVrh.15, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, and AAVrh14, for example, AAV3-3 (SEQ ID NO: 217 in WO2005033321), AAV1 (SEQ ID NOs: 219 and 202 in WO2005033321), AAV106.1 / hu.37 (SEQ ID NOs: 219 and 202 in WO2005033321), and AAVrh.15, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, and AAVrh14. SEQ ID NO: 10), AAV114.3 / hu.40 (SEQ ID NO: 11 of WO2005033321), AAV127.2 / hu.41 (SEQ ID NOs: 6 and 8 of WO2005033321), AAV128.3 / hu.44 (SEQ ID NO: 81 of WO2005033321), AAV130.4 / hu.48 (SEQ ID NO: 78 of WO2005033321), AAV145.1 / hu.53 (SEQ ID NO: 14 of WO2005033321), AAV146.1 / hu.54 (SEQ ID NO: 14 of WO2005033321), AAV147.1 / hu.55 (SEQ ID NO: 14 of WO2005033321), AAV148.1 / hu.56 (SEQ ID NO: 14 of WO2005033321), AAV149.1 / hu.57 (SEQ ID NO: 14 of WO2005033321), AAV149.1 / hu.58 (SEQ ID NO: 14 of WO2005033321), AAV149.1 / hu.59 ... Sequence numbers 176 and 177), AAV145.6 / hu.56 (SEQ ID NOs: 168 and 192 in WO2005033321), AAV16.12 / hu.11 (SEQ ID NOs: 153 and 57 in WO2005033321), AAV16.8 / hu.10 (SEQ ID NOs: 156 and 56 in WO2005033321), AAV161.10 / hu.60 (SEQ ID NO: 170 in WO2005033321), AAV161.6 / hu.61 (SEQ ID NO: 174 in WO2005033321), AAV1-7 / rh.48 (SEQ ID NO: 32 in WO2005033321), AAV1-8 / rh.49 (SEQ ID NOs: 103 and 25 in WO2005033321), AAV2 (SEQ ID NOs: 211 and 221 in WO2005033321), AAV2-15 / rh.62 (SEQ ID NOs: 33 and 114 in WO2005033321), AAV2-3 / rh.61 (SEQ ID NO: 21 in WO2005033321), AAV2-4 / rh.50 (SEQ ID NOs: 23 and 108 in WO2005033321), AAV2-5 / rh.51 (SEQ ID NOs: 104 and 22 in WO2005033321), AAV3.1 / hu.6 (SEQ ID NOs: 5 and 84 in WO2005033321), AAV3.1 / hu.9 (SEQ ID NOs: 155 and 58 in WO2005033321), AAV3-11 / rh.53 (SEQ ID NOs: 186 and 176 in WO2005033321), AAV3-3 (SEQ ID NO: 200 in WO2005033321), AAV 33.12 / hu.17 (SEQ ID NO: 4 in WO2005033321), AAV33.4 / hu.15 (SEQ ID NO: 50 in WO2005033321), AAV33.8 / hu.16 (SEQ ID NO: 51 in WO2005033321), AAV3-9 / rh.52 (SEQ ID NOs: 96 and 18 in WO2005033321), AAV4-19 / rh.55 (SEQ ID NO: 117 in WO2005033321), AAV4-4 (SEQ ID NOs: 201 and 218 in WO2005033321), AAV4-9 / rh.54 (SEQ ID NO: 116 in WO2005033321), AAV5 (SEQ ID NOs: 199 and 216 in WO2005033321), AAV52.1 / hu.20 (SEQ ID NO: 63 in WO2005033321), AAV52 / hu.19 (SEQ ID NO: 133 in WO2005033321), AAV5-22 / rh.58 (SEQ ID NO: 27 in WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 105 in WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 26 in WO2005033321), AAV58.2 / hu.25 (SEQ ID NO: 49 in WO2005033321), AAV6 (WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 106 in WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 26 in WO2005033321), AAV58.2 / hu.25 (SEQ ID NO: 49 in WO2005033321), AAV6 (WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 107 in WO2005033321), AAV5-3 / rh.57 (SEQ ID NO: 109 ... 3321), AAV7 (SEQ ID NOs: 222 and 213 of WO2005033321), AAV7.3 / hu.7 (SEQ ID NO: 55 of WO2005033321), AAV8 (SEQ ID NOs: 223 and 214 of WO2005033321), AAVH-1 / hu.1 (SEQ ID NO: 46 of WO2005033321), AAVH-5 / hu.3 (SEQ ID NO: 44 of WO2005033321), AAVhu.1 (SEQ ID NO: 144 of WO2005033321), AAVhu.10 (SEQ ID NO: 156 of WO2005033321), AAVhu.11 (SEQ ID NO: 153 in WO2005033321), AAVhu.12 (SEQ ID NO: 59 in WO2005033321), AAVhu.13 (SEQ ID NO: 129 in WO2005033321), AAVhu.14 / AAV9 (SEQ ID NOs: 123 and 3 in WO2005033321), AAVhu.15 (SEQ ID NO: 147 in WO2005033321), AAVhu.16 (SEQ ID NO: 148 in WO2005033321), AAVhu.17 (SEQ ID NO: 83 in WO2005033321), AAVhu.18 (SEQ ID NO: 149 in WO2005033321), AAVhu.19 (SEQ ID NO: 133 in WO2005033321), AAVhu.2 (SEQ ID NO: 143 in WO2005033321), AAVhu.20 (SEQ ID NO: 134 in WO2005033321), AAVhu.21 (SEQ ID NO: 135 in WO2005033321), AAVhu.22 (SEQ ID NO: 138 in WO2005033321), AAVhu.23.2 (SEQ ID NO: 137 in WO2005033321), AAVhu.24 (SEQ ID NO: 136 in WO2005033321), AAVhu.25 (SEQ ID NO: 146 in WO2005033321) , AAVhu.27 (SEQ ID NO: 140 in WO2005033321), AAVhu.29 (SEQ ID NO: 132 in WO2005033321), AAVhu.3 (SEQ ID NO: 145 in WO2005033321), AAVhu.31 (SEQ ID NO: 121 in WO2005033321), AAVhu.32 (SEQ ID NO: 122 in WO2005033321), AAVhu.34 (SEQ ID NO: 125 in WO2005033321), AAVhu.35 (SEQ ID NO: 164 in WO2005033321), AAVhu.37 (SEQ ID NO: 88 in WO2005033321), AA Vhu.39 (SEQ ID NO: 102 in WO2005033321), AAVhu.4 (SEQ ID NO: 141 in WO2005033321), AAVhu.40 (SEQ ID NO: 87 in WO2005033321), AAVhu.41 (SEQ ID NO: 91 in WO2005033321), AAVhu.42 (SEQ ID NO: 85 in WO2005033321), AAVhu.43 (SEQ ID NO: 160 in WO2005033321), AAVhu.44 (SEQ ID NO: 144 in WO2005033321), AAVhu.45 (SEQ ID NO: 127 in WO2005033321), AAVhu.AAVhu.46 (SEQ ID NO: 159 in WO2005033321), AAVhu.47 (SEQ ID NO: 128 in WO2005033321), AAVhu.48 (SEQ ID NO: 157 in WO2005033321), AAVhu.49 (SEQ ID NO: 189 in WO2005033321), AAVhu.51 (SEQ ID NO: 190 in WO2005033321), AAVhu.52 (SEQ ID NO: 191 in WO2005033321), AAVhu.53 (SEQ ID NO: 186 in WO2005033321), AAVhu.54 (SEQ ID NO: 188 in WO2005033321), AAVhu. AVhu.55 (SEQ ID NO: 187 in WO2005033321), AAVhu.56 (SEQ ID NO: 192 in WO2005033321), AAVhu.57 (SEQ ID NO: 193 in WO2005033321), AAVhu.58 (SEQ ID NO: 194 in WO2005033321), AAVhu.6 (SEQ ID NO: 84 in WO2005033321), AAVhu.60 (SEQ ID NO: 184 in WO2005033321), AAVhu.61 (SEQ ID NO: 185 in WO2005033321), AAVhu.63 (SEQ ID NO: 195 in WO2005033321) ), AAVhu.64 (SEQ ID NO: 196 in WO2005033321), AAVhu.66 (SEQ ID NO: 197 in WO2005033321), AAVhu.67 (SEQ ID NO: 198 in WO2005033321), AAVhu.7 (SEQ ID NO: 150 in WO2005033321), AAVhu.8 (SEQ ID NO: 12 in WO2005033321), AAVhu.9 (SEQ ID NO: 155 in WO2005033321), AAVLG-10 / rh.40 (SEQ ID NO: 14 in WO2005033321), AAVLG-4 / rh.38 (SEQ ID NO: 19 ...8 (SEQ ID NO: 12 in WO2005033321), AAVhu.9 (SEQ ID NO: 155 in WO2005033321), AAVLG-10 / rh.40 (SEQ ID NO: 14 in WO2005033321), AAVLG-4 / rh.38 (SEQ ID NO: 199 321), AAVLG-4 / rh.38 (SEQ ID NO: 7 in WO2005033321), AAVN721-8 / rh.43 (SEQ ID NO: 163 in WO2005033321), AAVN721-8 / rh.43 (SEQ ID NO: 43 in WO2005033321), AAVpi.1 (SEQ ID NO: 28 in WO2005033321), AAVpi.2 (SEQ ID NO: 30 in WO2005033321), AAVpi.3 (SEQ ID NO: 29 in WO2005033321), AAVrh.38 (SEQ ID NO: 86 in WO2005033321), AAVrh.40 (SEQ ID NO: 92 in WO2005033321), AAVrh.43 (SEQ ID NO: 163 in WO2005033321), AAVrh.44 (SEQ ID NO: 34 in WO2005033321), AAVrh.45 (SEQ ID NO: 41 in WO2005033321), AAVrh.47 (SEQ ID NO: 38 in WO2005033321), AAVrh.48 (SEQ ID NO: 115 in WO2005033321), AAVrh. 49 (SEQ ID NO: 103 in WO2005033321), AAVrh.50 (SEQ ID NO: 108 in WO2005033321), AAVrh.51 (SEQ ID NO: 104 in WO2005033321), AAVrh.52 (SEQ ID NO: 96 in WO2005033321), AAVrh.53 (SEQ ID NO: 97 in WO2005033321), AAVrh.55 (SEQ ID NO: 37 in WO2005033321), AAVrh. AAVrh.56 (SEQ ID NO: 152 in WO2005033321), AAVrh.57 (SEQ ID NO: 105 in WO2005033321), AAVrh.58 (SEQ ID NO: 106 in WO2005033321), AAVrh.59 (SEQ ID NO: 42 in WO2005033321), AAVrh.60 (SEQ ID NO: 31 in WO2005033321), AAVrh.61 (SEQ ID NO: 107 in WO2005033321), AAVrh. h.62 (SEQ ID NO: 114 in WO2005033321), AAVrh.64 (SEQ ID NO: 99 in WO2005033321), AAVrh.65 (SEQ ID NO: 35 in WO2005033321), AAVrh.68 (SEQ ID NO: 16 in WO2005033321), AAVrh.69 (SEQ ID NO: 39 in WO2005033321), AAVrh.70 (SEQ ID NO: 20 in WO2005033321), AAVrh.72 (SEQ ID NO: 9 of WO2005033321), or variants thereof, the contents of which are incorporated herein by reference in their entirety. Non-limiting examples of variants include SEQ ID NOs: 13, 15, 17, 19, 24, 36, 40, 45, 47, 48, 51-54, 60-62, 64-77, 79, 80, 82, 89, 90, 93-95, 98, 100, 101, 109-113, 118-120, 124, 126, 131, 139, 142, 151, 154, 158, 161, 162, 165-183, 202, 204-212, 215, 219, and 224-236 of WO2005033321, the contents of which are incorporated herein by reference in their entirety.

[0487] In some embodiments, the AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may have, at positions other than the five consecutive amino acids corresponding to positions 586 to 594 numbered relative to SEQ ID NO: 138, a sequence as described in International Publication No. WO2015168666, the contents of which are incorporated by reference in their entirety, such as, but not limited to, AAVrh8R (SEQ ID NO: 9 of WO2015168666), the A586R mutant of AAVrh8R (SEQ ID NO: 10 of WO2015168666), the R533A mutant of AAVrh8R (SEQ ID NO: 11 of WO2015168666), or variants thereof.

[0488] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has an amino acid sequence other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, e.g., AAVhE1.1 (SEQ ID NO: 44 of US9233131), AAVhEr1.5 (SEQ ID NO: 45 of US9233131), AAVhER1.14 (SEQ ID NO: 46 of US9233131), AAVhER1.15 (SEQ ID NO: 47 of US9233131), AAVhER1.16 (SEQ ID NO: 48 of US9233131), AAVhER1.17 (SEQ ID NO: 49 of US9233131), AAVhER1.18 (SEQ ID NO: 50 of US9233131), AAVhER1.19 (SEQ ID NO: 51 of US9233131), AAVhER1.20 (SEQ ID NO: 52 of US9233131), AAVhER1.21 (SEQ ID NO: 53 of US9233131), AAVhER1.22 (SEQ ID NO: 54 of US9233131), AAVhER1.23 (SEQ ID NO: 55 of US9233131), AAVhER1.24 (SEQ ID NO: 56 of US9233131), AAVhER1.25 (SEQ ID NO: 57 of US9233131), AAVhER1.26 (SEQ ID NO: 58 of US9233131), AAVhER1.27 (SEQ ID NO: 59 of US9233131), AAVhER1 131), AAVhEr1.8 (SEQ ID NO: 47 of US9233131), AAVhEr1.16 (SEQ ID NO: 48 of US9233131), AAVhEr1.18 (SEQ ID NO: 49 of US9233131), AAVhEr1.35 (SEQ ID NO: 50 of US9233131), AAVhEr1.7 (SEQ ID NO: 51 of US9233131), AAVhEr1.36 (SEQ ID NO: 52 of US9233131), ), AAVhEr2.29 (SEQ ID NO: 53 of US9233131), AAVhEr2.4 (SEQ ID NO: 54 of US9233131), AAVhEr2.16 (SEQ ID NO: 55 of US9233131), AAVhEr2.30 (SEQ ID NO: 56 of US9233131), AAVhEr2.31 (SEQ ID NO: 58 of US9233131), AAVhEr2.36 (SEQ ID NO: 57 of US9233131), AAVhER The AAV vector may be or may have a sequence as described in U.S. Pat. No. 9,233,131, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAVhEr3.1.23 (SEQ ID NO: 53 of U.S. Pat. No. 9,233,131), AAVhEr3.1 (SEQ ID NO: 59 of U.S. Pat. No. 9,233,131), AAV2.5T (SEQ ID NO: 42 of U.S. Pat. No. 9,233,131), or variants thereof.

[0489] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has a sequence similar to that of AAV-PAEC (SEQ ID NO: 1 in US20150376607), AAV-LK01 (SEQ ID NO: 2 in US20150376607), AAV-LK02 (SEQ ID NO: 3 in US20150376607), AAV-LK03 (SEQ ID NO: 4 in US20150376607), AAV-LK04 (SEQ ID NO: 5 in US20150376607), ...LK05 (SEQ ID NO: 6 in US20150376607), AAV-LK06 (SEQ ID NO: 7 in US20150376607), AAV-LK07 (SEQ ID NO: 8 in US20150376607), AAV-LK08 (SEQ ID NO: 9 in US20150376607), AAV-LK09 (SEQ ID NO: 10 in US20150376607), AAV-LK10 (SEQ ID NO: 11 in US20150376607), AAV-LK11 (S AAV-LK05 (SEQ ID NO: 5 in US20150376607), AAV-LK06 (SEQ ID NO: 7 in US20150376607), AAV-LK07 (SEQ ID NO: 8 in US20150376607), AAV-LK08 (SEQ ID NO: 9 in US20150376607), AAV-LK09 (SEQ ID NO: 10 in US20150376607), AAV-LK10 (SEQ ID NO: 11 in US20150376607), AAV-LK11 (SEQ ID NO: 12 in US20150376607), AAV-LK12 (SEQ ID NO: 13 in US20150376607), AAV-LK13 (SEQ ID NO: 14 in US20150376607), AAV-LK14 (SEQ ID NO: 15 in US20150376607), AAV-LK15 (SEQ ID NO: 16 in US20150376607), AAV-LK16 (SEQ ID NO: 17 in US20150376607), AAV-LK17 (SEQ ID NO: 18 in US20150376607), AAV-LK18 (SEQ ID NO: 19 in US20150376607), AAV-LK19 (SEQ ID NO: 20 in US20150376607), AAV-LK20 (SEQ ID NO: 21 in US20150376607), AAV-LK21 (SEQ ID NO: 22 in US20150376607), AAV-LK 0376607), AAV-LK13 (SEQ ID NO: 14 of US20150376607), AAV-LK14 (SEQ ID NO: 15 of US20150376607), AAV-LK15 (SEQ ID NO: 16 of US20150376607), AAV-LK16 (SEQ ID NO: 17 of US20150376607), AAV-LK17 (SEQ ID NO: 18 of US20150376607), AAV-LK18 (SEQ ID NO: 19 of US20150376607), AAV-LK19 (SEQ ID NO: 20 of US20150376607), AAV-PAEC2( US20150376607), AAV-PAEC4 (SEQ ID NO: 21 in US20150376607), AAV-PAEC6 (SEQ ID NO: 23 in US20150376607), AAV-PAEC7 (SEQ ID NO: 24 in US20150376607), AAV-PAEC8 (SEQ ID NO: 25 in US20150376607), AAV-PAEC11 (SEQ ID NO: 26 in US20150376607), AAV-PAEC12 (SEQ ID NO: 27 in US20150376607), or variants thereof,It may be or have an arrangement as described in U.S. Patent Publication No. US20150376607, the contents of which are incorporated herein by reference in their entirety.

[0490] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Pat. No. 9,163,261, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAV-2-pre-miRNA-101 (SEQ ID NO: 1 of U.S. Pat. No. 9,163,261) or a variant thereof.

[0491] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Patent Publication No. US20150376240, the contents of which are incorporated by reference in their entirety, such as, but not limited to, AAV-8h (SEQ ID NO: 6 of US20150376240), AAV-8b (SEQ ID NO: 5 of US20150376240), AAV-h (SEQ ID NO: 2 of US20150376240), AAV-b (SEQ ID NO: 1 of US20150376240), or a variant thereof.

[0492] In some embodiments, the AAV capsid polypeptide, e.g., the AAV capsid variant, or the parent AAV capsid, contains a sequence other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, e.g., AAV SM10-2 (SEQ ID NO: 22 in US20160017295), AAVshuffle100-1 (SEQ ID NO: 23 in US20160017295), AAVshuffle100-3 (SEQ ID NO: 24 in US20160017295), AAVshuffle100-7 (SEQ ID NO: 25 in US20160017295), AAVshuffle10-2 (SEQ ID NO: 34 in US20160017295), AAVshuffle10-6 (SEQ ID NO: 35 in US20160017295), AAVshuffle10-8 (SEQ ID NO: 36 in US20160017295), AAVshuffle100-2 (SEQ ID NO: 37 in US20160017295), AAV The sequence may be or may have a sequence described in U.S. Patent Publication No. US20160017295, the contents of which are incorporated by reference in their entirety, such as, but not limited to, SM10-1 (SEQ ID NO: 38 of US20160017295), AAV SM10-8 (SEQ ID NO: 39 of US20160017295), AAV SM100-3 (SEQ ID NO: 40 of US20160017295), AAV SM100-10 (SEQ ID NO: 41 of US20160017295), or variants thereof.

[0493] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Patent Publication No. US20150238550, the contents of which are incorporated by reference in their entirety, such as, but not limited to, BNP61 AAV (SEQ ID NO: 1 of US20150238550), BNP62 AAV (SEQ ID NO: 3 of US20150238550), BNP63 AAV (SEQ ID NO: 4 of US20150238550), or a variant thereof.

[0494] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has a sequence similar to that of AAVrh.50 (SEQ ID NO: 108 of US20150315612), AAVrh.43 (SEQ ID NO: 163 of US20150315612), AAVrh.62 (SEQ ID NO: 1 of US20150315612), AAVrh.70 (SEQ ID NO: 1 of US20150315612), AAVrh.80 (SEQ ID NO: 1 of US20150315612), AAVrh.90 (SEQ ID NO: 1 of US20150315612), AAVrh.100 (SEQ ID NO: 1 of US20150315612), AAVrh.110 (SEQ ID NO: 1 of US20150315612), AAVrh.120 (SEQ ID NO: 1 of US20150315612), AAVrh.130 (SEQ ID NO: 1 of US20150315612), AAVrh.140 (SEQ ID NO: 1 of US20150315612), AAVrh.150 (SEQ ID NO: 1 of US20150315612), AAVrh.160 (SEQ ID NO: 1 of US20150315612), AAVrh.170 (SEQ ID NO: 1 of US20150315612), AAVrh.180 (SEQ ID NO: 1 of US20150315612), AAVrh.190 (SEQ ID NO: 1 of US20150315612), AAVrh.20 (SEQ 14), AAVrh.48 (SEQ ID NO: 115 in US20150315612), AAVhu.19 (SEQ ID NO: 133 in US20150315612), AAVhu.11 (SEQ ID NO: 153 in US20150315612), AAVhu.53 (SEQ ID NO: 186 in US20150315612), AAV4-8 / rh.64 (SEQ ID NO: 15 in US20150315612), AAVLG-9 / hu.39 (SEQ ID NO: 24 ... ), AAV54.5 / hu.23 (SEQ ID NO: 60 in US20150315612), AAV54.2 / hu.22 (SEQ ID NO: 67 in US20150315612), AAV54.7 / hu.24 (SEQ ID NO: 66 in US20150315612), AAV54.1 / hu.21 (SEQ ID NO: 65 in US20150315612), AAV54.4R / hu.27 (SEQ ID NO: 64 in US20150315612), AAV46.2 / hu.28 (US20 The vector may be or may have a sequence as described in U.S. Patent Publication No. US20150315612, the contents of which are incorporated by reference in their entirety, such as, but not limited to, AAV46.6 / hu.29 (SEQ ID NO: 68 of US20150315612), AAV46.6 / hu.29 (SEQ ID NO: 69 of US20150315612), AAV128.1 / hu.43 (SEQ ID NO: 80 of US20150315612), or variants thereof.

[0495] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, may have, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, a sequence as described in International Publication No. WO2015121501, the contents of which are incorporated by reference in their entirety, such as, but not limited to, true AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501), "UPenn AAV10" (SEQ ID NO: 8 of WO2015121501), "Japanese AAV10" (SEQ ID NO: 9 of WO2015121501), or a variant thereof.

[0496] According to the present disclosure, AAV capsid polypeptides, e.g., AAV capsid variants, or parent AAV capsids, may be selected from or derived from various species. In some embodiments, the AAV capsid polypeptide, e.g., AAV capsid variants, or parent AAV capsids may be avian AAV (AAAV) at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138. In some embodiments, the AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may be or have a sequence as described in U.S. Pat. No. 9,238,800, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, AAAV (SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Pat. No. 9,238,800), or a variant thereof, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138.

[0497] In some embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be or have a sequence as described in U.S. Pat. No. 9,193,769, the contents of which are incorporated by reference in their entirety, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, such as, but not limited to, BAAV (SEQ ID NOs: 1 and 6 of U.S. Pat. No. 9,193,769), or variants thereof. In some embodiments, an AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may be a BAAV serotype comprising a sequence as described in U.S. Pat. No. 7,427,396, the contents of which are incorporated by reference in their entirety, such as, but not limited to, BAAV (SEQ ID NOs: 5 and 6 of U.S. Pat. No. 7,427,396), or variants thereof, at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138.

[0498] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be a caprine AAV. In some embodiments, the AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may be or have a caprine AAV serotype that includes, at positions other than the five consecutive amino acids corresponding to positions 586-594, numbered relative to SEQ ID NO: 138, a sequence as described in U.S. Pat. No. 7,427,396, the contents of which are incorporated herein by reference in their entirety, such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Pat. No. 7,427,396) or a variant thereof.

[0499] In other embodiments, an AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, may be engineered as a hybrid AAV from two or more serotypes, e.g., parent serotypes. In some embodiments, the AAV serotype, parent AAV capsid polypeptide, or AAV capsid variant may be AAV2G9, which includes sequences from AAV2 and AAV9 at positions other than the five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, e.g., the AAV2G9 AAV serotype may be or have a sequence as described in U.S. Patent Publication No. US20160017005, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has a sequence similar to that described in Pulicherla et al. (Molecular The serotype may be generated by an AAV9 capsid library with mutations at amino acids 390 to 627 (VP1 numbering) as described by J. Med. Therapy, 19(6):1070-1078 (2011). The serotypes and corresponding nucleotide and amino acid substitutions are: AAV9.1 (G1594C; D532H), AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y), AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G, C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1 203A, G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T, A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A172 0T;N457H, T574S), AAV9.14(T1340A, T1362C, T1560C, G1713A;L447H), AAV9.16(A1775T;Q592L), AAV9.24(T1507C, T1521G;W503R) , AAV9.26 (A1337G, A1769C; Y446C, Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D), AAV9.35 (A1289T, T1450A) , C1494T, A1515T, C1794A, G1816A; Q430L, Y484N, N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S), AAV9. 44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T; N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W5 09R, L517V), 9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I), AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A; Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S 469R, A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A; F535L), AAV9. 58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H), AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T;L511I), AAV9.65(C1335T, T1530C, C1568A;A523D), AAV9.68(C1510A;P504T), A AV9.80(G1441A,;G481R), AAV9.83(C1402A, A1500T;P468T, E500D), AAV9.87(T1464C, T1468C;S 490P), AAV9.90(A1196T;Y399F), AAV9.91(T1316G, A1583T, C1782G, T1806C;L439R, K528I), AAV The nucleotide sequence may be, but is not limited to, AAV9.93 (A1273G, A1421G, A1638C, C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R, T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

[0500] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant or parent AAV capsid, has an amino acid sequence other than five consecutive amino acids corresponding to positions 586-594 numbered relative to SEQ ID NO: 138, e.g., AAVF1 / HSC1 (SEQ ID NOs: 2 and 20 of WO2016049230), AAVF2 / HSC2 (SEQ ID NOs: 3 and 21 of WO2016049230), AAVF3 / HSC3 (SEQ ID NOs: 3 and 22 of WO2016049230), AAVF4 / HSC5 (SEQ ID NOs: 3 and 43 of WO2016049230), AAVF5 / HSC6 (SEQ ID NOs: 3 and 44 of WO2016049230), AAVF6 / HSC7 (SEQ ID NOs: 3 and 45 of WO2016049230), AAVF7 / HSC8 (SEQ ID NOs: 3 and 46 of WO2016049230), AAVF8 / HSC9 (SEQ ID NOs: 3 and 47 of WO2016049230), AAVF9 / HSC10 (SEQ ID NOs: 3 and 48 of WO2016049230), AAVF11 / HSC12 (SEQ ID NOs: 3 and 49 of WO2016049230), AAVF12 / HSC13 (SEQ ID NOs: 3 and 49 of WO2016049230), AAVF13 / HSC14 (SEQ ID NOs: 3 and 49 of WO2016049230), AAVF14 / HSC15 ( 0), AAVF4 / HSC4 (SEQ ID NOs: 6 and 23 of WO2016049230), AAVF5 / HSC5 (SEQ ID NOs: 11 and 25 of WO2016049230), AAVF6 / HSC6 (SEQ ID NOs: 7 and 24 of WO2016049230), AAVF7 / HSC7 (SEQ ID NOs: 8 and 27 of WO2016049230), AAVF8 / HSC8 (SEQ ID NOs: 9 and 28 of WO2016049230), AAVF9 / HSC9 (WO AAVF11 / HSC11 (SEQ ID NOs: 10 and 29 in WO2016049230), AAVF11 / HSC11 (SEQ ID NOs: 4 and 26 in WO2016049230), AAVF12 / HSC12 (SEQ ID NOs: 12 and 30 in WO2016049230), AAVF13 / HSC13 (SEQ ID NOs: 14 and 31 in WO2016049230), AAVF14 / HSC14 (SEQ ID NOs: 15 and 32 in WO2016049230), AAVF15 / HSC15 (SEQ ID NOs: 16 and 33 in WO2016049230), AAVF16 / HSC16 (SEQ ID NOs: 17 and 34 in WO2016049230), AAVF17 / HSC17 (SEQ ID NOs: 18 and 35 in WO2016049230), AAVF18 / HSC18 (SEQ ID NOs: 19 and 36 in WO2016049230), AAVF19 / HSC19 (SEQ ID NOs: 20 and 37 in WO2016049230), AAVF19 / HSC19 (SEQ ID NOs: 21 and 22 in WO2016049230), AAVF19 / HSC20 (SEQ ID NOs: 23 and 24 in WO2016049230), AAVF19 / HSC20 (SEQ ID NOs: 25 and 36 in WO2016049230), AAVF19 / HSC21 (SEQ ID NOs: The AAVF16 / HSC16 (SEQ ID NOs: 16 and 33), AAVF16 / HSC16 (SEQ ID NOs: 17 and 34 of WO2016049230), AAVF17 / HSC17 (SEQ ID NOs: 13 and 35 of WO2016049230), or variants or derivatives thereof, may be or have a sequence as described in International Publication No. WO2016049230, the contents of which are incorporated herein by reference in their entirety.

[0501] In some embodiments, the AAV capsid polypeptide, e.g., an AAV capsid variant, or parent AAV capsid, has a sequence similar to that of AAV CBr-El (SEQ ID NOs: 13 and 87 of US8734809), AAV CBr-E2 (SEQ ID NOs: 14 and 88 of US8734809), AAV CBr-E3 (SEQ ID NOs: 15 and 89 of US8734809), AAV CBr-E4 (SEQ ID NOs: 16 and 90 of US8734809), AAV CBr-E5 (SEQ ID NOs: 17 and 91 of US8734809), AAV CBr-e5 (SEQ ID NOs: 18 and 92 of US8734809), AAV CBr-E6 (SEQ ID NOs: 19 and 93 of US8734809), AAV CBr-E7 (SEQ ID NOs: 19 and 94 of US8734809), AAV CBr-E8 (SEQ ID NOs: 20 and 21 of US8734809), AAV CBr-E9 (SEQ ID NOs: 22 and 23 of US8734809), AAV CBr-E10 (SEQ ID NOs: 23 and 24 of US8734809), AAV CBr-E11 (SEQ ID NOs: 13 and 87 of US8734809), AAV CBr-E2 (SEQ ID NOs: 14 and 88 of US8734809), AAV CBr-E3 (SEQ ID NOs: 15 and 89 of US8734809), AAV CBr-E4 (SEQ ID NOs: 16 and 90 of US8734809), AAV CBr- CBr-E7 (SEQ ID NOs: 20 and 94 in US8734809), AAV CBr-E8 (SEQ ID NOs: 21 and 95 in US8734809), AAV CLv-D1 (SEQ ID NOs: 22 and 96 in US8734809), AAV CLv-D2 (SEQ ID NOs: 23 and 97 in US8734809), AAV CLv-D3 (SEQ ID NOs: 24 and 98 in US8734809), AAV CLv-D4 (SEQ ID NOs: 25 and 99 in US8734809), AAV CLv-D5 (SEQ ID NOs: 26 and 100 in US8734809), AAV CLv-D6 (SEQ ID NOs: 27 and 101 in US8734809), AAV CLv-D7 (SEQ ID NOs: 28 and 102 in US8734809), AAV CLv-D8 (SEQ ID NOs: 29 and 103 in US8734809), AAV CLv-E1 (SEQ ID NOs: 13 and 87 in US8734809), AAV CLv-R1 (SEQ ID NOs: 30 and 104 in US8734809), AAV CLv-R2 (SEQ ID NOs: 31 and 105 in US8734809), AAV CLv-R3 (SEQ ID NOs: 32 and 106 in US8734809), AAV CLv-R4 (SEQ ID NOs: 33 and 107 in US8734809), AAV CLv-R5 (SEQ ID NOs: 34 and 108 in US8734809), AAV CLv-R6 (SEQ ID NOs: 35 and 109 in US8734809), AAV CLv-R7 (SEQ ID NOs: 36 and 110 in US8734809), AAV CLv-R8 (SEQ ID NOs: X and X in US8734809), AAVCLv-R9 (SEQ ID NOs: X and X in US8734809), AAV CLg-F1 (SEQ ID NOs: 39 and 113 in US8734809), AAV CLg-F2 (SEQ ID NOs: 40 and 114 in US8734809), AAV CLg-F3 (SEQ ID NOs: 41 and 115 in US8734809), AAV CLg-F4 (SEQ ID NOs: 42 and 116 in US8734809), AAV CLg-F5 (SEQ ID NOs: 43 and 117 in US8734809), AAV CLg-F6 (SEQ ID NOs: 43 and 117 in US8734809), AAV CLg-F7 (SEQ ID NOs: 44 and 118 in US8734809), AAV CLg-F8 (SEQ ID NOs: 43 and 117 in US8734809), AAV CSp-1 (SEQ ID NOs: 45 and 119 in US8734809), AAV CSp-10 (SEQ ID NOs: 46 and 120 in US8734809), AAV CSp-11 (SEQ ID NOs: 47 and 121 in US8734809), AAV CSp-2 (SEQ ID NOs: 48 and 122 in US8734809), AAV CSp-3 (SEQ ID NOs: 49 and 123 in US8734809), AAV CSp-4 (SEQ ID NOs: 50 and 124 in US8734809), AAV CSp-6 (SEQ ID NOs: 51 and 125 in US8734809), AAV CSp-7 (SEQ ID NOs: 52 and 126 in US8734809), AAV CSp-8 (SEQ ID NOs: 53 and 127 in US8734809), AAV CSp-9 (SEQ ID NOs: 54 and 128 in US8734809), AAV CHt-2 (SEQ ID NOs: 55 and 129 in US8734809), AAV CHt-3 (SEQ ID NOs: 56 and 130 in US8734809), AAV CKd-1 (SEQ ID NOs: 57 and 131 in US8734809), AAV CKd-10 (SEQ ID NOs: 58 and 132 in US8734809), AAV CKd-2 (SEQ ID NOs: 59 and 133 in US8734809), AAV CKd-3 (SEQ ID NOs: 60 and 134 in US8734809), AAV CKd-4 (SEQ ID NOs: 61 and 135 in US8734809), AAV CKd-6 (SEQ ID NOs: 62 and 136 in US8734809), AAV CKd-7 (SEQ ID NOs: 63 and 137 in US8734809), AAV CKd-8 (SEQ ID NOs: 64 and 138 in US8734809), AAV CLv-1 (SEQ ID NOs: 35 and 139 in US8734809), AAV CLv-12 (SEQ ID NOs: 66 and 140 in US8734809), AAV CLv-13 (SEQ ID NOs: 67 and 141 in US8734809), AAV CLv-2 (SEQ ID NOs: 68 and 142 in US8734809), AAV CLv-3 (SEQ ID NOs: 69 and 143 in US8734809), AAV CLv-4 (SEQ ID NOs: 70 and 144 in US8734809), AAV CLv-6 (SEQ ID NOs: 71 and 145 in US8734809), AAV CLv-8 (SEQ ID NOs: 72 and 146 in US8734809), AAV CKd-B1 (SEQ ID NOs: 73 and 147 in US8734809), AAV CKd-B2 (SEQ ID NOs: 74 and 148 in US8734809), AAV CKd-B3 (SEQ ID NOs: 75 and 149 in US8734809), AAV CKd-B4 (SEQ ID NOs: 76 and 150 in US8734809), AAV CKd-B5 (SEQ ID NOs: 77 and 151 in US8734809), AAV CKd-B6 (SEQ ID NOs: 78 and 152 in US8734809), AAV CKd-B7 (SEQ ID NOs: 79 and 153 in US8734809), AAV CKd-B8 (SEQ ID NOs: 80 and 154 in US8734809), AAV CKd-H1 (SEQ ID NOs: 81 and 155 in US8734809), AAV CKd-H2 (SEQ ID NOs: 82 and 156 in US8734809), AAV CKd-H3 (SEQ ID NOs: 83 and 157 in US8734809), AAV CKd-H4 (SEQ ID NOs: 84 and 158 in US8734809), AAV CKd-H5 (SEQ ID NOs: 85 and 159 in US8734809), AAVCKd-H6 (SEQ ID NOs: 77 and 151 in US8734809), AAV CHt-1 (SEQ ID NOs: 86 and 160 in US8734809), AAV CLv1-1 (SEQ ID NO: 171 in US8734809), AAV CLv1-2 (SEQ ID NO: 172 in US8734809), AAV CLv1-3 (SEQ ID NO: 173 in US8734809), AAV CLv1-4 (SEQ ID NO: 174 in US8734809), AAV The sequence may be or may have a sequence as described in U.S....

Claims

1. An AAV capsid mutant, (a) the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or (b) comprising at least 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; Optionally, the capsid variant comprises: (i) the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; and / or (ii) the AAV capsid variant comprising an amino acid sequence having at least one, two, or three modifications, but not more than 30, 20, or 10 modifications, relative to the amino acid sequence of SEQ ID NO:

138.

2. 2. The AAV capsid variant of claim 1, wherein the amino acid sequence is located immediately after position 586, 588, or 589 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO:

138.

3. (i) the five contiguous amino acids comprise PLNGA (SEQ ID NO:3679); (ii) the 6 contiguous amino acids comprise PLNGAV (SEQ ID NO: 3680); (iii) the seven contiguous amino acids comprise PLNGAVH (SEQ ID NO: 3681); (iv) the eight contiguous amino acids comprise PLNGAVHL (SEQ ID NO: 3682); and / or (v) the 9 contiguous amino acids comprise PLNGAVHLY (SEQ ID NO: 3648); 3. The AAV capsid variant of claim 1 or 2, optionally wherein the amino acid sequence of (i), (ii), (iii), (iv) or (v) is located immediately after position 586 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:

138.

4. (i) the five contiguous amino acids comprise YSTDE (SEQ ID NO:3691) or YSTDV (SEQ ID NO:3700); (ii) the six consecutive amino acids comprise YSTDER (SEQ ID NO: 3692) or YSTDVR (SEQ ID NO: 3701); and / or (iii) the seven contiguous amino acids comprise YSTDVRM (SEQ ID NO:3650), YSTDERM (SEQ ID NO:3657), or YSTDERK (SEQ ID NO:3658); 3. The AAV capsid variant of claim 1 or 2, optionally wherein the amino acid sequence of (i), (ii) or (iii) is located immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:

138.

5. (i) the five contiguous amino acids comprise IVMNS (SEQ ID NO:3694); (ii) the six consecutive amino acids comprise IVMNSL (SEQ ID NO: 3695); and / or (iii) the seven contiguous amino acids comprise IVMNSLK (SEQ ID NO: 3651); 3. The AAV capsid variant of claim 1 or 2, optionally wherein the amino acid sequence of (i), (ii) or (iii) is located immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:

138.

6. (i) the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648), in which the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) is located immediately after position 586 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; (ii) the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654), wherein the amino acid sequence of GGTLAVVSL (SEQ ID NO: 3654) occurs immediately after position 586 relative to the reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; (iii) the amino acid sequence of IVMNSLK (SEQ ID NO: 3651), wherein the amino acid sequence of IVMNSLK (SEQ ID NO: 3651) occurs immediately after position 588 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO: 138; or (iv) the amino acid sequence of any of SEQ ID NOs: 3649, 3650, 3652, 3653, or 3655-3659, wherein the amino acid sequence of any of said sequences is located immediately after position 589 relative to a reference sequence numbered according to the amino acid sequence of SEQ ID NO:

138.

7. (i) the capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleotide sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (ii) the capsid variant comprises an amino acid sequence encoded by a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660-3671; (iii) the nucleotide sequence encoding the capsid variant comprises the nucleotide sequence of any one of SEQ ID NOs: 3660-3671, or a nucleotide sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (iv) The AAV capsid variant of any one of claims 1 to 6, wherein the nucleotide sequence encoding the capsid variant comprises a nucleotide sequence containing at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of the nucleotide sequence of any of SEQ ID NOs: 3660-3671.

8. (i) the capsid variant further comprises a substitution at position K449, numbered according to SEQ ID NO: 138, e.g., a K449R substitution; (ii) the capsid variant further comprises a modification, e.g., an insertion, substitution, and / or deletion, in loop I, II, IV, and / or VI; (iii) the capsid variant comprises an amino acid residue other than "A" at position 587 and / or an amino acid residue other than "Q" at position 588, numbered according to SEQ ID NO: 138; (iv) the capsid variant comprises an amino acid sequence having at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, relative to the amino acid sequence of SEQ ID NO: 138; (v) the capsid variant comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; (vi) the capsid variant comprises an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 137, or a sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; and / or (vii) The AAV capsid variant of any one of claims 1 to 7, wherein the nucleotide sequence encoding the capsid variant comprises the nucleotide acid sequence of SEQ ID NO: 137, or a sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto.

9. (i) a VP1 protein, a VP2 protein, a VP3 protein, or a combination thereof; (ii) an amino acid sequence corresponding to positions 138 to 743 of any one of SEQ ID NOs: 3636 to 3647, e.g., VP2, or a sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; (iii) an amino acid sequence corresponding to positions 203 to 743 of any one of SEQ ID NOs: 3636 to 3647, e.g., VP3, or a sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; (iv) an amino acid sequence of any one of SEQ ID NOs: 3636-3647, or an amino acid sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; (v) an amino acid sequence having at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, of the amino acid sequence of any one of SEQ ID NOs: 3636-3647; (vi) an amino acid sequence encoded by the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto.

10. 10. The AAV capsid variant of any one of claims 1 to 9, wherein the nucleotide sequence encoding the capsid variant comprises the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto, and optionally, the nucleotide sequence encoding the capsid variant is codon-optimized.

11. An AAV capsid mutant, (i) the amino acid sequence of any one of claims 1 to 3 or 6 to 10, further comprising an amino acid sequence that is at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 3636; or (ii) The AAV capsid variant, comprising the amino acid sequence of SEQ ID NO: 3636.

12. An AAV capsid mutant, (i) the amino acid sequence of any one of claims 1, 2, 4, or 6-10, further comprising an amino acid sequence that is at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) identical to SEQ ID NOs: 3638 or 3645-3646; or (ii) The AAV capsid mutant comprising the amino acid sequence of SEQ ID NO: 3638 or 3645-3646.

13. An AAV capsid mutant, (i) the amino acid sequence of any one of claims 1, 2, 5-10, further comprising an amino acid sequence that is at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) identical to SEQ ID NO: 3639; or (ii) the AAV capsid variant comprising the amino acid sequence of SEQ ID NO: 3639.

14. 14. The AAV capsid variant of any one of claims 1 to 13, comprising the amino acid sequence of any one of SEQ ID NOs: 3636 to 3647, or an amino acid sequence that is at least 95% identical thereto.

15. 1. An AAV capsid variant comprising a parent amino acid sequence having an insert, e.g., a targeting peptide, wherein the insert comprises: (a) the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or (b) the AAV capsid variant comprising at least 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659.

16. (i) the parent sequence comprises the amino acid sequence of SEQ ID NO: 138, or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (ii) the parent sequence comprises an amino acid sequence that contains at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, e.g., substitutions, relative to the amino acid sequence of SEQ ID NO: 138; (iii) the parent sequence contains a substitution at position K449, e.g., a K449R substitution; (iv) the parent sequence comprises an amino acid sequence encoded by the nucleotide acid sequence of SEQ ID NO: 137, or a nucleotide acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and / or (v) the AAV capsid variant of Claim 15, wherein the nucleotide sequence encoding the parent sequence comprises the nucleotide acid sequence of SEQ ID NO: 137, or a nucleotide acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

17. 17. The AAV capsid variant of claim 15 or 16, wherein the insert comprises an amino acid sequence selected from PLNGAVHLY (SEQ ID NO:3648), GGTLAVVSL (SEQ ID NO:3654), IVMNSLK (SEQ ID NO:3651), RDSPKGW (SEQ ID NO:3649), YSTDVRM (SEQ ID NO:3650), RESPRGL (SEQ ID NO:3652), SFNDTRA (SEQ ID NO:3653), YGLPKGP (SEQ ID NO:3655), or STGTLRL (SEQ ID NO:3656), and optionally, the insert is inserted immediately after position 586, 588, or 589 in the parent amino acid sequence.

18. (i) an amino acid other than "A" at position 587 and / or an amino acid other than "Q" at position 588 of said parent sequence; or (ii) The AAV capsid mutant of any one of claims 15 to 17, comprising a deletion at position 587 and / or a deletion at position 588 of the parent amino acid sequence.

19. (i) an insert comprising the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) inserted immediately after position 586 of the parent amino acid sequence and a deletion of the amino acids "AQ" at positions 587-588 of the parent amino acid sequence; (ii) an insert comprising the amino acid sequence of IVMNSLK (SEQ ID NO: 3651), inserted immediately after position 588 of the parent amino acid sequence; (iii) the AAV capsid variant of any one of claims 15 to 18, comprising an insert comprising an amino acid sequence selected from RDSPKGW (SEQ ID NO: 3649), YSTDVRM (SEQ ID NO: 3650), RESPRGL (SEQ ID NO: 3652), SFNDTRA (SEQ ID NO: 3653), YGLPKGP (SEQ ID NO: 3655), or STGTLRL (SEQ ID NO: 3656), inserted immediately after position 589 of the parent amino acid sequence.

20. have increased targeting for CNS cells or tissues (e.g., brain cells, brain tissue, spinal cord cells, or spinal cord tissue) and / or muscle cells or tissues compared to the targeting of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138, and optionally (i) the brain cells or tissue are cells or tissue of the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus; (ii) the spinal cord cells or tissue are cervical, thoracic, and / or lumbar cells or tissue; and / or (iii) The AAV capsid variant of any one of claims 1 to 19, wherein the muscle cell or tissue is a cardiac muscle (e.g., atrial or ventricular muscle region), quadriceps muscle, and / or diaphragm muscle cell or tissue.

21. The capsid variant (i) is at least about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, or 400-fold enriched in the brain relative to the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 4; (ii) transduces a brain region selected from, for example, the dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, and putamen, wherein the level of transduction is at least 5, 10, 50, 100, 200, 500, 1,000, 2,000, 5,000, or 10,000-fold greater than the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 5, e.g., an immunohistochemistry assay, a qRT-PCR, or an RT-ddPCR assay; (iii) delivers elevated levels of payload to brain regions, optionally wherein the levels of payload are at least 500, 1,000, 2,000, 5,000, or 10,000 times higher compared to a reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., a qRT-PCR or RT-ddPCR assay, and optionally wherein the brain regions include the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus; (iv) delivers elevated levels of payload to spinal regions, optionally wherein the levels of payload are increased by at least 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 fold compared to a reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., a qRT-PCR assay, and optionally wherein the spinal regions include the cervical, thoracic, and / or lumbar regions; and / or (v) the AAV capsid variant of any one of claims 1 to 20, wherein the AAV capsid variant delivers elevated levels of viral genomes to brain regions, optionally wherein the level of viral genomes is increased by at least 5, 10, 20, 30, 40, or 50 fold compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., a qRT-PCR or RT-ddPCR assay, and optionally wherein the brain regions comprise the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

22. 22. The AAV capsid variant of any one of claims 1-2, 5, 6-10, 13-17 or 19-21, which exhibits preferential transduction in brain regions compared to said transduction in the dorsal root ganglia (DRG).

23. The capsid variant is (i) is enriched in the brain by at least about 300-fold, or 400-fold, relative to the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 4; (ii) transduces a brain region selected from, for example, the dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, and putamen, wherein the level of transduction is at least 500, 1,000, 2,000, 5,000, or 10,000-fold greater than the reference sequence of SEQ ID NO: 138, as measured, for example, by an assay such as that described in Example 5, e.g., an immunohistochemistry assay, a qRT-PCR, or an RT-ddPCR assay; (iii) delivers elevated levels of payload to brain regions, optionally wherein the levels of the payload are increased by at least 500, 1,000, 2,000, 5,000, or 10,000 fold compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., a qRT-PCR or an RT-ddPCR assay, and optionally wherein the brain regions include the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus; (iv) delivers elevated levels of payload to spinal regions, optionally wherein the levels of payload are increased by at least 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 fold compared to a reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., a qRT-PCR assay, and optionally wherein the spinal regions include the cervical, thoracic, and / or lumbar regions; and / or (v) the AAV capsid variant of any one of claims 1-3, 6-11, 14-21, which delivers elevated levels of viral genomes to brain regions, optionally wherein the level of viral genomes is increased by at least 5, 10, 20, 30, 40, or 50 fold compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., a qRT-PCR or RT-ddPCR assay, and optionally wherein the brain region comprises the frontal cortex, sensory cortex, motor cortex, putamen, thalamus, cerebellar cortex, dentate nucleus, caudate nucleus, and / or hippocampus.

24. (i) the AAV capsid variant has increased tropism for muscle cells or tissue, e.g., cardiac tissue, relative to the tropism of a reference sequence comprising the amino acid sequence of SEQ ID NO: 138; and / or (ii) the AAV capsid variant of any one of claims 1, 2, 5, 6-10, 13-17, or 19-22, wherein the variant delivers elevated levels of payload to muscle regions, optionally wherein the levels of payload are elevated by at least 10, 15, 20, 30, or 40 fold compared to the reference sequence of SEQ ID NO: 138, e.g., as measured by an assay (e.g., as described in Example 5), e.g., an IHC assay or an RT-ddPCR assay, and optionally wherein the muscle regions comprise myocardium (e.g., atrial or ventricular muscle regions), quadriceps muscle, and / or diaphragm muscle regions.

25. A polynucleotide encoding a polypeptide, such as an AAV capsid variant according to any one of claims 1 to 24.

26. 26. The polynucleotide of claim 25, comprising the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto.

27. a peptide, e.g., a targeting peptide, (i) the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or (ii) comprising at least 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; The peptide, for example, a targeting peptide.

28. a peptide, e.g., a targeting peptide, (i) an amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648) or at least 5, 6, 7, 8, or 9 contiguous amino acids derived from the amino acid sequence of PLNGAVHLY (SEQ ID NO: 3648); (ii) an amino acid sequence of IVMNSLK (SEQ ID NO: 3651) or at least 5, 6, 7, 8, or 9 contiguous amino acids derived from the amino acid sequence of IVMNSLK (SEQ ID NO: 3651); (iii) the amino acid sequence of YSTDVRM (SEQ ID NO:3650), YSTDERM (SEQ ID NO:3657), or YSTDERK (SEQ ID NO:3658), or at least 5, 6, 7, 8, or 9 contiguous amino acids derived from the amino acid sequence YSTDVRM (SEQ ID NO:3650), YSTDERM (SEQ ID NO:3657), or YSTDERK (SEQ ID NO:3658); or (iv) comprising an amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659, or at least 5, 6, 7, 8, or 9 consecutive amino acids derived from an amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; The peptide, for example, a targeting peptide.

29. A polynucleotide encoding an AAV capsid variant, (a) the amino acid sequence of any of SEQ ID NOs: 1725-3622 or 3648-3659; or (b) the polynucleotide comprising at least 5, 6, 7, 8, or 9 consecutive amino acids from the amino acid sequence of any one of SEQ ID NOs: 1725-3622 or 3648-3659.

30. (i) the AAV capsid variant comprises the amino acid sequence of any one of SEQ ID NOs: 3636-3647, or an amino acid sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto; (ii) the AAV capsid variant comprises an amino acid sequence having at least one, two, or three modifications, but no more than 30, 20, or 10 modifications, of the amino acid sequence of any one of SEQ ID NOs: 3636-3647; and / or (iii) The polynucleotide of claim 29, wherein the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 3623-3635, or a nucleotide sequence having at least 90% (e.g., at least about 95, 96, 97, 98, or 99%) sequence identity thereto.

31. An AAV particle comprising an AAV capsid variant according to any one of claims 1 to 24, an AAV capsid variant encoded by a polynucleotide according to any one of claims 25 to 26 or 29 to 30, or an AAV capsid variant comprising a peptide, for example a targeting peptide, according to any one of claims 27 to 28.

32. 32. The AAV particle of claim 31, comprising a nucleotide sequence encoding a payload, optionally wherein the encoded payload comprises a therapeutic protein or a functional variant thereof; an antibody or antibody fragment; an enzyme; a component of a gene editing system; an RNAi agent (e.g., dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA); or a combination thereof.

33. (i) the therapeutic protein or functional variant thereof, e.g., recombinant protein, is associated with (e.g., is aberrantly expressed in) a neurological or neurodegenerative disorder, a muscular or neuromuscular disorder, or a neuro-oncological disorder, optionally wherein the therapeutic protein or functional variant thereof is apolipoprotein E (APOE) (e.g., ApoE2, ApoE3, and / or ApoE4); human survival of single motor neuron (SMN) 1 or SMN2; glucocerebrosidase (GBA1); aromatic L-amino acid decarboxylase (AADC); aspartoacylase (ASPA); tripeptidyl peptidase I (CLN2); beta-galactosidase (GLB1); N-sulfoglucosamine sulfohydrolase (SGSH); N-acetyl-alpha-glucosaminidase (NAGLU); iduronate 2-sulfatase (IDS); intracellular cholesterol transporter (NPC1); gigaxonin (GAN); or a combination thereof; (ii) the antibody or antibody-binding fragment (a) a CNS-related target, e.g., an antigen associated with a neurological or neurodegenerative disorder, e.g., β-amyloid, APOE, tau, SOD1, TDP-43, huntingtin (HTT), and / or synuclein; (b) a muscle- or neuromuscular-associated target, e.g., an antigen associated with a muscle disorder or a neuromuscular disorder; or (c) binds to a neuro-oncology-associated target, e.g., an antigen associated with a neuro-oncology disorder, e.g., HER2, or EGFR (e.g., EGFRvIII); (iii) the enzyme comprises a meganuclease, zinc finger nuclease, TALEN, recombinase, integrase, base editor, Cas9, or a fragment thereof; (iv) the components of the gene editing system include one or more components of a CRISPR-Cas system, optionally wherein the one or more components of the CRISPR-Cas system include Cas9, e.g., a Cas9 ortholog or Cpf1, and a single guide RNA (sgRNA), wherein (a) the sgRNA is located upstream (5') of the cas9 enzyme; and / or (b) the sgRNA is located downstream (3') of the cas9 enzyme; and / or (v) the AAV particles of claim 32, wherein the RNAi agent (e.g., dsRNA, siRNA, shRNA, pre-miRNA, pri-miRNA, miRNA, stRNA, lncRNA, piRNA, or snoRNA) modulates, e.g., inhibits, expression of a CNS-related gene, mRNA, and / or protein, optionally wherein the CNS-related gene is selected from SOD1, MAPT, APOE, HTT, C9ORF72, TDP-43, APP, BACE, SNCA, ATXN1, ATXN3, ATXN7, SCN1A-SCN5A, SCN8A-SCN11A, or a combination thereof.

34. a viral genome comprising a promoter operably linked to the nucleic acid sequence encoding the payload, optionally wherein the promoter is selected from the group consisting of human elongation factor 1 α-subunit (EF1α), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC), neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), intercellular adhesion molecule 2 (ICAM-2), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), Ca2+ / calmodulin-dependent protein kinase I 34. The AAV particle of any one of claims 31 to 33, wherein the AAV particle is selected from a promoter selected from the group consisting of a promoter of an excitatory amino acid transporter 2 (EAAT2), a promoter of an amino acid sequence selected from the group consisting of ...

35. the viral genome further comprises: (i) a polyA signal sequence; (ii) an inverted terminal repeat (ITR) sequence, optionally wherein the ITR sequence is located 5' to the encoded payload and / or wherein the ITR sequence is located 3' to the encoded payload; (iii) enhancers, Kozak sequences, intronic regions, and / or exonic regions; (iv) a miR binding site, e.g., a miR binding site that regulates, e.g., reduces, expression of the payload encoded by the viral genome in cells or tissues in which the corresponding miRNA is expressed; and / or 35. The AAV particle of claim 34, comprising: (v) a nucleotide sequence encoding a Rep protein, e.g., a nonstructural protein, wherein the Rep protein comprises a Rep78 protein, a Rep68 protein, a Rep52 protein, and / or a Rep40 protein, and optionally the Rep78 protein, the Rep68 protein, the Rep52 protein, and / or the Rep40 protein is encoded by at least one Rep gene.

36. The viral genome (i) at least 1-5 copies, e.g., at least 1, 2, 3, 4, or 5 copies, of the miR binding site; (ii) at least three copies of a miR binding site, optionally where all three copies contain the same miR binding site, or where at least one, two, or all copies contain different miR binding sites; and / or (iii) at least four copies of a miR binding site, optionally, where all four copies contain the same miR binding site, or where at least one, two, three, or all copies contain different miR binding sites.

37. The miR binding site comprises a miR122 binding site, a miR183 binding site, a miR-142-3p, or a combination thereof, optionally wherein: (i) the miR122 binding site comprises the nucleotide sequence of SEQ ID NO: 3672, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of SEQ ID NO: 3672; (ii) the miR183 binding site comprises the nucleotide sequence of SEQ ID NO: 3675, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of SEQ ID NO: 3675; and / or (iii) the miR-142-3p binding site comprises the nucleotide sequence of SEQ ID NO: 3674, or a nucleotide sequence substantially identical thereto (e.g., having at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% sequence identity); or a nucleotide sequence having at least one, two, three, four, five, six, or seven modifications, but no more than ten modifications, of SEQ ID NO: 3674.

38. The viral genome: (i) is single-stranded, and / or (ii) 38. The AAV particle according to any one of claims 34 to 37, further comprising a nucleic acid sequence encoding the AAV capsid mutant according to any one of claims 1 to 24.

39. 39. The AAV capsid variant, polynucleotide, peptide, or AAV particle of any one of claims 1 to 38, which is isolated, e.g., recombinant.

40. 40. A vector comprising a polynucleotide encoding the AAV capsid variant of any one of claims 1 to 24 or 39, a polynucleotide of any one of claims 25-26, 29-30 or 39, or a polynucleotide encoding a peptide, such as a targeting peptide, of any one of claims 27-28 or 39.

41. A cell, e.g., a host cell, optionally (i) the cell is a mammalian cell or an insect cell; (ii) the cell is a cell of a brain or spinal cord region, optionally a cell of the frontal cortex, sensory cortex, motor cortex, caudate nucleus, dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, putamen, cervical spinal cord region, thoracic spinal cord region, and / or lumbar spinal cord region; and / or (iii) A cell, e.g., a host cell, comprising the AAV capsid variant of any one of claims 1 to 24 or 39, the polynucleotide of any one of claims 25 to 26, 29 to 30 or 39, the peptide of any one of claims 27 to 28 or 39, the AAV particle of any one of claims 31 to 39, or the vector of claim 40, which is a neuron, a sensory neuron, a motor neuron, an astrocyte, or a muscle cell (e.g., a cardiac, diaphragm, or quadriceps cell).

42. 1. A method of producing AAV particles, comprising: (i) providing a host cell containing a viral genome; (ii) incubating the host cell under conditions suitable for encapsulating the viral genome in an AAV capsid variant of any one of claims 1 to 24 or 39, an AAV capsid variant encoded by a polynucleotide of any one of claims 25 to 26, 29 to 30 or 39, or an AAV capsid variant comprising a peptide, e.g., a targeting peptide, of any one of claims 27 to 28 or 39; The method, whereby the AAV particles are produced.

43. A pharmaceutical composition comprising an AAV particle according to any one of claims 31 to 39, an AAV particle comprising a capsid variant according to any one of claims 1 to 24 or 39, or an AAV particle comprising a peptide according to any one of claims 27 to 28 or 39, and a pharmaceutically acceptable excipient.

44. 10. A method of delivering a payload to a cell or tissue (e.g., a CNS cell, CNS tissue, muscle cell, or muscle tissue), comprising administering an effective amount of the pharmaceutical composition of claim 43, an AAV particle of any one of claims 31-39, an AAV particle comprising a capsid variant of any one of claims 1-24 or 39, or an AAV particle comprising a peptide of any one of claims 27-28 or 39.

45. (i) the cell or tissue is a cell or tissue of a brain region or a spinal cord region, and optionally a cell or tissue of the frontal cortex, sensory cortex, motor cortex, caudate nucleus, dentate nucleus, cerebellar cortex, cerebral cortex, brainstem, hippocampus, thalamus, putamen, cervical spinal cord region, thoracic spinal cord region, and / or lumbar spinal cord region; (ii) the cell is a neuron, sensory neuron, motor neuron, astrocyte, or muscle cell (e.g., a cardiac, diaphragm, or quadriceps cell); and / or (iii) The method of claim 44, wherein the cell or tissue is in a subject, and optionally the subject has, has been diagnosed with, or is at risk of having a neurological, neurodegenerative, muscular, neuromuscular, or neuro-oncological disorder.

46. 44. A method of treating a subject having or diagnosed as having a neurological, neurodegenerative, muscular, neuromuscular, or neuro-oncological disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 43, an AAV particle of any one of claims 31 to 39, an AAV particle comprising a capsid variant of any one of claims 1 to 24 or 39, or an AAV particle comprising a peptide of any one of claims 27 to 28 or 39.

47. 47. The method of claim 46, wherein treating comprises preventing progression of said disease or disorder in said subject, optionally wherein said subject is a human.

48. The AAV particles (i) intramuscularly, intravenously, intracerebrally, intrathecally, intracerebroventricularly, via intraparenchymal administration, or via intracisternal injection (ICM); (ii) via focused ultrasound (FUS), e.g., FUS in conjunction with intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS in conjunction with intravenous administration; and / or (iii) The method of any one of claims 45 to 47, wherein the method is administered to the subject intravenously.

49. 44. The pharmaceutical composition of claim 43, an AAV particle of any one of claims 31 to 39, an AAV particle comprising a capsid variant of any one of claims 1 to 24 or 39, or an AAV particle comprising a polypeptide of any one of claims 27 to 28 or 39, for use in a method of delivering a payload to a cell or tissue.

50. 44. The pharmaceutical composition of claim 43, the AAV particle of any one of claims 31 to 39, the AAV particle comprising the capsid variant of any one of claims 1 to 24 or 39, or the AAV particle comprising the polypeptide of any one of claims 27 to 28 or 39, for use in a method for treating a neurological, neurodegenerative, muscular, neuromuscular, or neuro-oncological disorder.

51. 44. A pharmaceutical composition according to claim 43, an AAV particle according to any one of claims 31 to 39, an AAV particle comprising a capsid variant according to any one of claims 1 to 24 or 39, or an AAV particle comprising a polypeptide according to any one of claims 27 to 28 or 39, for use in the manufacture of a medicament.

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