Recombinant adeno-associated viruses and uses thereof

Abstract

The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins modified to contain amino acid sequences that confer and / or enhance desired properties. In particular, the present invention provides modified capsid proteins containing peptide inserts from heterologous proteins inserted into or near the variable region IV (VR-IV) of the viral capsid such that the insert is surface-exposed on the AAV particle. The present invention also provides capsid proteins that target rAAVs to target tissues, particularly capsid proteins containing peptides derived from erythropoietin or dynein inserted into the surface-exposed variable region and that target rAAVs to neural tissues, including retinal tissue and / or the central nervous system, and deliver therapeutic agents for treating neurological and / or ocular disorders.

Description

[Technical field]

[0001] 0. Sequence List This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII file, created on March 29, 2020, is named 38013_0002P1_SL.txt and is 637,866 bytes in size.

[0002] The present invention relates to recombinant adeno-associated viruses (rAAVs) having capsid proteins modified to include amino acid sequences that confer and / or improve desired properties. In particular, the present invention provides modified capsid proteins that include a peptide insert from a heterologous protein inserted into or near variable region IV (VR-IV) or, alternatively, into or near variable region VIII (VR-VIII) of the viral capsid such that the insert is surface-exposed on the AAV particle. The present invention also provides capsid proteins that direct rAAV to target tissues, particularly capsid proteins that include peptides derived from, for example, erythropoietin or dynein, inserted into the surface-exposed variable region to target and / or improve transduction of rAAV to neural tissues, including the retina and central nervous system, and deliver therapeutic agents to treat neurological disorders. [Background technology]

[0003] The use of adeno-associated viruses (AAV) as gene delivery vectors is a promising avenue for the treatment of many unmet patient needs. Dozens of naturally occurring AAV capsids have been reported, and surveys of the natural diversity of AAV sequences in primate tissues have identified over 100 variants distributed across multiple clades. AAV belongs to the Parvoviridae family and is a single-stranded DNA virus with a relatively small genome and simple genetic components. AAV establishes latent infection without helper virus. The AAV genome typically has Rep and Cap genes flanked by terminal inverted repeats (ITRs), which function as replication and packaging signals for vector generation. The capsid proteins form a capsid that can carry genomic DNA and determine tissue tropism for delivering DNA to target cells.

[0004] Due to the promise of low pathogenicity and long-term targeted gene expression, recombinant AAV (rAAV) has been used as a gene transfer vector with therapeutic sequences packaged in various capsids. Such vectors have been used in preclinical gene therapy research, and more than 20 gene therapy products are currently under clinical development. Recombinant AAV, such as AAV9, has demonstrated desirable neurotropic properties, and clinical trials using recombinant AAV9 for the treatment of CNS diseases are ongoing. However, attempts to improve the neurotropic properties of rAAV in human subjects have met with limited success.

[0005] For example, there remains a need for rAAV vectors with improved neurotropic properties for use in crossing the blood-brain barrier to deliver therapeutic agents in the treatment of disorders associated with the central nervous system and the eye, particularly the retina. There is also a need for rAAV vectors with improved tissue-specific targeting and / or improved tissue-specific transduction to deliver therapeutic agents. Summary of the Invention

[0006] Recombinant adeno-associated viruses (rAAVs) are provided having capsid proteins modified to include amino acid sequences that confer and / or enhance desired properties, such as tissue targeting, transduction, and / or integration of the rAAV genome. In particular, the present invention provides modified capsid proteins that include one or more peptide inserts from a heterologous protein inserted into or near variable region IV (VR-IV) or into or near variable region VIII (VR-VIII) of the viral capsid such that the inserts are surface-exposed on the AAV particle. In certain embodiments, the insertion is immediately after the amino acid residue corresponding to one of amino acids 451-461 of the AAV9 capsid protein (SEQ ID NO:118, as numbered in FIG. 8), including after amino acid 454 of AAV9 capsid (i.e., between amino acids 454 and 455), or, in the capsid protein of a different AAV type, after the residue corresponding to amino acid 454 of AAV9 (e.g., SEQ ID NOs:110-117 or 119-121), where "corresponding" means aligned with the sequence alignment in FIG. 8, or, in the case of an AAV type not included in FIG. 8, with a similar amino acid sequence alignment of the AAV9 capsid protein (SEQ ID NO:118) and AAV capsid proteins as would be well known in the art. Thus, the invention provides modified capsid proteins comprising a peptide insertion from a heterologous protein inserted immediately after or near the amino acid corresponding to the amino acid residue at position 454 of AAV9, as numbered in FIG. 8. In additional specific embodiments, the insertion is immediately after (i.e., between amino acids 588 and 589) the amino acid residue corresponding to amino acid 588 of the AAV9 capsid protein (SEQ ID NO:118, numbered in FIG. 8), or after a residue corresponding to amino acid 588 of AAV9 in the capsid protein of a different AAV type (e.g., SEQ ID NOs:110-117 or 119-121).The capsid protein can be an AAV9 capsid protein, but can also be any AAV capsid protein, such as AAV serotype 1 (SEQ ID NO:110); AAV serotype 2 (SEQ ID NO:111); AAV serotype 3 (SEQ ID NO:112); AAV serotype 4 (SEQ ID NO:113); AAV serotype 5 (SEQ ID NO:114); AAV serotype 6 (SEQ ID NO:115); 451-461 of the AAV7 capsid (SEQ ID NO:116); 451-461 of the AAV8 capsid (SEQ ID NO:117); AAV serotype 9 (SEQ ID NO:118); AAV serotype 9e (SEQ ID NO:119); AAV serotype rh10 (SEQ ID NO:120); AAV serotype rh20 (SEQ ID NO:121); and AAV serotype hu.37 (SEQ ID NO:122), AAV serotype rh39 (SEQ ID NO:124), and AAV serotype rh74 (SEQ ID NO:123 or SEQ ID NO:154) (see FIG. 8).

[0007] Also provided are capsid proteins that direct rAAV to target tissues, in particular capsid proteins that contain peptides derived from erythropoietin or dynein (including axonemal or cytoplasmic dynein) or peptides that promote tissue targeting and / or cellular uptake and / or integration of the rAAV genome, which peptides are inserted into the surface-exposed variable regions to target the rAAV to neural tissues, including the central nervous system, and retinal tissues, and deliver therapeutic agents to treat neurological and ocular disorders. These peptides are advantageously inserted into the amino acid sequence of the capsid protein such that the inserted peptides are surface-exposed when the capsid protein is incorporated into an AAV particle. These peptides are inserted immediately after one of amino acids 262-273; 451-461; or 585-593 of the AAV9 capsid (SEQ ID NO:118, see Figure 8 for alignment), or immediately after one of the amino acids corresponding to those amino acids, or immediately after the amino acid residue corresponding to the first codon encoding VP2, i.e., amino acid 138 of the AAV9 capsid and the amino acid corresponding to position 138 of the AAV9 capsid (SEQ ID NO:118, see Figure 8 for alignment). In certain embodiments, the inserted peptide is at least 4 consecutive amino acids of one of the axonemal dynein heavy chain peptides KMQVPFQ (SEQ ID NO:1); TLAAPFK (SEQ ID NO:2); QQAAPSF (SEQ ID NO:3); RYNAPFK (SEQ ID NO:4); LKLPPIV (SEQ ID NO:5); PFIKPFE (SEQ ID NO:6); or TLSLPWK (SEQ ID NO:7), or alternatively, 5, 6, 7, 8, 9, 10 or 11 consecutive amino acids of QEQLERALNSS (SEQ ID NO:8), a non-linear epitope of erythropoietin designated ARA290.

[0008] Modified capsid proteins are provided that include peptides that target specific tissues, including those that promote or increase cellular uptake and / or integration of the rAAV genome, where the peptides are inserted into a surface-exposed variable region of the capsid protein. In certain embodiments, the peptides are targeted to bone (e.g., at least 4 consecutive amino acids or at least 7 or 8 consecutive amino acids of DDDDDDDD (SEQ ID NO: 9)), brain (at least 4 amino acids or at least 7 consecutive amino acids or 7 consecutive amino acids of LSRLDA (SEQ ID NO: 10) or 7, 8, or 9 consecutive amino acids of CLSSRLDAC (SEQ ID NO: 11)), kidney (at least 4 or 5 consecutive amino acids of peptide CLPVASC (SEQ ID NO: 12) or LPVAS (SEQ ID NO: 13) or peptide CLPVASC (SEQ ID NO: 12) or LPVAS (SEQ ID NO: 13)), muscle (at least 4 or 5 consecutive amino acids of peptide ASSLNIA (SEQ ID NO: 14)), or 6 or 7 consecutive amino acids of peptide CLPVASC (SEQ ID NO: 12) or LPVAS (SEQ ID NO: 13)). The peptide may be a peptide of 4, 5, 6, or 7 consecutive amino acids or ASSLNIA (SEQ ID NO: 14), a retinal cell (at least 4 consecutive amino acids or 5, 6, or 7 consecutive amino acids of LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16)), or derived from the transferrin receptor (at least 4 consecutive amino acids or at least 7 consecutive amino acids or 7 consecutive amino acids of HAIYPRH (SEQ ID NO: 17), THRPPMWSPVWP (SEQ ID NO: 18), RTIGPSV (SEQ ID NO: 19), or CRTIGPSVC (SEQ ID NO: 20)). In some embodiments, the peptide is CLPVASC (SEQ ID NO: 12), or ASSLNIA (SEQ ID NO: 14), and a capsid containing this peptide, for example inserted after position 454 of the capsid AAV9, preferentially targets the rAAV having the capsid to the kidney compared to the liver. In other embodiments, the inserted peptide is at least 4 consecutive amino acids or at least 7 or 8 consecutive amino acids, or is the peptide SITLVKSTQTV (SEQ ID NO: 21) or TILSRSTQTG (SEQ ID NO: 22) or QAVRTSL (SEQ ID NO: 23) or QAVRTSH (SEQ ID NO: 24).In some embodiments, the peptide is 12 or fewer consecutive amino acids. In other embodiments, modified capsids are provided having one or more amino acid substitutions that may improve tropism, transduction, or reduce immunoneutralizing activity. Such amino acid modifications include A269S of AAV8 and corresponding substitutions in other AAV-type capsids, S263F / S269T / A273T of AAV9 and corresponding substitutions in other AAV-type capsids, W530R or Q474A of AAV9 and corresponding substitutions in other AAV-type capsids. Capsids having these amino acid substitutions may further have a substitution of NNN (asparagine) with AAA (alanine) at 498-500 of the AAV8 capsid, or a substitution of NNN (asparagine) with AAA (alanine) at 496-498 of the AAV9 capsid, or corresponding substitutions in other AAV-type capsids.

[0009] Also provided are modified capsid proteins that promote transduction of rAAV in one or more tissues, including one or more cell types, upon systemic, intravenous, intrathecal, intranasal, intraperitoneal, or intravitreal administration, the capsid protein being inserted into a surface-exposed variable region (VR) of the capsid, e.g., VR-I, VR-IV, or VR-VIII, or after the first amino acid of VP2, e.g., immediately after residue 138 of the AAV9 capsid (amino acid sequence of SEQ ID NO: 118) or immediately after the corresponding residue of another AAV capsid, or alternatively, a peptide modified with one or more of the amino acid substitutions described herein, and transduction of the AAV having the modified capsid in at least one tissue is increased upon said administration, compared to transduction of the AAV having the corresponding unmodified capsid. In certain embodiments, transduction is measured by detection of the transgene, such as GFP fluorescence.

[0010] In some embodiments, rAAVs are provided that incorporate the modified capsids described herein, including rAAVs with genomes that include transgenes of therapeutic interest. Packaging cells are provided for producing the rAAVs described herein. Methods of treatment by delivery of the modified rAAVs described herein, and pharmaceutical compositions that include the modified rAAVs described herein are also provided. Methods of producing rAAVs with modified capsids described herein are also provided.

[0011] The invention is illustrated by the examples set forth below of the construction of rAAV9 capsids modified with peptide inserts designed based on the human axonemal dynein heavy chain tail, ARA290, and other tissue targeting or homing peptides, and capsids modified with amino acid substitutions.

[0012] 3.1.Embodiment 1. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insert of at least 4 and at most 20 contiguous amino acids from a heterologous protein that is not a capsid protein, said peptide insert immediately following an amino acid residue corresponding to one of amino acids 451-461 of the AAV9 capsid protein of Figure 8 (SEQ ID NO: 118), and wherein said peptide insert is surface-exposed when said capsid protein is packaged as an AAV particle.

[0013] 2. The rAAV capsid protein of embodiment 1, wherein said capsid protein is from at least one AAV serotype: AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), or serotype rh74 (AAVrh74).

[0014] 3. The peptide insert is in the sequence shown in FIG. 450 to 459 of the AAV1 capsid amino acid sequence (SEQ ID NO:110); 449 to 458 of the AAV2 capsid amino acid sequence (SEQ ID NO:111); 449 to 459 of the AAV3 capsid amino acid sequence (SEQ ID NO:112); 443 to 453 of the AAV4 capsid amino acid sequence (SEQ ID NO: 113); 442 to 445 of the AAV5 capsid amino acid sequence (SEQ ID NO:114); 450 to 459 of the AAV6 capsid amino acid sequence (SEQ ID NO: 115); 451 to 461 of the AAV7 capsid amino acid sequence (SEQ ID NO: 116); 451 to 461 of the AAV8 capsid amino acid sequence (SEQ ID NO: 117); 451 to 461 of the AAV9 capsid amino acid sequence (SEQ ID NO:118); 452 to 461 of the AAV9e capsid amino acid sequence (SEQ ID NO:119); 452 to 461 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 120); 452 to 461 of the AAVrh20 capsid amino acid sequence (SEQ ID NO:121); 452 to 461 of the AAVhu.37 capsid amino acid sequence (SEQ ID NO:122); 452 to 461 of the AAVrh74 capsid amino acid sequence (SEQ ID NO:123 or SEQ ID NO:154); or 452 to 461 of the AAVrh39 capsid amino acid sequence (SEQ ID NO: 124) The rAAV capsid protein of embodiment 2, wherein the rAAV capsid protein is present immediately after one of the amino acid residues in

[0015] 4. The rAAV capsid protein of embodiment 3, wherein the peptide insert is located immediately following one of amino acid residues I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid, or immediately following amino acid residues in the AAV capsid that correspond to amino acids I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid (SEQ ID NO: 118) according to the amino acid numbering aligned in FIG. 8.

[0016] 5. The rAAV capsid protein of any preceding embodiment, wherein said heterologous protein is a homing domain, a neutralizing antibody epitope, or a purification tag.

[0017] 6. The homing domain comprises: Neural tissue homing domain; axonemal or cytoplasmic dynein homing domains; bone homing domain; kidney-homing domain; muscle homing domain; endothelial cell homing domain; integrin receptor binding domain; transferrin receptor binding domain; a tumor cell targeting domain; or Retinal cell homing domain 6. The rAAV capsid protein of embodiment 5,

[0018] 7. The rAAV capsid protein of embodiment 6, wherein the peptide insert comprises or consists of a dynein or dynein homing peptide of at least 4 or at least 7 consecutive amino acids of the amino acid sequence SITLVKSTQTV (SEQ ID NO:21), TILSRSTQTG (SEQ ID NO:22), VVMVGEKPITITQHSVETEG (SEQ ID NO:25), RSSEEDKSTQTT (SEQ ID NO:26), KMQVPFQ (SEQ ID NO:1), LKLPPIV (SEQ ID NO:5), PFIKPFE (SEQ ID NO:6), TLSLPWK (SEQ ID NO:7), QQAAPSF (SEQ ID NO:3), RYNAPFK (SEQ ID NO:4), TLAVPFK (SEQ ID NO:27), TLAAPFK (SEQ ID NO:2), LGETTRP (SEQ ID NO:15), or LALGETTRP (SEQ ID NO:16).

[0019] 8. The rAAV capsid protein of embodiment 6, wherein the peptide insert from the transferrin receptor binding domain comprises at least 4 or at least 7 contiguous amino acids of the amino acid sequence RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20).

[0020] 9. The rAAV capsid protein of embodiment 6, wherein the peptide insert from the retinal cell homing domain comprises the amino acid sequence LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16).

[0021] 10. The rAAV capsid protein of embodiment 9, wherein the LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16) peptide insert is present in the AAV8 capsid protein or the AAV9 capsid protein.

[0022] 11. The rAAV capsid protein of any of the preceding embodiments, wherein the peptide insert is present in an AAV9 capsid protein after amino acid S454 of said AAV9 capsid protein (SEQ ID NO: 118) or after a residue in an AAV capsid protein that corresponds to S454 of said AAV9 capsid protein according to the amino acid numbering aligned in FIG. 8.

[0023] 12. The rAAV capsid protein of any of the above embodiments, wherein said capsid protein is not said AAV2 capsid protein.

[0024] 13. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to any of the above embodiments, or encoding an amino acid sequence sharing at least 80% identity therewith.

[0025] 14. A packaging cell capable of expressing the nucleic acid of embodiment 13 to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

[0026] 15. A rAAV vector comprising a capsid protein according to any one of embodiments 1 to 12.

[0027] 16. The rAAV vector of embodiment 15, further comprising a transgene.

[0028] 17. A pharmaceutical composition comprising an rAAV vector according to embodiment 15 or 16 and a pharma- ceutical acceptable carrier.

[0029] 18. A method for delivering a transgene to a cell, the method comprising contacting the cell with an rAAV vector of embodiment 16, wherein the transgene is delivered to the cell.

[0030] 19. A method for delivering a transgene to a target tissue, or a target cell or its cell matrix, of a subject in need thereof, the method comprising administering to the subject an rAAV vector described in embodiment 16, wherein the transgene is delivered to the target tissue of the subject.

[0031] 20. A pharmaceutical composition for use in delivering a transgene to a cell, the composition comprising an rAAV vector described in embodiment 16, and the cell being contacted with the vector.

[0032] 21. A pharmaceutical composition for use in delivering a transgene to a target tissue in a subject in need thereof, the pharmaceutical composition comprising the rAAV vector of embodiment 16, the peptide insert being a homing peptide, and the vector being administered to the subject.

[0033] 22. The method or pharmaceutical composition for use according to embodiments 18 to 21, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, or intravitreally.

[0034] 23. The target tissue, or the target cell or its cell matrix, nervous tissue, said vector comprising said peptide insert from said nervous tissue homing domain; bone, said vector comprising said peptide insert from said bone-homing domain; the kidney, and the vector comprises the peptide insert from the kidney-homing domain; muscle, said vector comprising said peptide insert from said muscle-homing domain; an endothelial cell, said vector comprising said peptide insert from said endothelial cell homing domain; a cell expressing an integrin receptor, said vector comprising said peptide insert from said integrin receptor binding domain; a tumor cell expressing a transferrin receptor, said vector comprising said peptide insert from said transferrin receptor binding domain; a tumor cell, and said vector comprises a peptide insert from said tumor cell targeting domain; or a retinal cell, the vector comprising the peptide insert from the retinal cell homing domain. The method or pharmaceutical composition for use according to any one of embodiments 18 to 21, comprising:

[0035] 24. A recombinant adeno-associated virus (rAAV) capsid protein, comprising: A neural tissue homing protein or domain, with the proviso that the peptide insert does not contain the sequence TLAVPFK (SEQ ID NO:27); axonemal or cytoplasmic dynein homing domains; bone homing domain; kidney-homing domain; muscle homing domain; endothelial cell homing domain; integrin receptor binding domain; a transferrin receptor binding domain (with the proviso that the peptide insert does not contain the sequence RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20); A tumor cell targeting domain; and Retinal cell homing domain, with the proviso that the peptide insert does not contain the sequence LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16) and a peptide insertion of at least 4 and up to 20 consecutive amino acids from a heterologous protein or domain selected from the group consisting of: The recombinant adeno-associated virus (rAAV) capsid protein, wherein the peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle.

[0036] 25. The rAAV capsid protein of embodiment 24, wherein the neural tissue homing protein or retinal cell homing domain is a human axonemal dynein (HAD) heavy chain tail.

[0037] 26. The rAAV capsid protein of embodiment 25, wherein the peptide insert comprises at least 4 and up to 12 consecutive amino acids from the dimerization domain of the HAD heavy chain tail.

[0038] 27. The peptide insert is (as shown in Figures 7A-7M): (aa1-1542 of DYH1_HUMAN UniProtKB-Q9P2D7) (SEQ ID NO: 97); (aa1-1764 of DYH2_HUMAN UniProtKB-Q9P225) (SEQ ID NO: 98); (aa 1 to 1390 of DYH3_HUMAN UniProtKB-Q8TD57) (SEQ ID NO: 99); (aa1 to 1941 of DYH5_HUMAN UniProtKB-Q8TE73) (SEQ ID NO: 100); (aa1 to 1433 of DYH6_HUMAN UniProtKB-Q9C0G6) (SEQ ID NO: 101); (aa1 to 1289 of DYH7_HUMAN UniProtKB-Q8WXX0) (SEQ ID NO: 102); (aa1 to 1807 of DYH8_HUMAN UniProtKB-Q96JB1) (SEQ ID NO: 103); (aa1 to 1831 of DYH9_HUMAN UniProtKB-Q9NYC9) (SEQ ID NO: 104); (aa1 to 1793 of DYH10_HUMAN UniProtKB-Q8IVF4) (SEQ ID NO: 105); (aa1 to 1854 of DYH11_HUMAN UniProtKB-Q96DT5) (SEQ ID NO: 106); (aa1 to 1214 of DYH12_HUMAN UniProtKB-Q6ZR08) (SEQ ID NO: 107); (aa 1 to 200 of DYH14_HUMAN UniProtKB-Q0VDD8) (SEQ ID NO: 108); or (aa1 to 1794 of DYH17_HUMAN UniProtKB-Q9UFH2) (SEQ ID NO: 109) 27. The rAAV capsid protein of embodiment 26, comprising at least 4 and at most 12 consecutive amino acids from the group consisting of:

[0039] 28. The rAAV capsid protein of embodiment 27, wherein the peptide insert comprises at least 4 and up to 12 consecutive amino acids from residues 1-200 of any one of the axonemal dynein heavy chain sequences (Figures 7A-7M).

[0040] 29. The rAAV capsid protein of embodiment 27, wherein the peptide insert comprises 7 consecutive amino acids from any one of the axonemal dynein heavy chain sequences of Figures 7A-7M.

[0041] 30. The rAAV capsid protein of embodiment 28, wherein the peptide insert comprises seven consecutive amino acids from residues 1-200 of any one of the dynein heavy chain sequences (Figures 7A-7M).

[0042] 31. The peptide insert is KMQVPFQ(SEQ ID NO:1); TLAAPFK (SEQ ID NO:2); QQAAPSF (SEQ ID NO:3); RYNAPFK (SEQ ID NO:4); LKLPPIV (SEQ ID NO:5); PFIKPFE (SEQ ID NO:6); or TLSLPWK (SEQ ID NO: 7) 26. The rAAV capsid protein of embodiment 25, comprising at least four consecutive amino acids of one of:

[0043] 32. The peptide insert is KMQVPFQ(SEQ ID NO:1); TLAAPFK (SEQ ID NO:2); QQAAPSF (SEQ ID NO:3); RYNAPFK (SEQ ID NO:4); LKLPPIV (SEQ ID NO:5); PFIKPFE (SEQ ID NO:6); or TLSLPWK (SEQ ID NO: 7) 26. The rAAV capsid protein of embodiment 25, comprising a peptide from one of:

[0044] 33. The rAAV capsid protein of embodiment 32, wherein the peptide insert comprises the amino acid sequence TLAAPFK (sequence number 2).

[0045] 34. The rAAV capsid protein of embodiment 24, wherein the neural tissue homing protein is a mouse axonemal dynein (MAD) heavy chain tail.

[0046] 35. The rAAV capsid protein of embodiment 24, wherein the neural tissue homing domain is an EPO (erythropoietin) domain that binds to a natural repair receptor and is not erythropoietin, or a conformational analog of said domain.

[0047] 36. The rAAV capsid protein of embodiment 35, wherein the peptide insert comprises at least 4 and up to 11 consecutive amino acids from QEQLERALNSS (sequence number 8).

[0048] 37. The rAAV capsid protein of embodiment 36, wherein the peptide insert is the ARA290 sequence QEQLERALNSS (sequence number 8).

[0049] 38. The rAAV capsid protein of embodiment 24, wherein the neural tissue homing protein is a brain-homing domain having an SRL (serine-arginine-lysine) motif.

[0050] 39. The rAAV capsid protein of embodiment 38, wherein the peptide insert from the brain-homing domain comprises at least 7 consecutive amino acids of the amino acid sequence LSSRLDA (sequence number 10) or CLSSRLDAC (sequence number 11).

[0051] 40. The rAAV capsid protein of embodiment 24, wherein the axonemal or cytoplasmic dynein homing domain is a dynein light chain homing domain.

[0052] 41. The rAAV capsid protein of embodiment 40, wherein the peptide insert from the dynein light chain homing domain is one of SITLVKSTQTV (sequence number 21), TILSRSTQTG (sequence number 22), VVMVGEKPITITQHSVETEG (sequence number 25), or RSSEEDKSTQTT (sequence number 26).

[0053] 42. The rAAV capsid protein of embodiment 24, wherein the bone homing protein is a hydroxyapatite (HA) binding domain.

[0054] 43. The rAAV capsid protein of embodiment 42, wherein the peptide insert from the hydroxyapatite (HA) binding domain is at least 6 amino acid residues of the sequence DDDDDDDD (sequence number 9).

[0055] 44. The rAAV capsid protein of embodiment 24, wherein the kidney-homing domain is the amino acid sequence CLPVASC (sequence number 12).

[0056] 45. The rAAV capsid protein of embodiment 44, wherein the peptide insert from the kidney-homing domain is the amino acid sequence LPVAS (sequence number 13) or CLPVASC (sequence number 12).

[0057] 46. ​​The rAAV capsid protein of embodiment 24, wherein the peptide insert from the muscle-homing domain is the amino acid sequence ASSLNIA (sequence number 14).

[0058] 47. The rAAV capsid protein of embodiment 24, wherein the peptide insert is the amino acid sequence QAVRTSL (sequence number 23) or QAVRTSH (sequence number 24).

[0059] 48. The rAAV capsid protein of embodiment 24, wherein the peptide insert from the endothelial cell homing domain is the amino acid sequence SIGYPLP (sequence number 28).

[0060] 49. The rAAV capsid protein of embodiment 24, wherein the peptide insert from the integrin binding domain has the amino acid sequence CDCRGDCFC (sequence number 29).

[0061] 50. The rAAV capsid protein of embodiment 24, wherein the transferrin receptor binding domain is a transferrin domain, or a conformational analog thereof, or an iron mimetic.

[0062] 51. The rAAV capsid protein of embodiment 50, wherein the peptide insertion from the transferrin domain comprises at least 4 consecutive amino acids and up to 12 consecutive amino acids from the sequence HAIYPRH (sequence number 17) or THRPPMWSPVWP (sequence number 18).

[0063] 52. The rAAV capsid protein of embodiment 51, wherein the peptide insert is the amino acid sequence HAIYPRH (sequence number 17) or THRPPMWSPVWP (sequence number 18).

[0064] 53. The rAAV capsid protein of embodiment 24, wherein the peptide insert from the tumor cell targeting domain is the amino acid sequence NGRAHA (sequence number 30).

[0065] 54. The peptide insert comprises the amino acid residues (shown in FIG. 8): 138; 262-272; 450-459; or 585-593 of the AAV1 capsid amino acid sequence (SEQ ID NO:110); 138; 262-272; 449-458; or 584-592 of the AAV2 capsid amino acid sequence (SEQ ID NO:111); 138; 262-272; 449-459; or 585-593 of the AAV3 capsid amino acid sequence (SEQ ID NO:112); 137; 256-262; 443-453; or 583-591 of the AAV4 capsid amino acid sequence (SEQ ID NO:113); 137; 252-262; 442-445; or 574-582 of the AAV5 capsid amino acid sequence (SEQ ID NO:114); AAV6 capsid amino acid sequence (SEQ ID NO: 115): 138; 262-272; 450-459; 585-593; 138; 263-273; 451-461; 586-594 of the AAV7 capsid amino acid sequence (SEQ ID NO: 116); AAV8 capsid amino acid sequence (SEQ ID NO: 117): 138; 263-274; 452-461; 587-595; AAV9 capsid amino acid sequence (SEQ ID NO:118): 138; 262-273; 452-461; 585-593; AAV9e capsid amino acid sequence (SEQ ID NO: 119): 138; 262-273; 452-461; 585-593; 138; 263-274; 452-461; 587-595 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 120); 138; 263-274; 452-461; 587-595 of the AAVrh20 capsid amino acid sequence (SEQ ID NO:121); 138; 263-274; 452-461; 587-595 of the AAVhu37 capsid amino acid sequence (SEQ ID NO:122); 138; 263-274; 452-461; 587-595 of the AAVrh74 capsid amino acid sequence (SEQ ID NO:123 or SEQ ID NO:154); or AAVrh39 capsid amino acid sequence (SEQ ID NO: 124) 138; 263-274; 452-461; 587-595 54. The rAAV capsid protein of any one of embodiments 24 to 53, wherein the rAAV capsid protein is present immediately after one of the following:

[0066] 55. A recombinant AAV capsid protein comprising the amino acid sequence TLAAPFK (SEQ ID NO: 2) inserted between amino acid residues 588 to 589 of the AAV9 capsid (SEQ ID NO: 118) or correspondingly between amino acid residues 588 to 589 of the AAV9 capsid as aligned in FIG. 8.

[0067] 56. A recombinant AAV capsid protein comprising the amino acid sequence TLAAPFK (SEQ ID NO: 2) inserted immediately after one of amino acids I451 to L461, or S268 of the AAV9 capsid (SEQ ID NO: 118) or corresponding to one of amino acids I451 to L461, or S268 of the AAV9 capsid as aligned in FIG. 8.

[0068] 57. A recombinant AAV capsid protein comprising the amino acid sequence QEQLERALNSS (SEQ ID NO:8) inserted between amino acid residues 588 to 589 of the AAV9 capsid (SEQ ID NO:118) or correspondingly between amino acid residues 588 to 589 of the AAV9 capsid as aligned in FIG. 8.

[0069] 58. A recombinant AAV capsid protein comprising the amino acid sequence QEQLERALNSS (SEQ ID NO:8) inserted immediately after one of amino acids I451 to L461, or S268 of the AAV9 capsid (SEQ ID NO:118) or corresponding to one of amino acids I451 to L461, or S268 of the AAV9 capsid as aligned in FIG. 8.

[0070] 59. The rAAV capsid protein of any of embodiments 24-54, wherein the peptide insert is located immediately following an amino acid residue corresponding to one of amino acids 451-461, S268 or Q588 of the AAV9 capsid protein (sequence number 118) as aligned in FIG. 8.

[0071] 60. The rAAV capsid protein of embodiment 59, wherein the peptide insertion is located immediately following one of amino acids 451 to 461 of the AAV9 capsid protein (sequence number 118).

[0072] 61. The rAAV capsid protein of any of embodiments 24 to 53, wherein the peptide insertion is present in the eighth variable region (VR-VIII).

[0073] 62. The rAAV capsid protein of any of embodiments 24 to 61, wherein the capsid protein is not the AAV2 capsid protein.

[0074] 63. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to any one of embodiments 24 to 62, or encoding an amino acid sequence sharing at least 80% identity therewith.

[0075] 64. A packaging cell capable of expressing the nucleic acid of embodiment 63 to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

[0076] 65. A rAAV vector comprising a capsid protein according to any one of embodiments 24 to 62.

[0077] 66. The rAAV vector of embodiment 65, further comprising a transgene.

[0078] 67. A pharmaceutical composition comprising an rAAV vector according to embodiment 65 or 66 and a pharma- ceutical acceptable carrier.

[0079] 68. A method for delivering a transgene to a cell, the method comprising contacting the cell with an rAAV vector described in embodiment 66, wherein the transgene is delivered to the cell.

[0080] 69. A method for delivering a transgene to a target tissue of a subject in need thereof, the method comprising administering to the subject an rAAV vector described in embodiment 66, wherein the transgene is delivered to the subject.

[0081] 70. A pharmaceutical composition for use in delivering a transgene to a cell, the pharmaceutical composition comprising an rAAV vector described in embodiment 66, and the transgene being delivered to the cell.

[0082] 71. A pharmaceutical composition for use in delivering a transgene to a target tissue in a subject in need thereof, the pharmaceutical composition comprising an rAAV vector described in embodiment 66, wherein the transgene is delivered to the target tissue.

[0083] 72. The method or pharmaceutical composition for use according to embodiments 68 to 71, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, or intravitreally.

[0084] 73. The method or pharmaceutical composition for use according to embodiments 68 to 71, wherein the vector is administered via lumbar puncture or via the cisterna magna.

[0085] 74. A recombinant adeno-associated virus (rAAV) capsid protein, comprising a peptide insertion of at least four consecutive amino acids from one of TLAVPFK (SEQ ID NO:27), RTIGPSV (SEQ ID NO:19), CRTIGPSVC (SEQ ID NO:20), LGETTRP (SEQ ID NO:15), or LALGETTRP (SEQ ID NO:16);

[0086] The recombinant adeno-associated virus (rAAV) capsid protein, wherein the peptide insert is located immediately following the amino acid residue corresponding to amino acid 268, 454 or 588 of the AAV9 capsid protein, as aligned in FIG.

[0087] 75. The rAAV capsid protein of embodiment 74, wherein the capsid protein is not the AAV2 capsid protein.

[0088] 76. The rAAV capsid protein of embodiment 74, wherein the peptide insert comprises the amino acid sequence TLAVPFK (sequence number 27) between amino acid residues 454 and 455 of the AAV9 capsid protein (sequence number 118).

[0089] 77. The rAAV capsid protein of embodiment 74, wherein the peptide insert comprises the amino acid sequence TLAVPFK (SEQ ID NO: 27) immediately following one of amino acid residues 262 to 273 of the AAV9 capsid protein (SEQ ID NO: 118).

[0090] 78. The rAAV capsid protein of embodiment 74, wherein the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) between amino acid residues 454 to 455 of the AAV8 capsid protein (sequence number 117).

[0091] 79. The rAAV capsid protein of embodiment 78, wherein the peptide insert comprises the amino acid sequence LALGETTRP (sequence number 16).

[0092] 80. The rAAV capsid protein of embodiment 74, wherein the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) inserted between amino acid residues 590 to 591 of the AAV8 capsid protein (sequence number 117).

[0093] 81. The rAAV capsid protein of embodiment 80, wherein the peptide insert comprises the amino acid sequence LALGETTRP (sequence number 16).

[0094] 82. The rAAV capsid protein of embodiment 74, wherein the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) immediately following amino acid residues 263 to 274 of the AAV8 capsid protein (sequence number 117).

[0095] 83. The rAAV capsid protein of embodiment 82, wherein the peptide insert comprises the amino acid sequence LALGETTRP (sequence number 16).

[0096] 84. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to any one of embodiments 74 to 83, or encoding an amino acid sequence sharing at least 80% identity therewith.

[0097] 85. A packaging cell capable of expressing the nucleic acid of embodiment 84 to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

[0098] 86. A rAAV vector comprising a capsid protein according to any one of embodiments 74 to 83.

[0099] 87. The rAAV vector of embodiment 86, further comprising a transgene.

[0100] 88. A pharmaceutical composition comprising an rAAV vector according to embodiment 86 or 87 and a pharma- ceutical acceptable carrier.

[0101] 89. A method for delivering a transgene to a cell, the method comprising contacting the cell with an rAAV vector of embodiment 86 or 87, wherein the transgene is delivered to the cell.

[0102] 90. A method for delivering a transgene to a target tissue of a subject in need thereof, the method comprising administering to the subject an rAAV vector described in embodiment 86 or 87, wherein the transgene is delivered to the target tissue.

[0103] 91. A pharmaceutical composition for use in delivering a transgene to a cell, the pharmaceutical composition comprising an rAAV vector described in embodiment 86 or 87, and the transgene being delivered to the cell.

[0104] 92. A pharmaceutical composition for use in delivering a transgene to a target tissue in a subject in need thereof, the pharmaceutical composition comprising an rAAV vector described in embodiment 86 or 87, wherein the transgene is delivered to the target tissue.

[0105] 93. The method or pharmaceutical composition for use according to embodiments 89 to 92, wherein the target tissue is a retinal cell and the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) or LALGETTRP (sequence number 16).

[0106] 94. The method or pharmaceutical composition for use according to embodiments 89 to 93, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, or intravitreally.

[0107] 95. The method or pharmaceutical composition for use according to embodiments 89 to 93, wherein the vector is administered via lumbar puncture or via the cisterna magna.

[0108] 96. A recombinant AAV capsid protein comprising one or more amino acid substitutions relative to a wild-type or unmodified capsid protein, wherein the rAAV capsid protein is an AAV8 capsid protein having an A269S amino acid substitution (SEQ ID NO: 117), or an AAV9 capsid protein having S263G / S269R / A273T substitutions, or a W503R or Q474A substitution (SEQ ID NO: 118), or a corresponding substitution in a capsid protein of another AAV type capsid.

[0109] 97. The recombinant AAV capsid protein of embodiment 96, further comprising 498-NNN / AAA-500 for the AAV8 capsid protein or 496-NNN / AAA-498 for the AAV9 capsid protein (sequence number 118), or a corresponding substitution in the capsid protein of another AAV type capsid.

[0110] 98. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to embodiment 96 or 97, or encoding an amino acid sequence sharing at least 80% identity therewith.

[0111] 99. A packaging cell capable of expressing the nucleic acid described in embodiment 98 to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

[0112] 100. A rAAV vector comprising any of the capsid proteins of embodiments 96 or 97.

[0113] 101. The rAAV vector of embodiment 100, further comprising a transgene.

[0114] 102. A pharmaceutical composition comprising an rAAV vector according to embodiment 100 or 101 and a pharma- ceutically acceptable carrier.

[0115] 103. A method for delivering a transgene to a cell, the method comprising contacting the cell with an rAAV vector described in embodiment 101, wherein the transgene is delivered to the cell.

[0116] 104. A method for delivering a transgene to a target tissue of a subject in need thereof, the method comprising administering to the subject an rAAV vector described in embodiment 101, wherein the transgene is delivered to the target tissue.

[0117] 105. A pharmaceutical composition for use in delivering a transgene to a cell, the pharmaceutical composition comprising an rAAV vector described in embodiment 101, and the transgene being delivered to the cell.

[0118] 106. A pharmaceutical composition for use in delivering a transgene to a target tissue of a subject in need thereof, the pharmaceutical composition comprising an rAAV vector described in embodiment 101, and the transgene being delivered to the target tissue.

[0119] 107. The method or pharmaceutical composition for use according to embodiments 102 to 106, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, or intravitreally.

[0120] 108. The method or pharmaceutical composition for use according to embodiments 102 to 106, wherein the vector is administered via lumbar puncture or via the cisterna magna. [Brief description of the drawings]

[0121] [Figure 1] FIG. 1 shows a comparison of the capsid amino acid sequence including the adeno-associated virus type 9 VR-IV loop (AAV9 VR-IV) from residues L447 to R476 (residues 451-459 are shown in brackets) with corresponding regions in other AAVs. The figure discloses SEQ ID NOs: 87-92, 88, and 93-96, respectively, in order of appearance. [Diagram 2] FIG. 1 shows a protein model of the AAV capsid structure, showing the capsid variable regions VR-IV, VR-V, and VR-VIII. The box highlights the loop region of VR-IV that provides the surface-exposed amino acids as represented in the model. [Diagram 3] High packaging efficiency (titer) in terms of genome copies per mL (GC / mL) is shown for wild-type AAV9 and eight candidate modified rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097), each of which contains a FLAG insert immediately following a different site in the AAV9 VR-IV from residues I451 to Q458. All vectors were packaged with the luciferase transgene in 10 mL cultures; error bars represent standard error of the mean. [Figure 4] Figure 1 demonstrates surface exposure of the lVR-IV loop FLAG insert in each of eight candidate modified rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097), as confirmed by immunoprecipitation of packaged vectors by binding to anti-FLAG resin. [Diagram 5] Figure 1 shows transduction efficiency in Lec2 cells transduced with a capsid vector carrying the luciferase gene (as a transgene) packaged into either wild-type AAV9 (9-luc) or each of eight candidate modified (FLAG peptide inserted) rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097); transduction activity is expressed as percent luciferase activity (A), taking the activity of 9-luc as 100%, or as relative light units (RLU) per microgram of protein (B). [Figure 6A]1 shows a bar graph illustrating that inserts of various peptide lengths and compositions immediately after S454 of AAV9 can affect the efficiency of AAV particle production in packaging cells. Ten peptides of various compositions and lengths were inserted after S454 in AAV9 VR-IV. qPCR was performed on harvested supernatants of transfected suspension HEK293 cells 5 days after transfection. The results shown in the bar graph demonstrate that insert quality affects the ability of AAV particles to be produced and secreted by HEK293 cells, as indicated by the overall yield (titer). (Error bars represent standard error of the average peptide length marked in brackets on the Y-axis). [Figure 6B] Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 6C] Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 6D]Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 6E] Fluorescence images of transduced cell cultures of the following cell lines are shown: (6B) Lec2 cell line, (6C) HT-22 cell line, (6D) hCMEC / D3 cell line, and (6E) C2C12 cell line. AAV9 wild type and S454 inserted homing peptide capsids containing a GFP transgene were used to transduce the aforementioned cell lines. The P1 vector was not included in the images due to extremely low transduction efficiency, and the P8 vector was not included due to low titer. AAV9.S454.FLAG showed low transduction levels in all cell types tested. [Figure 7A] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7B] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7C] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7D] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7E] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7F] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7G]1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7H] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7I] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7J] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7K] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7L] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 7M] 1 shows the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14, and 17. [Figure 8-1] Alignment of AAV1-9e, rh10, rh20, rh39, and rh74 version 1 and version 2 with insertion sites for heterologous peptides after the VP2 start codon and in or near variable region 1 (VR-I), variable region 4 (VR-IV), and variable region 8 (VR-VIII) (all highlighted in gray), with the specific insertion site in variable region 8 (VR-VIII) of each capsid protein indicated by the symbol "#" (after amino acid residue 588 according to the AAV9 amino acid numbering). [Figure 8-2] This is a continuation of Figure 8-1. [Figure 8-3] This is a continuation of Figure 8-1. [Figure 8-4] This is a continuation of Figure 8-1. [Figure 8-5] This is a continuation of Figure 8-1. [Figure 9]1 shows the amino acid sequence for the recombinant AAV9 vector containing a peptide insert of ARA290 between Q588 and A589 (SEQ ID NO: 153); the ARA290 insert is shown in bold. [Figure 10] GFP (green fluorescent protein) transgene copies in mouse brain cells following administration of the AAV vectors: AAV9; AAV.PHP.eB, also referred to herein as AAV9e (AAV9 with the peptide TLAVPFK (sequence number 27) inserted between positions 588 and 589 and the modifications A587D / A588G); AAV.hDyn (AAV9 with TLAAPFK (sequence number 2) between positions 588 and 589); AAV.PHP.S (AAV9 with the peptide QAVRTSL (sequence number 23) between positions 588 and 589); and AAV.PHP.SH (AAV9 with the peptide QAVRTSH (sequence number 24) inserted between positions 588 and 589). [Figure 11] 1 shows the amino acid sequences for recombinant AAV9 vectors containing TLAAPFK (SEQ ID NO:2) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the TLAAPFK (SEQ ID NO:2) inserts shown in bold. [Figure 12] 1 shows the amino acid sequences for recombinant AAV9 vectors containing the KMQVPFQ (SEQ ID NO: 1) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the KMQVPFQ (SEQ ID NO: 1) inserts shown in bold. [Figure 13] 1 shows the amino acid sequences for recombinant AAV9 vectors containing peptide inserts of QQAAPSF (SEQ ID NO: 3) between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the QQAAPSF (SEQ ID NO: 3) inserts shown in bold. [Figure 14]1 shows the amino acid sequences for recombinant AAV9 vectors containing RYNAPFK (SEQ ID NO: 4) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the RYNAPFK (SEQ ID NO: 4) inserts shown in bold. [Figure 15] 1 shows the amino acid sequences for recombinant AAV9 vectors containing LKLPPIV (SEQ ID NO:5) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the LKLPPIV (SEQ ID NO:5) inserts shown in bold. [Figure 16] 1 shows the amino acid sequences for recombinant AAV9 vectors containing PFIKPFE (SEQ ID NO: 6) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the PFIKPFE (SEQ ID NO: 6) inserts shown in bold. [Figure 17] 1 shows the amino acid sequences for recombinant AAV9 vectors containing the TLSLPWK (SEQ ID NO: 7) peptide inserts between Q588 and A589 (A), between S268 and S269 in VR-III (B), and between S454 and G455 in VR-IV (C), with the TLSLPWK (SEQ ID NO: 7) inserts shown in bold. [Figure 18] 1 shows the amino acid sequences for recombinant AAV8 vectors containing the LGETTRP (SEQ ID NO: 15) peptide inserts between N590 and T591 (A), between A269 and T270 in VR-III (B), and between T453 and T454 in VR-IV (C), with the LGETTRP (SEQ ID NO: 15) inserts shown in bold. [Figure 19]1 shows the amino acid sequences for recombinant AAV8 vectors containing the LALGETTRP (SEQ ID NO: 16) peptide inserts between N590 and T591 (A), between A269 and T270 in VR-III (B), and between T453 and T454 in VR-IV (C), with the LALGETTRP (SEQ ID NO: 16) inserts shown in bold. [Figure 20] Shown is an in vitro Transwell assay for AAV vectors crossing the blood-brain barrier (BBB) ​​cell layer (A) and results showing that AAV.hDyn (indicated by inverted triangles) crosses the BBB cell layer of the assay faster than AAV9 (squares) and faster and to a greater extent than AAV2 (circles) (B). [Figure 21] Figure 8 shows the results of next generation sequencing (NGS) analysis of brain gDNA from mice administered pools of modified and native capsids intravenously, revealing the relative abundance in mouse tissues of the different capsids in the pools. Three different pools were injected into mice. The dotted lines indicate which vectors were pooled together. Parental AAV9 was included in each pool as a control (pool 1: BC01, pool 2: BC31, pool 3: BC01). Barcodes for each capsid in the pools are listed in Tables 8a-8c. [Figure 22] Figure 1 shows the in vivo transduction profile of AAV.hDyn in female C57Bl / 6 mice, showing copy number / microgram gDNA in brain (A), liver (B), heart (C), lung (D), kidney (E), skeletal muscle (F), sciatic nerve (G), and ovary (H) in naive mice or mice injected with either AAV9 or AAV.hDyn, with AAV.hDyn demonstrating increased brain biodistribution compared to AAV9. [Figure 23] Images of immunohistochemical staining of brain sections from the striatum (A), hippocampus (B), and cortex (C) showing the distribution of GFP from AAV.hDyn throughout the brain reveal global transduction of the brain with the engineered vector. [Figure 24]1 shows the in vivo kidney to liver transduction efficiency ratios of genetically modified AAV9 vectors containing a homing peptide insert immediately following amino acid 454. Details of the homing peptides used in this study are outlined in Table 8. [Diagram 25] 1 shows the amino acid sequence for a recombinant AAV9 vector containing a TLAVPFK (SEQ ID NO:27) peptide insert between S454 and G455 of VR-IV, with the TLAVPFK (SEQ ID NO:27) insert shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0122] Recombinant adeno-associated viruses (rAAVs) are provided having capsid proteins modified to include amino acid sequences that confer and / or enhance desired properties, such as tissue targeting, transduction, and integration of the rAAV genome. In particular, modified capsid proteins are provided that include peptide inserts of 4-20, or 7 contiguous amino acids, and in embodiments, 12 or fewer contiguous amino acids, from a heterologous protein in or near the variable region IV (VR-IV) of the viral capsid, such that the peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle. Also provided are recombinant capsid proteins, and rAAVs containing them, that have peptides inserted therein that target specific tissues and / or promote rAAV cellular uptake, transduction, and / or genome integration, such as from the dimerization domain of the heavy chain tail region of human axonemal dynein and others described herein (see Tables 1A and 1B).

[0123] Modified capsids having one or more amino acid substitutions that facilitate transduction and / or tissue tropism as described herein are also provided. Recombinant vectors containing the capsid proteins are also provided, along with pharmaceutical compositions thereof, nucleic acids encoding the capsid proteins, and methods of making and using the capsid proteins and rAAV vectors with modified capsids for targeted delivery, improved transduction and / or treatment of disorders associated with target tissues. In particular, compositions containing rAAV and methods of using capsid proteins containing peptides derived from erythropoietin or dynein or related to dynein to target rAAV to neural tissues, including the retina and / or central nervous system, and to facilitate delivery of therapeutic agents for treating neurological disorders and / or disorders of the eye, particularly the retina, are provided. Compositions containing rAAV containing peptide inserts that target or home to target tissues, such as bone, kidney, muscle, lung, retina, and heart, and methods of using the same are also provided.

[0124] 5.1.Definition The term "AAV" or "adeno-associated virus" refers to a dependoparvovirus within the Parvoviridae genus of viruses. The AAV may be derived from a naturally occurring "wild-type" virus, a rAAV genome packaged in a capsid containing a capsid protein encoded by a naturally occurring cap gene, and / or a rAAV genome packaged in a capsid containing a capsid protein encoded by a non-naturally occurring capsid cap gene. Examples of the latter include rAAVs having capsid proteins that contain peptide inserts within the amino acid sequence of the naturally occurring capsid.

[0125] The term "rAAV" refers to "recombinant AAV." In some embodiments, a recombinant AAV has an AAV genome in which some or all of the rep and cap genes have been replaced with heterologous sequences.

[0126] The term "rep-cap helper plasmid" refers to a plasmid that provides viral rep and cap gene functions and assists in the production of AAV from rAAV genomes that lack functional rep and / or cap gene sequences.

[0127] The term "cap gene" refers to a nucleic acid sequence that encodes a capsid protein that forms or helps form the capsid coat of a virus. In the case of AAV, the capsid protein can be VP1, VP2, or VP3.

[0128] The term "rep gene" refers to a nucleic acid sequence that encodes nonstructural proteins necessary for viral replication and biogenesis.

[0129] As used herein, the terms "nucleic acid" and "nucleotide sequence" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA / RNA molecules, and analogs of DNA or RNA molecules. Such analogs may be generated using nucleotide analogs, including, but not limited to, inosine or tritylated bases. Such analogs may also include DNA or RNA molecules containing modified backbones that confer beneficial attributes to the molecule, such as, for example, nuclease resistance or increased ability to cross cell membranes. A nucleic acid or nucleotide sequence may be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions, may contain triple-stranded portions, but is preferably double-stranded DNA.

[0130] As used herein, the terms "subject," "host," and "patient" are used interchangeably. As used herein, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), or, in certain embodiments, a human.

[0131] As used herein, the term "therapeutic agent" refers to any agent that can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is related to the function provided by the transgene. As used herein, "therapeutically effective amount" refers to the amount of agent (e.g., the amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder when administered to a subject suffering from the same. Furthermore, a therapeutically effective amount in reference to an agent of the present invention means that the amount of agent alone or in combination with other therapies provides at least one therapeutic benefit in the treatment or management of the disease or disorder.

[0132] As used herein, the term "prophylactic agent" refers to any agent that can be used in preventing, delaying, or slowing the progression of a disease or disorder, where the disease or disorder is related to the function provided by the transgene. As used herein, "prophylactically effective amount" refers to an amount of prophylactic agent (e.g., the amount of product expressed by the transgene) that provides at least one prophylactic benefit in preventing or delaying a target disease or disorder when administered to a subject predisposed thereto. A prophylactically effective amount can also refer to an amount of agent sufficient to prevent or delay the onset of a target disease or disorder; or slow the progression of a target disease or disorder; an amount sufficient to delay or minimize the onset of a target disease or disorder; or an amount sufficient to prevent or delay recurrence or spread. A prophylactically effective amount can also refer to an amount of agent sufficient to prevent or delay the worsening of symptoms of a target disease or disorder. Furthermore, a prophylactically effective amount in reference to a prophylactic agent of the present invention means that the amount of prophylactic agent alone or in combination with other agents provides at least one prophylactic benefit in preventing or delaying a disease or disorder.

[0133] The preventive agent of the present invention can be administered to a subject who is "predisposed" to the target disease or disorder. A subject who is "predisposed" to a disease or disorder is one who shows symptoms related to the development of the disease or disorder, or has genetic makeup, environmental exposure, or other risk factors for such a disease or disorder, but the symptoms are not yet at a level that can be diagnosed as the disease or disorder. For example, a patient who has a family history of a disease related to the missing gene (provided by the transgene) may qualify as being predisposed. In addition, a patient who has a latent tumor that persists after removal of the primary tumor may qualify as being predisposed to tumor recurrence.

[0134] "Central nervous system" ("CNS"), as used herein, refers to the nervous tissues reached by a circulating agent after crossing the blood-brain barrier, including, for example, the brain, optic nerves, cranial nerves, and spinal cord. The CNS also includes the central canal of the spinal cord and the cerebrospinal fluid that fills the ventricles of the brain.

[0135] 5.2. Recombinant AAV Capsids and Vectors One aspect relates to a capsid protein of a recombinant adeno-associated virus (rAAV), which is a capsid protein modified to include a peptide insert from a heterologous protein that is not an AAV protein, and the peptide insert is surface-exposed when packaged as an AAV particle. In some embodiments, the peptide insert is present in the variable region IV (VR-IV) of the AAV9 capsid, or the corresponding region for another type of AAV capsid (i.e., between two amino acids without deleting any capsid amino acids) (see alignment in Figure 8). In some embodiments, the peptide insert is present in the variable region VIII (VR-VIII) of the AAV9 capsid, or the corresponding region for another type of AAV capsid (i.e., between two amino acids without deleting any capsid amino acids) (see alignment in Figure 8). In some embodiments, the peptide insert is from a heterologous protein or domain (other than an AAV capsid protein or domain) that directs the rAAV particle to a target tissue and / or facilitates rAAV uptake, transduction and / or genome integration. Nucleic acids encoding the modified capsid proteins and variants thereof, packaging cells for expressing the nucleic acids to generate rAAV vectors, rAAV vectors further comprising a transgene, and pharmaceutical compositions of the rAAV vectors, as well as methods of using the rAAV vectors to deliver a transgene to a target cell type or tissue in a subject in need thereof, are also provided.

[0136] In various embodiments, the target tissue may be neural tissue, bone, kidney, muscle, eye / retina, or endothelial tissue, or a specific receptor or tumor, and the peptide insert is derived from a heterologous protein or domain that specifically recognizes and / or binds to the tissue, or, for example, one or more specific cell types, for example, within the target tissue, or its cellular matrix. In particular, peptides derived from erythropoietin or dynein, particularly those that bind to or are related to the heavy chain dimerization domain of axonemal dynein or cytoplasmic dynein, or cytoplasmic dynein inserted into any surface-exposed variable region, may target rAAV to neural tissue, including crossing the blood-brain barrier to the CNS and delivering therapeutic agents to treat neurological disorders.

[0137] 5.2.1 rAAV Vectors with Peptide Inserts The inventors have surprisingly discovered suitable locations for peptide insertions in and near the AAV9 capsid VR-IV loop (see FIG. 2) as well as in corresponding regions on the VR-IV loop of capsids of other AAV types. Previous studies have investigated potential locations in various AAVs, but none have identified the AAV9 VR-IV as suitable for this purpose (e.g., Wu et al, 2000, “Mutational Analysis of the Adeno-Associated Virus Type 2(AAV2)Capsid Gene and Construction of AAV2 Vectors with Altered Tropism,” J of Virology 74(18):8635-8647; Lochrie et al, 2006, “Adeno-associated virus(AAV)capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5,” Virology 353:68-82; Shi and Bartlett, 2003, “RGD Inclusion in VP3 Provides Adeno-Associated Virus Type 2(AAV2)-Based Vectors with a Heparan Sulfate-Independent Cell Entry Mechanism,” Molecular Therapy 7(4):515525-;Nicklin et al.,2001,“Efficient and Selective AAV2-Mediated Gene Transfer Directed to Human Vascular Endothelial Cells” Molecular Therapy 4(2):174-181;Grifman et al.,2001,“Incorporation of Tumor-Targeting Peptides into Recombinant Adeno-associated Virus Capsids,” Molecular Therapy 3(6):964-975;Girod et al.1999,“Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2,” Nature Medicine 3(9):1052-1056;Douar et al. al., 2003, “Deleterious effect of peptide insertions in a permissive site of the AAV2 capsid,” Virology 309:203-208; and Ponnazhagan, et al. 2001, J. of Virology 75(19):9493-9501). .

[0138] Thus, rAAV vectors are provided that carry peptide inserts at novel insertion points, particularly in surface-exposed variable regions in the capsid coat, particularly in or near variable region IV of the capsid protein. In some embodiments, the rAAV capsid protein comprises a peptide insert immediately following (i.e., connected by a peptide bond to the C-terminus) an amino acid residue corresponding to one of amino acids 451-461 of the AAV9 capsid protein (amino acid sequence SEQ ID NO: 118, see FIG. 8 for alignment of capsid protein amino acid sequences of other AAV serotypes with the amino acid sequence of the AAV9 capsid), and said peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle. The peptide insert should not delete any residues of the AAV capsid protein. Typically, the peptide insert is present in a variable (less conserved) region of the capsid protein compared to other serotypes, and in a surface-exposed loop.

[0139] A peptide insertion described as being inserted "at" a given site refers to an insertion immediately following (i.e., with a peptide bond to its carboxy group) the residue normally found at that site in the wild-type virus. For example, an insertion at Q588 in AAV9 means that the peptide insertion occurs between Q588 and the consecutive amino acid (A589) in the AAV9 wild-type capsid protein sequence (SEQ ID NO: 118). In embodiments, there is no deletion of amino acid residues at or near the insertion point (within 5, 10, 15 residues or within the structural loop that is the site of the insertion).

[0140] In certain embodiments, the capsid protein is an AAV9 capsid protein and the insertion is immediately following at least one of amino acid residues 451-461. In certain embodiments, the peptide insertion is immediately following amino acid I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid (amino acid sequence SEQ ID NO:118). In certain embodiments, the peptide is inserted between residues S454 and G455 of the AAV9 capsid protein or between residues corresponding to S454 and G455 of an AAV capsid protein other than the AAV9 capsid protein (amino acid sequence SEQ ID NO:118).

[0141] In other embodiments, modified capsid proteins are provided that contain a targeting peptide heterologous to the capsid protein inserted into the AAV capsid protein such that the heterologous peptide is surface exposed when incorporated into an AAV vector. Such peptides are preferably from the human axonemal dynein (HAD) heavy chain tail, or those listed in Tables 1A and 1B below, or other targeting peptides for specific tissue types.

[0142] In other embodiments, the capsid protein is from at least one AAV type selected from AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), and serotype rh74 (AAVrh74, versions 1 and 2) (see FIG. 8 ), and wherein the insertion is present immediately following an amino acid residue corresponding to at least one of amino acid residues 451-461. An alignment of these different AAV serotypes shows the "corresponding" amino acid residues in the different capsid amino acid sequences such that the "corresponding" amino acid residues are arranged in the same positions in the alignment as the residues in the reference sequence, as shown in Figure 8. In some particular embodiments, the peptide inserts are located at 450-459 of the AAV1 capsid (SEQ ID NO:110); 449-458 of the AAV2 capsid (SEQ ID NO:111); 449-459 of the AAV3 capsid (SEQ ID NO:112); 443-453 of the AAV4 capsid (SEQ ID NO:113); 442-445 of the AAV5 capsid (SEQ ID NO:114); 450-459 of the AAV6 capsid (SEQ ID NO:115); 451-461 of the AAV7 capsid (SEQ ID NO:116); 452-463 of the AAV8 capsid (SEQ ID NO:117); 453-454 of the AAV9 capsid (SEQ ID NO:118); 454-455 of the AAV10 capsid (SEQ ID NO:119); 455-456 of the AAV20 capsid (SEQ ID NO:120); 456-457 of the AAV30 capsid (SEQ ID NO:121); 457-458 of the AAV40 capsid (SEQ ID NO:122); 459-459 of the AAV50 capsid (SEQ ID NO:123); 451-463 of the AAV70 capsid (SEQ ID NO:124); 458-459 of the AAV80 capsid (SEQ ID NO:125); 459- 452 to 461 of the AAVrh20 capsid (SEQ ID NO:121); 452 to 461 of the AAVhu.37 (SEQ ID NO:122); 452 to 461 of the AAVrh74 (SEQ ID NO:123 or SEQ ID NO:154); or 452 to 461 of the AAVrh39 (SEQ ID NO:124).In certain embodiments, the rAAV capsid protein comprises a peptide insertion immediately following (i.e., at its C-terminus) amino acid 588 of the AAV9 capsid protein (having the amino acid sequence of SEQ ID NO:118, see FIG. 8), which is surface-exposed when the capsid protein is packaged as an AAV particle. In other embodiments, the rAAV capsid protein has a peptide insertion that is not immediately following amino acid 588 of AAV9 or corresponding to amino acid 588 of AAV9.

[0143] In other embodiments, the capsid protein is a targeting peptide comprising at least 4 contiguous amino acids, or at least 7 contiguous amino acids, or exactly 7 contiguous amino acids, but in embodiments no more than 12 contiguous amino acids, of Tables 1A and 1B, or a functional fragment thereof, of AAV serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype 9 (AAV9), serotype 10 (AAV10), serotype 11 (AAV11), serotype 12 (AAV12), serotype 13 (AAV13), serotype 14 (AAV14), serotype 15 (AAV15), serotype 16 (AAV16), serotype 17 (AAV17), serotype 18 (AAV18), serotype 19 (AAV19), serotype 20 (AAV20), serotype 21 (AAV21), serotype 22 (AAV22), serotype 23 (AAV23), serotype 24 (AAV24), serotype 25 (AAV25), serotype 26 (AAV26), serotype 27 (AAV27), serotype 28 (AAV28), serotype 29 (AAV29), serotype 30 (AAV30), serotype 31 (AAV31), serotype 32 (AAV32), serotype 33 (AAV33), serotype 34 (AAV34), s 8), serotype rh8 (AAVrh8), serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype rh39 (AAVrh39), serotype hu.37 (AAVhu.37), and serotype rh74 (AAVrh74, versions 1 and 2) (see FIG. 8), and the peptide is inserted into the capsid protein at any point such that the peptide is surface exposed when incorporated into the AAV vector.In certain embodiments, the peptide is selected from the group consisting of 138; 262-272; 450-459; or 585-593 of the AAV1 capsid (SEQ ID NO: 110); 138; 262-272; 449-458; or 584-592 of the AAV2 capsid (SEQ ID NO: 111); 138; 262-272; 449-459; or 585-593 of the AAV3 capsid (SEQ ID NO: 112); and 137; 256-257 of the AAV4 capsid (SEQ ID NO: 113). 62; 443-453; or 583-591; 137; 252-262; 442-445; or 574-582 of the AAV5 capsid (SEQ ID NO: 114); 138; 262-272; 450-459; 585-593 of the AAV6 capsid (SEQ ID NO: 115); 138; 263-273; 451-461; 586-594 of the AAV7 capsid (SEQ ID NO: 116); 138; 263-274 of the AAV8 capsid (SEQ ID NO: 117) 138 of AAV9 capsid (SEQ ID NO: 118); 262-273; 452-461; 585-593; 138 of AAV9e capsid (SEQ ID NO: 119); 262-273; 452-461; 585-593; 138 of AAVrh10 capsid (SEQ ID NO: 120); 263-274; 452-461; 587-595; 138 of AAVrh20 capsid (SEQ ID NO: 121); 263-274; 45 8) of the AAVrh39 capsid (SEQ ID NO:124), or after 138;263-274;452-461;587-595 (as numbered in FIG. 8) of the AAVrh74 capsid (SEQ ID NO:123 or SEQ ID NO:154).

[0144] In some embodiments, the capsid protein is from an AAV other than serotype AAV2. In some embodiments, the peptide insertion is not present immediately following the amino acid residues corresponding to amino acids 570 or 611 of the AAV2 capsid protein. In some embodiments, the peptide insertion is not present between the amino acid residues corresponding to amino acids 587-588 of the AAV2 capsid protein (see US 2014 / 0294771 to Schaffer et al.). In some embodiments, an insertion of bone 1 peptide having the amino acid sequence DDDDDDDD (SEQ ID NO:9) is not present immediately following amino acid 138 of the AAV2 capsid protein (see Almeciga-Diaz et al., 2018, Pediatr Res. 84:545).

[0145] AAV vectors comprising modified capsids are also provided. In some embodiments, the AAV vectors are non-replicative and do not contain nucleotide sequences encoding rep or cap proteins (which are provided by packaging cells in the manufacture of rAAV vectors). In some embodiments, the AAV-based vectors comprise components from one or more serotypes of AAV. In some embodiments, the AAV-based vectors provided herein include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.e B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or a combination of two or more thereof.In some embodiments, the AAV-based vectors provided herein are selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particle, or a combination of two or more thereof.In some embodiments, the rAAV particles are, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. , AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AA V.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.P HP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV AAV capsid proteins that are at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2 and / or VP3 sequences of an AAV capsid serotype selected from AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or derivatives, modifications, or pseudotypes thereof. These modified AAV vectors can include a genome that includes a transgene encoding a therapeutic protein.

[0146] In certain embodiments, the recombinant AAV for use in the compositions and methods herein is Anc80 or Anc80L65 (see, e.g., Zinn et al., 2015, Cell Rep. 12(6):1056-1068, which is incorporated by reference in its entirety). In certain embodiments, the recombinant AAV for use in the compositions and methods herein is AAV.7m8 (including variants thereof) (see, e.g., US9,193,956; US9,458,517; US9,587,282; US2016 / 0376323, and WO2018 / 075798, each of which is incorporated by reference in its entirety). In certain embodiments, the AAV for use in the compositions and methods herein is any AAV disclosed in US9,585,971, e.g., AAV-PHP.B. In certain embodiments, the AAV for use in the compositions and methods herein is an AAV2 / Rec2 vector or an AAV2 / Rec3 vector having a hybrid capsid sequence derived from AAV8 and serotypes cy5, rh20, or rh39 (see, e.g., Issa et al., 2013, PLoS One 8(4):e60361, which is incorporated herein by reference for these vectors). In certain embodiments, the AAV for use in the compositions and methods herein is an AAV disclosed in any of the following, each of which is incorporated herein by reference in its entirety: US,282,199; US7,906,111; US8,524,446; US8,999,678; US8,628,966; US8,927,514; US8,734,809; US9,284,357; US9,409,953; US9,169,299; US9,193,956; US9,458,517; US9,587,282; US2015 / 0374803; US2015 / 0126588; US2017 / 0067908; US2013 / 0224836; US2016 / 0215024; US2017 / 0051257; PCT / US2015 / 034799; and PCT / EP2015 / 053335.In some embodiments, the rAAV particles are disclosed in accordance with the following patents and patent applications, each of which is incorporated by reference in its entirety: U.S. Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; U.S. Patent Application Publication Nos. 2015 / 0374803; 2015 / 0126588; 2017 / 006 7908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; and International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335; or any of the other International Patent Application Nos. PCT / EP2015 / 053335, which are at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical capsid proteins, to the VP1, VP2 and / or VP3 sequences of the AAV capsid disclosed in any of the following International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335.

[0147] In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in U.S. Patent No. 9,840,719 and WO2015 / 013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO2014 / 172669, such as AAV rh.74, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise the AAV2 / 5 capsid, as described in Georgiadis et al., 2016, Gene Therapy 23:857-862 and Georgiadis et al., 2018, Gene Therapy 25:450, each of which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise any of the AAV capsids disclosed in WO2017 / 070491, such as AAV2tYF, which is incorporated by reference in its entirety. In some embodiments, the rAAV particles are capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9):418, which is incorporated by reference in its entirety. In some embodiments, the rAAV particles comprise any AAV capsid disclosed in U.S. Patent Nos. 8,628,966; US 8,927,514; US 9,923,120, and WO 2016 / 049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16 (each of which is incorporated by reference in its entirety).

[0148] In some embodiments, the rAAV particles are prepared using the methods described in International Application Publication Nos. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2 of the '051 publication), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of the '321 publication), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of the '888 publication), WO 2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of the '689 publication), WO 200 No. 9 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of the '964 publication), WO2010 / 127097 (see, e.g., SEQ ID NOs: 5-38 of the '097 publication), and WO2015 / 191508 (see, e.g., SEQ ID NOs: 80-294 of the '508 publication), and U.S. Application Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of the '924 publication) (the contents of each of which are incorporated by reference in their entirety herein).In some embodiments, the rAAV particles are prepared by the methods described in International Application Publication Nos. WO 2003 / 052051 (see, e.g., SEQ ID NO: 2 of the '051 publication), WO 2005 / 033321 (see, e.g., SEQ ID NOs: 123 and 88 of the '321 publication), WO 03 / 042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO 2006 / 068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of the '888 publication), WO 2006 / 110689 (see, e.g., SEQ ID NOs: 5-38 of the '689 publication), WO 2009 / 104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24, and 31 of the '964 ...52051 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO 2009 / 1062051 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of the '397 publication), WO 2009 / 1052051 (see, e.g., SEQ ID NOs: 2, 81 and / or VP3 sequences of the AAV capsid disclosed in U.S. Patent Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of the '924 publication).

[0149] In additional embodiments, the rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsid is a rAAV2 / 8 or rAAV2 / 9 pseudotyped AAV capsid. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001)).

[0150] In certain embodiments, single-stranded AAV (ssAAV) may be used. In certain embodiments, self-complementary vectors, such as scAAV, may be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, 8(16):1248-1254; US 6,596,535; US 7,125,717; and US 7,456,683, each of which is incorporated herein by reference in its entirety).

[0151] Typically, a peptide insert is a sequence of contiguous amino acids from a heterologous protein or domain thereof. The inserted peptide is typically long enough to retain a particular biological sample function, property, or characteristic of the protein or domain from which it is derived. The inserted peptide is typically short enough to allow the capsid protein to form a coat similarly or substantially similarly to the native capsid protein without the insert. In preferred embodiments, the peptide insert is about 4 to about 30 amino acid residues in length, about 4 to about 20, about 4 to about 15, about 5 to about 10, or about 7 amino acids in length. The peptide sequence for insertion is at least 4 amino acids in length and can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide sequence is 16, 17, 18, 19, or 20 amino acids in length. In embodiments, the peptide is no more than 7, 10, or 12 amino acids in length.

[0152] A "peptide insertion from a heterologous protein" in an AAV capsid protein refers to an amino acid sequence that is introduced into the capsid protein and is not native to any AAV serotype capsid. Non-limiting examples include peptides of human proteins in an AAV capsid protein.

[0153] In some embodiments, the peptide insert is from a homing protein or homing domain thereof or a targeting protein or targeting domain thereof. A "homing domain" or "homing protein" is a domain or protein that preferentially or selectively targets a particular cell type, tissue type, organ, tumor type, etc., including the cellular matrix of a particular cell type, compared to other cells, tissues, organs, or tumors. In the context of the present invention, a peptide from a homing protein or domain can provide a peptide for insertion into a capsid protein to form part of the capsid coat or AAV vector, which can then direct the capsid, coat, or vector to target a particular cell type, tissue type, organ, tumor type, etc., or promote uptake and / or integration of the AAV genome. Non-limiting examples of homing proteins or domains include neural tissue homing domains, axonemal or cytoplasmic dynein homing domains, bone homing domains, kidney homing domains, muscle homing domains, endothelial cell homing domains, retinal cell homing domains, domains that target specific cell receptors, such as integrin receptor binding domains and transferrin receptor binding domains, tumor cell targeting domains, targeting peptides from other viruses, and the like. As used herein, the terms "homing" and "targeting" are used interchangeably. These peptides may also, or alternatively, facilitate cellular uptake, transduction, and / or genomic integration of rAAV in cells of the target tissue.

[0154] Examples of peptides for use as peptide inserts as any of the AAV capsid sites described herein are provided in Tables 1A-1B below, including at least a 4 amino acid contiguous portion thereof, or a 7 amino acid contiguous portion thereof, in some embodiments, 12 or fewer contiguous amino acids, that have the functional attributes of the peptide. See also, e.g., Laakkonen and Vuorinen, 2010, "Homing peptides as targeted delivery vehicles," Integrative Biology, 2:326-337 (review article). In certain embodiments, recombinant AAV capsids and AAV vectors are modified to include a peptide from any of Tables 1A and 1B below, or at least a 4, 5, 6, or 7 amino acid contiguous portion thereof, inserted into the AAV capsid sequence in such a way that the peptide insert is presented. In other embodiments, the peptide is inserted after amino acid residues at positions 138, 262-273, 451-461, or 585-593 of the amino acid sequence of the AAV9 capsid (SEQ ID NO:118) or the corresponding positions in any other AAV serotype (see Figure 8 for capsid sequence alignment). [Table 1A] [Table 1B] TIFF0007619958000003.tif52168 [Table 2] [Table 3A] TIFF0007619958000006.tif251169TIFF0007619958000007.tif188167 [Table 3B] TIFF0007619958000009.tif254168TIFF0007619958000010.tif195168

[0155] For example, a rAAV vector containing a transgene encoding glial derived growth factor (GDGF) is used to treat / prevent / manage Parkinson's disease. Typically, the rAAV vector is administered systemically. For example, the rAAV vector can be provided by intravenous, intrathecal, intranasal, and / or intraperitoneal administration.

[0156] In certain embodiments, the rAAV of the present invention is utilized for delivery to a target tissue or a target cell type (associated with the disorder or disease to be treated / prevented), including a cellular matrix associated with the target cell type. A disease or disorder associated with a particular tissue or cell type is one that affects the particular tissue or cell type more than other tissues of the body, or where the effects or symptoms of the disorder are manifested in a particular tissue or cell type. A method of delivering a transgene to a target tissue of a subject in need thereof includes administering to the subject an rAAV whose peptide insert is a homing peptide. In the case of Parkinson's disease, for example, an rAAV vector can be used that includes a peptide insert that directs the rAAV to neural tissue, in particular, the peptide insert facilitates the rAAV to cross the blood-brain barrier to the CNS. Such peptide inserts include those derived from neural tissue homing domains, such as the "EPO peptide" or "HAD peptide" described herein.

[0157] For example, capsid proteins containing EPO peptides can be used to retarget AAV to the CNS and cross the blood-brain barrier. Capsid proteins containing EPO peptides can further have a protective effect on CNS tissue, for example, EPO inserts bind to natural repair receptors, activate IRR biological switches, suppress inflammation, and / or initiate CNS repair. In some embodiments, rAAV containing the EPO peptides of the invention are used for one or more of the following disorders: organ ischemic injury, stroke, myocardial infarction, kidney injury, kidney disease, brain injury, kidney ischemia, limb ischemia, autoimmune encephalomyelitis, autoimmune neuritis, multiple sclerosis, Guillain-Barre syndrome, neuropathic pain, diabetic complications such as diabetic retinopathy and diabetic autonomic neuropathy, and sarcoidosis.

[0158] For diseases or disorders associated with nervous tissue, rAAV vectors containing peptide inserts from nervous tissue homing domains, such as those described herein, may be used. Diseases / disorders associated with nervous tissue include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis (ALS), Batten disease, Batten juvenile NCL, Canavan disease, chronic pain, Friedreich's ataxia, glioblastoma multiforme, Huntington's disease, late infantile neuronal ceroid lipofuscinosis (LINCL), lysosomal storage disorders, Leber's congenital amaurosis, multiple sclerosis, Parkinson's disease, Pompe disease, Rett syndrome, spinal cord injury, spinal muscular atrophy (SMA), stroke, and traumatic brain injury. The vector may further contain a transgene for therapeutic / prophylactic benefit to subjects suffering from or at risk of developing a disease or disorder (see Tables 3A-3B).

[0159] For bone-related diseases or disorders, rAAV vectors that contain peptide inserts from bone-homing domains, such as those described herein, can be used.

[0160] For diseases or disorders related to the kidney, rAAV vectors that contain peptide inserts from kidney-homing domains, such as those described herein, can be used.

[0161] For muscle-related diseases or disorders, rAAV vectors that contain peptide inserts from muscle-homing domains, such as those described herein, can be used.

[0162] For diseases or disorders associated with endothelial cells, rAAV vectors containing peptide inserts from endothelial cell homing domains, such as those described herein, can be used.

[0163] For diseases or disorders associated with integrin receptors or cells expressing specific integrin receptors, rAAV vectors containing peptide inserts from integrin receptor binding domains, such as those described herein, can be used.

[0164] For diseases or disorders associated with the transferrin receptor or cells expressing the transferrin receptor, rAAV vectors that contain peptide inserts from the transferrin receptor binding domain, such as those described herein, can be used.

[0165] For tumor-related diseases or disorders, rAAV vectors containing peptide inserts from the tumor cell targeting domain can be used.

[0166] For diseases or disorders related to the retina or eye, rAAV vectors containing peptide inserts from the retinal cell homing domain, including HAD peptides, can be used. The peptide inserts enhance retinal tropism and guide the rAAV to target the subject's eye or retina and cross the blood-ocular barrier. The term "retinal cells" refers to one or more of the cell types found in or near the retina, including amacrine cells, bipolar cells, horizontal cells, Muller glial cells, photoreceptor cells (e.g., rods and cones), retinal ganglion cells (e.g., midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, and photosensitive ganglion cells), retinal pigment epithelium, endothelial cells of the inner limiting membrane, and the like.

[0167] Typically, when the rAAV vector contains a peptide insert for retinal cell homing, the vector is administered by in vivo injection, for example, by direct injection into the eye. For example, the rAAV containing a peptide insert for improving retinal tropism can be administered intravitreally. In some embodiments, the rAAV for improving retinal tropism is administered by intraocular injection, for example, via the pars plana into the vitreous or aqueous humor of the eye. In some embodiments, the rAAV for improving retinal tropism is administered by periocular injection or subconjunctival injection. One advantage of the rAAV vector with a peptide insert for retinal cell homing is that the subject can avoid surgery, for example, avoid surgery to implant a therapeutic agent that is instead delivered by injection. In certain embodiments, the therapeutic agent is delivered by the rAAV vector described herein by intravitreal injection to provide a therapeutically effective amount for treating eye-related diseases or disorders, particularly diseases or disorders related to the retina of the subject. In more embodiments, treatment is achieved after a single intravitreal injection, no more than two intravitreal injections, no more than three intravitreal injections, no more than four intravitreal injections, no more than five intravitreal injections, or no more than six intravitreal injections.

[0168] Diseases / disorders related to the eye or retina are referred to as "ophthalmic diseases". Non-limiting examples of ophthalmic diseases include anterior ischemic optic neuropathy; acute macular neuroretinopathy; Bardet-Biedl syndrome; Behcet's disease; branch retinal vein occlusion; central retinal vein occlusion; congenital choroideremia; choroidal neovascularization; chorioretinal degeneration; cone-rod dystrophies; color vision disorders (e.g., achromatopsia, protanopia, deuteranopia, and tritanopia); congenital stationary night blindness; diabetic uveitis; epiretinal membrane disorders; hereditary macular degeneration; histoplasmosis; macular degeneration (e.g., acute macular degeneration, nonexudative age-related macular degeneration, exudative age-related macular degeneration); diabetic retinopathy; edema ( For example, macular edema, cystoid macular edema, diabetic macular edema); glaucoma; Leber's congenital amaurosis; Leber's hereditary optic neuropathy; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular trauma; ocular tumors; proliferative vitreoretinopathy (PVR); retinopathy of prematurity; retinoschisis; retinitis pigmentosa; retinal artery occlusive disease, retinal detachment, Stargardt disease (fundus flaviforme); sympathetic ophthalmia; uveal diffusion; uveitic retinal disease; Usher syndrome; Vogt-Koyanagi-Harada (VKH) syndrome; or posterior ocular conditions associated with ocular laser or photodynamic therapy.

[0169] The rAAV vector of the present invention can also facilitate the delivery, particularly targeted delivery, of oligonucleotides, drugs, contrast agents, inorganic nanoparticles, liposomes, and antibodies to target cells or tissues.The rAAV vector can also facilitate the delivery, particularly targeted delivery, of non-coding DNA, RNA, or oligonucleotides to target tissues.

[0170] The agents can be provided in pharma- ceutically acceptable compositions known in the art and / or described herein, and the rAAV molecules of the invention can be administered alone or in combination with other prophylactic and / or therapeutic agents.

[0171] The dosage and frequency of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage and frequency will typically vary according to factors specific to each patient, depending on the particular therapeutic or prophylactic agent being administered, the severity and type of disease, the route of administration, and the age, weight, response, and medical history of the patient, and should be determined according to the judgment of the practitioner and each patient's circumstances. Suitable regimens will be determined by considering such factors and will be described, for example, in the literature and in the Physician's Desk Reference (56 th The dosages may be selected by one skilled in the art according to the dosages recommended in The American Journal of Clinical Oncology, vol. 13, no. 1, ed., 2002. Prophylactic and / or therapeutic agents may be administered repeatedly. Several aspects of the procedure may vary, such as the temporal regimen for administering prophylactic or therapeutic agents, and whether such agents are administered separately or as a mixture.

[0172] The amount of an agent of the invention that will be effective can be determined by standard clinical techniques. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. For any agent used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Doses can be determined based on the IC 50 The animal model can be designed to achieve a circulating plasma concentration range that includes (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0173] The preventive and / or therapeutic agents and their combinations can be tested in suitable animal model systems before use in humans. Such animal model systems include, but are not limited to, rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system well known in the art can be used. Such model systems are widely used and well known to those skilled in the art. In some embodiments, animal model systems for CNS pathology based on rats, mice, or other small mammals other than primates are used.

[0174] Once the prophylactic and / or therapeutic agents of the present invention are tested in animal models, they can be tested in clinical trials to establish their effectiveness.The establishment of clinical trials can be carried out according to the general methodology known to those skilled in the art, and the optimal dosage and administration route of the agent of the present invention as well as the toxicity profile can be established.For example, clinical trials can be designed to test the rAAV molecules of the present invention for efficacy and toxicity in human patients.

[0175] The toxicity and efficacy of the prophylactic and / or therapeutic agents of the present invention can be evaluated, for example, by LD 50 (lethal dose for 50% of the population) and ED 50 To determine the dose that is therapeutically effective in 50% of the population, a standard pharmaceutical procedure can be used in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the ratio LD 50 / ED 50 The therapeutic index may be expressed as: Prophylactic and / or therapeutic agents that exhibit a high therapeutic index are preferred. Prophylactic and / or therapeutic agents that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such agents to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0176] The rAAV molecules of the invention are typically administered for a time and in an amount effective to obtain the desired therapeutic and / or prophylactic benefit. Data obtained from cell culture assays and animal studies can be used in designing ranges and / or schedules for the dosage of prophylactic and / or therapeutic agents for use in humans. Dosages of such agents are administered at an ED with little or no toxicity. 50 The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0177] The therapeutically effective dosage of a rAAV vector for a patient is typically about 1×10 9 ~Approx. 1×10 16 Genomic rAAV vector, or approximately 1 × 10 10 ~Approx. 1×10 15 , about 1×10 12 ~Approx. 1×10 16 , or approximately 1 × 10 14 ~Approx. 1×10 16 A solution containing a concentration of AAV genome of about 0.1 ml to about 100 ml. The level of expression of the transgene can be monitored to determine / adjust dosage, frequency, scheduling, etc.

[0178] Treatment of a subject with a therapeutically or prophylactically effective amount of the agent of the present invention may include a single treatment or may include a series of treatments.For example, the pharmaceutical composition containing the agent of the present invention may be administered once a day, twice a day, or three times a day.In some embodiments, the agent may be administered once a day, every other day, once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once a year.It is also understood that the effective dosage of a given agent, for example, the effective dosage of an agent containing the dual antigen-binding molecule of the present invention, may increase or decrease during the course of treatment.

[0179] In some embodiments, ongoing treatment is indicated, e.g., on a long-term basis, such as in the ongoing treatment and / or management of a chronic disease or disorder, For example, in certain embodiments, an agent of the invention is administered for a predetermined period of time, e.g., at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, or for the remainder of the life of the subject in need thereof.

[0180] The rAAV molecules of the present invention can be administered alone or in combination with other prophylactic and / or therapeutic agents. Each prophylactic or therapeutic agent can be administered sequentially in any order at the same or different time points; however, if not administered at the same time, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered separately in any suitable form and by any suitable route.

[0181] In various embodiments, different prophylactic and / or therapeutic agents are administered less than 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 to about 3 hours apart, about 3 to about 4 hours apart, about 4 to about 5 hours apart, about 5 to about 6 hours apart, about 6 to about 7 hours apart, about 7 to about 8 hours apart, about 8 to about 9 hours apart, about 9 to about 10 hours apart, about 10 to about 11 hours apart, about 11 to about 12 hours apart, no more than 24 hours apart, or no more than 48 hours apart. In certain embodiments, two or more agents are administered during the same patient visit.

[0182] Methods of administering the agents of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous (including infusion or bolus injection)), epidural, and by absorption through epithelial or mucocutaneous or mucosal linings (e.g., intranasal, oral mucosa, rectal, and intestinal mucosa, etc.). In certain embodiments, such as when the transgene is intended to be expressed in the CNS, the vector is administered via lumbar puncture or via the cisterna magna.

[0183] In certain embodiments, the agents of the invention are administered intravenously and may be administered together with other biologically active agents.

[0184] In another specific embodiment, the agent of the present invention may be delivered in a sustained release formulation, for example, where the formulation provides extended release and therefore extended half-life of the administered agent. Controlled release systems suitable for use include, but are not limited to, diffusion-controlled, solvent-controlled, and chemically-controlled systems. Diffusion-controlled systems include, for example, reservoir devices in which the molecules of the present invention are encapsulated within the device such that the release of the molecules is controlled by permeation through a diffusion barrier. Common reservoir devices include, for example, membranes, capsules, microcapsules, liposomes, and hollow fibers. Monolithic (matrix) devices are another type of diffusion-controlled system, in which the dual antigen-binding molecules are dispersed or dissolved in a rate-controlling matrix (e.g., a polymer matrix). The agent of the present invention may be homogeneously dispersed throughout the rate-controlling matrix, and the rate of release is controlled by diffusion through the matrix. Suitable polymers for use in monolithic matrix devices include naturally occurring polymers, synthetic polymers, and synthetically modified natural polymers, as well as polymer derivatives.

[0185] Any technique known to one of skill in the art can be used to produce sustained release formulations containing one or more of the agents described herein. For example, U.S. Patent No. 4,526,938; PCT Publication WO91 / 05548; PCT Publication WO96 / 20698; Ning et al., “Intratumoral Radioimmunotheraphy of a Human Colon Cancer Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology,50:372 397,1995;Cleek et al.,“Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro.Intl.Symp.Control.Rel.Bioact.Mater.,24:853 854,1997; and Lam et al.,“Microencapsulation of Recombinant Humanized Monoclonal Antibody for See, "Local Delivery," Proc. Int'l. Symp. Control Rel. Bioact. Mater., 24:759 760, 1997, each of which is incorporated herein by reference in its entirety. In one embodiment, a pump can be used in a controlled release system (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng., 14:20, 1987; Buchwald et al., Surgery, 88:507, 1980; and Saudek et al., N. Engl. J. Med., 321:574, 1989).In another embodiment, polymeric materials can be used to achieve controlled release of drugs comprising the biantigen-binding molecule, or antigen-binding fragments thereof (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NY (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem., 23:61, 1983; also, Levy et al., Science, 228:190, 1985; During et al., Ann. Neurol., 25:351, 1989; Howard et al., J. Neurosurg., 7:161, 1989). 1:105,1989); U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; PCT Publication No. WO 99 / 15154; and PCT Publication No. WO 99 / 20253). In yet another embodiment, the controlled release system is placed in the vicinity of the therapeutic target (e.g., the affected joint), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)). Other controlled release systems are discussed in the review by Langer, Science, 249:1527 1533,1990.

[0186] rAAVs can also be used for scientific research, e.g., in vivo delivery transgenes for optogenetics, gene knockdown with miRNA, recombinase delivery for conditional gene deletion, gene editing with CRISPR, etc.

[0187] 5.5. Pharmaceutical Compositions and KitsThe invention further provides pharmaceutical compositions comprising a pharma- ceutically acceptable carrier and an agent of the invention, the agent comprising an rAAV molecule of the invention. In some embodiments, the pharmaceutical composition comprises rAAV combined with a pharma- ceutically acceptable carrier for administration to a subject. In one embodiment, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's complete and incomplete adjuvants), excipient, or vehicle with which the agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. Further examples of pharma- ceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides; proteins, e.g., serum albumin and gelatin; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, e.g., EDTA; sugar alcohols, e.g., mannitol or sorbitol; salt-forming counterions, e.g., sodium; and / or non-ionic surfactants, e.g., TWEEN™, polyethylene glycol (PEG), and PLURONICS™, as known in the art.The pharmaceutical compositions of the present invention may also contain, in addition to the above-mentioned ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives. These compositions may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0188] In certain embodiments of the invention, a pharmaceutical composition is provided for use in accordance with the methods of the invention, said pharmaceutical composition comprising a therapeutically and / or prophylactically effective amount of an agent of the invention together with a pharma- ceutically acceptable carrier.

[0189] In certain embodiments, the agents of the present invention are substantially pure (i.e., substantially free of substances that limit their effectiveness or produce undesirable side effects). In certain embodiments, the host or subject is an animal, e.g., a mammal, e.g., a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) and a primate (e.g., monkeys, e.g., cynomolgus monkeys and humans). In certain embodiments, the host is a human.

[0190] The present invention further provides a kit that can be used in the above method.In one embodiment, the kit comprises, for example, one or more agents of the present invention in one or more containers.In another embodiment, the kit further comprises, for example, one or more other preventive or therapeutic agents useful for treating pathology in one or more containers.

[0191] The invention also provides an agent of the invention packaged in a sealed container, such as an ampoule or sachet, indicating the amount of agent or active agent. In one embodiment, the agent is supplied as a dry, sterile, lyophilized powder or water-free concentrate in a sealed container, which can be reconstituted, for example, with water or saline, to the appropriate concentration for administration to a subject. Typically, the agent is supplied as a dry, sterile, lyophilized powder in a sealed container in a unit dosage of at least 5 mg, more often at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, or at least 75 mg. The lyophilized agent should be stored between 2-8°C in its original container, and the agent should be administered within 12 hours, usually within 6 hours, 5 hours, 3 hours, or 1 hour after being reconstituted. In an alternative embodiment, the agent of the invention is supplied in liquid form in a sealed container indicating the amount and concentration of the agent or active agent. Typically, the liquid form of the drug is supplied in a sealed container at at least 1 mg / ml, at least 2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / kg, or at least 25 mg / ml.

[0192] The compositions of the present invention include bulk drug compositions (e.g., non-pure or non-sterile compositions) useful in the manufacture of pharmaceutical compositions and pharmaceutical compositions (i.e., compositions suitable for administration to a subject or patient). Bulk drug compositions may be used, for example, in the preparation of unit dosage forms containing a prophylactically or therapeutically effective amount of an agent disclosed herein or a combination of such an agent and a pharma- ceutically acceptable carrier.

[0193] The present invention further provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the agents of the present invention. Also, one or more other prophylactic or therapeutic agents useful for treating the target disease or disorder may also be included in the pharmaceutical pack or kit. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical composition of the present invention. Optionally associated with such container(s) may be a notice in a form prescribed by a governmental authority regulating the manufacture, use or sale of a drug or biological product, which notice reflects the approval by the authority of the manufacture, use or sale for human administration.

[0194] Usually, the components of the composition of the present invention are supplied separately or mixed together in unit dosage form, for example as dry lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of drug or active agent.When the composition is administered by injection, it can be dispensed with an injection bottle containing sterile pharmaceutical grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed before administration. EXAMPLES

[0195] The following examples report the analysis of surface-exposed loops on the AAV9 capsid to identify candidates for capsid modification via insertional mutagenesis. As an example, the present invention describes the construction of a modified rAAV9 capsid to contain a 7-mer peptide designed based on the human axonemal dynein heavy chain tail. Briefly, three criteria were used to select surface loops that may be suitable for short peptide insertions: 1) minimal side chain interactions with adjacent loops; 2) variable sequence and structure between serotypes (lack of conserved sequences); and 3) potential to interfere with commonly targeted neutralizing antibody epitopes. A panel of peptide insertion mutants was constructed and individual mutants were screened for viable capsid assembly, peptide surface exposure, and potency. The top candidates were then used as templates for the insertion of homing peptides to test whether these peptide insertion points could be used to retarget rAAV vectors to tissues of interest. Further examples demonstrate improved transduction and tissue tropism for some of the modified AAV capsids described herein.

[0196] 6.1. Example 1 - Analysis of AAV9 Capsids Figures 1 and 2 show the analysis and protein model (Figure 2) of variable region 4 of adeno-associated virus type 9 (AAV9 VR-IV) with amino acid sequence comparison to VR-IV of other AAVs (Figure 1). As can be seen, AAV9 VR-IV is exposed on the surface at the tip or outer surface of the three-fold spike. Further analysis showed that there are several side chain interactions between VR-IV and VR-V and that the sequence and structure of VR-IV are variable among AAV serotypes, and furthermore, there is the potential to interfere with commonly targeted neutralizing antibody epitopes, thereby reducing the immunogenicity of the modified capsid.

[0197] 6.2. Example 2 - Construction of AAV9 mutants Eight AAV9 variants were constructed, each containing a heterologous peptide at a different insertion point in the VR-IV loop. The heterologous peptide was a FLAG tag inserted immediately following the following residues in vectors identified as pRGNX1090-1097, as shown in Table 4. [Table 4]

[0198] 6.3. Example 3 - Packaging Efficiency Analysis 3 shows high packaging efficiency in terms of genome copies per mL (GC / mL) for wild-type AAV9 and eight candidate rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097), each containing a FLAG insertion at a different site within the VR-IV of AAV9. All vectors were packaged with a luciferase transgene in 10 mL cultures to facilitate determination of which insertion points disrupted capsid packaging; error bars represent standard error of the mean.

[0199] As can be seen, all candidates package with high efficiency.

[0200] 6.4. Example 4 - Analysis of Surface FLAG Exposure Figure 4 shows the surface exposure of the FLAG insert in each of eight candidate rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097), as confirmed by immunoprecipitation of the transduction vectors by binding to anti-FLAG resin. Binding to anti-FLAG indicates an insertion point that allows the formation of a capsid that displays the peptide insert on its surface.

[0201] The transduced cells were lysed and centrifuged. 500 μL of cell culture supernatant was loaded onto 20 μL of agarose-FLAG beads and eluted with SDS-PAGE loading buffer which was also directly loaded onto the gel. For negative control, 293-ssc supernatant containing no FLAG insert was used.

[0202] As can be seen, 1090 had the lowest titer of the candidate vectors and showed the least protein pulldown. Extremely low titers were also observed with the positive control. It appears that not enough of the positive control was loaded for visualization on SDS-PAGE.

[0203] 6.5. Example 5 - Analysis of Transduction Efficiency Figures 5A-5B show transduction efficiency in Lec2 cells transduced with capsid vectors carrying the luciferase gene as a transgene packaged either in wild-type AAV9 (9-luc) or in each of the eight candidate rAAV9 vectors (1090, 1091, 1092, 1093, 1094, 1095, 1096, and 1097); activity is expressed as percent luciferase activity, taking the activity of 9-luc as 100% (Figure 5A), or as relative light units (RLU) per microgram of protein (Figure 5B).

[0204] CHO-derived Lec2 cells were grown in αMEM and 10% FBS. Approximately 2 × 10 Lec2 cells were 8 Lec2 cells were transduced with an MOI of GC vector (approximately 10,000 MOI) and treated with ViraDuctin reagent (Lec2 cells were transduced with an MOI of approximately 10,000 GC / cell but with 40 μg / mL zinc chloride (ZnCl 2 Similar results were observed for those treated with ; results not shown. Lec2 cells are a proline auxotroph derived from CHO.

[0205] As can be seen, the in vitro transduction efficiency is lower than that obtained using wild-type AAV9 (9-luc). Nevertheless, previous studies have shown that the introduction of homing peptides can significantly increase in vitro gene transfer in target cells, whereas it can decrease in vitro gene transfer in non-target cells (such as 293, Lec2, or HeLa) (see, for example, Nicklin et al. 2001; and Grifman et al. 2001).

[0206] 6.6. Example 6 - Analysis of packaging efficiency as a factor of inserted peptide composition and length Figure 6A shows a bar graph illustrating that inserts of various peptide lengths and compositions immediately after S454 of the AAV9 capsid (SEQ ID NO: 118) can affect the efficiency of AAV particle production in packaging cell lines. Ten peptides of various compositions and lengths were inserted after S454 (between residues 454 and 455) in AAV9 VR-IV. qPCR was performed on harvested supernatants of transfected suspension HEK293 cells 5 days after transfection. The results shown in the bar graph demonstrate that insert quality and length can affect the ability of AAV particles to be produced and packaged at high titers in 293 cells. (Error bars represent standard error of the average length of the peptides marked in brackets on the Y axis).

[0207] AAV9 vectors with capsid proteins containing homing peptides of the following peptide sequences (Table 5) at the S454 insertion site were studied: Suspension-adapted HEK293 cells were cultured at 1 × 10 in 10 mL of medium one day before transduction. 6 Cells were seeded at 1000 cells / mL. Triple plasmid DNA transfections were performed using PEIpro® (Polypus transfection) at a DNA:PEI ratio of 1:1.75. Five days after transfection, cells were spun down and the supernatants were harvested and stored at -80°C. [Table 5]

[0208] qPCR was performed on the supernatants of transfected suspension HEK293 cells harvested 5 days after transfection. Samples were subjected to DNase I treatment to remove residual plasmid or cellular DNA, and then heat treated to inactivate DNase I and denature capsids. Samples were titered via qPCR using TaqMan Universal PCR Master Mix, No AmpEraseUNG (ThermoFisherScientific) and primers / probes against the PolyA sequence packaged in the transgene construct. A standard curve was established using the RGX-501 vector BDS.

[0209] While peptide inserts immediately after S454 ranging from 5 to 10 amino acids in length generated AAV particles with reasonable titers, there may be an upper size limit, as significant packaging defects were observed for peptide inserts with a length of 12 amino acids.

[0210] 6.7. Example 7 - Homing peptides alter the transduction properties of AAV9 in vitro when inserted after S454. Figures 6B-E show fluorescent images of cell cultures of (Figure 6B) Lec2 cell line (sialic acid-deficient epithelial cell line), (Figure 6C) HT-22 cell line (neuronal cell line), (Figure 6D) hCMEC / D3 cell line (brain endothelial cell line), and (Figure 6E) C2C12 cell line (muscle cell line). The aforementioned cell lines were transduced using AAV9 wild-type and S454-inserted homing peptide capsids from Table 5 containing a GFP transgene.

[0211] Cell lines were incubated with 5–20 × 10 3 Cells were seeded in 96-well plates at 1 × 10 cells / well (depending on cell line). Cells were inoculated with AAV9-GFP vector (with or without insert) at 1 × 10 10Cells were transduced with 10 ...

[0212] The images show that when inserted after S454 in the AAV9 capsid protein compared to the unmodified AAV9 capsid, it can alter the transduction properties of AAV9 in vitro. P7 (TfR1 peptide, HAIYPRH (SEQ ID NO: 17)) showed the highest transduction rate for all cell lines, followed by P9 (TfR3 peptide, RTIGPSV (SEQ ID NO: 19)). P4 (kidney 1 peptide, LPVAS (SEQ ID NO: 13)) showed slightly higher transduction rates than that of AAV9 wild type for all cell types. Higher transduction rates were observed for P6 (muscle 1 peptide, ASSLNIA (SEQ ID NO: 14)) in brain endothelial hCMEC / D3 cell line and C2C12 muscle cell line cultures compared to Lec2 and HT-22 cell line cultures. P1 vector was not included in the images due to extremely low transduction efficiency, and P8 vector was not included due to low titer.

[0213] 6.8. Example 8 - Analysis of Human Axonemal Dynein (HAD) 7A-7M show the amino acid sequences for the heavy chain tail domains of human axonemal dyneins 1-12, 14 and 17, respectively.

[0214] 6.9. Example 9 - Analysis of AAV Capsids for Peptide Insertion Sites FIG. 8 shows an alignment of AAV1-9e, rh10, rh20, rh39, rh74 and hu.37 capsid sequences within the VP2 start codon, variable region 1 (VR-I), variable region 4 (VR-IV), and insertion sites for human axonemal dynein peptides in or near variable region 8 (VR-VIII) highlighted in grey, with the specific insertion site within variable region 8 (VR-VIII) of each capsid protein indicated by the symbol "#" (after amino acid residue 588 according to the AAV9 amino acid numbering).

[0215] 6.10. Example 10 - Construction of rAAV Capsids Containing ARA290 FIG. 9 shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of ARA290 between Q588 and A589 of the AAV9 capsid amino acid sequence (SEQ ID NO: 153).

[0216] 6.11. Example 11 - Comparison of AAV genome copies / μg genomic DNA for various vectors Figure 10 shows copies of the GFP (green fluorescent protein) transgene expressed in mouse brain cells following administration of the AAV vectors: AAV9; AAV.PHP.eB; AAV.hDyn (AAV9 having TLAAPFK (sequence number 2) between 588 and 589 with no other amino acid modifications to the capsid sequence); AAV.PHP.S; and AAV.PHP.SH (see Table 10).

[0217] AAV.PHP.B is a capsid with a TLAVPFK (SEQ ID NO: 27) insert in the AAV9 capsid with no other amino acid modifications to the capsid sequence. AAV.PHP.eB is a capsid with a TLAVPFK (SEQ ID NO: 27) insert in the AAV9 capsid with two amino acid modifications upstream of the capsid sequence of the PHP.B insert (see also Table 10). Table 6A summarizes the capsids utilized in the study. [Table 6A]

[0218] Materials and Methods AAV9, AAV.PHPeB, AAV.hDyn, AAV.PHP.S, and AAV.PHP.SH constructs encoding the GFP transgene were prepared and formulated in 1x PBS + 0.001% Pluronic. Female C57BL / 6 mice were randomized into treatment groups based on body weight on day 1. Five groups of female C57BL / 6 mice were intravenously administered AAV9.GFP, AAV.PHPeB.GFP, AAV.hDyn.GFP, AAV.PHP.S.GFP, or AAV.PHP.SH.GFP, respectively, according to Table 6B below. Dosing volume was 10 mL / kg (0.200 mL / 20 g mouse). Mice were 8-12 weeks old on the starting day. On day 15 post-dosing, animals were euthanized and peripheral tissues were collected, including brain tissue, liver, forelimb biceps, heart, kidney, lung, ovary, and sciatic nerve. [Table 6B]

[0219] Quantitative PCR (qPCR) was used to determine the number of vector genomes per μg of brain genomic DNA. Brain samples from injected mice were processed and genomic DNA was isolated using the Blood and Tissue Genomic DNA kit from Qiagen. qPCR assays were performed on a QuantStudio5 instrument (Life Technologies Inc) using a primer-probe combination specific for eGFP according to the standard curve method.

[0220] AAV vector genome copies per μg of brain genomic DNA were at least 1 log higher in mice administered AAV.hDyn compared to all other AAV serotypes: AAV9, AAV.PHPeB, PHP.S, and PHP.SH (see Figure 10). As seen in this study, GC / μg genomic DNA was highest in AAV.hDyn, an AAV9 capsid containing the "TLAAPFK" (SEQ ID NO: 2) peptide insert (a peptide from human axonemal dynein) between residues 588-589 of the AAV9 capsid. The study demonstrated transduction in mouse brain of an average of more than 1E04 GC / μg transgene in five mice systemically administered with AAV.hDyn carrying eGFP. However, other modified AAV9 capsids, including the vector AAV.PHPeB, which contains the "TLAVPFK" (SEQ ID NO: 27) sequence (a peptide from mouse dynein), demonstrated transduction in mouse brain at below 1E03GC / μg transgene upon systemic treatment.

[0221] 6.12. Example 12 - Use of tissue-homing rAAV vectors in methods of treatment Identify a disorder that can be treated / prevented by providing a nucleic acid (transgene) (see Tables 3A-3B). Identify a subject with a disorder associated with a target tissue. Administer the subject with a first amount of an rAAV vector of the invention, which vector contains a capsid protein with a peptide insert that homes to the target tissue and carries the transgene to be delivered. If necessary, administer a second or third dose of the vector to the subject until a therapeutically effective amount of the transgene is delivered to the target tissue and a therapeutic or prophylactic benefit is provided to the subject.

[0222] In some embodiments, methods are provided for administering a transgene to the retina, whereby an AAV.hDyn capsid encapsulating a transgene is administered intravenously, systemically or intravitreally.

[0223] 6.13. Example 13 - Construction of a rAAV Capsid Containing TLAAPFK (SEQ ID NO:2) 11A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLAAPFK (SEQ ID NO:2) between Q588 and A589 of VR-IIIV. The inserted peptide is in bold.

[0224] 11B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLAAPFK (SEQ ID NO:2) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0225] 11C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLAAPFK (SEQ ID NO:2) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0226] 6.14. Example 14 - Construction of rAAV capsid containing KMQVPFQ (SEQ ID NO:1) 12A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence KMQVPFQ (SEQ ID NO: 1) between Q588 and A589 of VR-VIII. The inserted peptide is in bold.

[0227] 12B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence KMQVPFQ (SEQ ID NO: 1) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0228] 12C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence KMQVPFQ (SEQ ID NO: 1) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0229] 6.15. Example 15 - Construction of a rAAV Capsid Containing QQAAPSF (SEQ ID NO:3) 13A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence QQAAPSF (SEQ ID NO:3) between Q588 and A589 of VR-VIII. The inserted peptide is in bold.

[0230] Figure 13B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence QQAAPSF (SEQ ID NO:3) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0231] 13C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence QQAAPSF (SEQ ID NO:3) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0232] 6.16. Example 16 - Construction of a rAAV capsid containing RYNAPFK (SEQ ID NO:4) 14A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence RYNAPFK (SEQ ID NO: 4) between Q588 and A589 of VR-VIII. The inserted peptide is in bold.

[0233] Figure 14B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence RYNAPFK (SEQ ID NO: 4) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0234] 14C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence RYNAPFK (SEQ ID NO:4) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0235] 6.17. Example 17 - Construction of a rAAV capsid containing LKLPPIV (SEQ ID NO:5) 15A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence LKLPPIV (SEQ ID NO:5) between Q588 and A589 of VR-VIII. The inserted peptide is in bold.

[0236] Figure 15B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence LKLPPIV (SEQ ID NO:5) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0237] Figure 15C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence LKLPPIV (SEQ ID NO:5) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0238] 6.18. Example 18 - Construction of a rAAV capsid containing PFIKPFE (SEQ ID NO:6) 16A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence PFIKPFE (SEQ ID NO:6) between Q588 and A589 of VR-VIII. The inserted peptide is in bold.

[0239] Figure 16B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence PFIKPFE (SEQ ID NO:6) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0240] Figure 16C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence PFIKPFE (SEQ ID NO:6) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0241] 6.19. Example 19 - Construction of a rAAV capsid containing TLSLPWK (SEQ ID NO:7) 17A shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLSLPWK (SEQ ID NO:7) between Q588 and A589 of VR-VIII. The inserted peptide is in bold.

[0242] Figure 17B shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLSLPWK (SEQ ID NO:7) between S268 and S269 of VR-III. The inserted peptide is in bold.

[0243] Figure 17C shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLSLPWK (SEQ ID NO:7) between S454 and G455 of VR-IV. The inserted peptide is in bold.

[0244] 6.20. Example 20 - Construction of rAAV capsids containing LGETTRP (SEQ ID NO: 15) 18A shows the amino acid sequence for a recombinant AAV8 vector capsid containing a peptide insert of amino acid sequence LGETTRP (SEQ ID NO: 15) between N590 and T591 of VR-VIII. The inserted peptide is in bold.

[0245] Figure 18B shows the amino acid sequence for a recombinant AAV8 vector capsid containing a peptide insert of amino acid sequence LGETTRP (SEQ ID NO: 15) between A269 and T270 of VR-III. The inserted peptide is in bold.

[0246] Figure 18C shows the amino acid sequence for a recombinant AAV8 vector capsid containing a peptide insert of amino acid sequence LGETTRP (SEQ ID NO: 15) between T453 and T454 of VR-IV. The inserted peptide is in bold.

[0247] 6.21. Example 21 - Construction of rAAV capsids containing LALGETTRP (SEQ ID NO: 16) 19A shows the amino acid sequence for a recombinant AAV8 vector capsid containing a peptide insert of amino acid sequence LALGETTRP (SEQ ID NO: 16) between N590 and T591 of VR-VIII. The inserted peptide is in bold.

[0248] Figure 19B shows the amino acid sequence for a recombinant AAV8 vector capsid containing a peptide insert of amino acid sequence LALGETTRP (SEQ ID NO: 16) between A269 and T270 of VR-III. The inserted peptide is in bold.

[0249] Figure 19C shows the amino acid sequence for a recombinant AAV8 vector capsid containing a peptide insert of amino acid sequence LALGETTRP (SEQ ID NO: 16) between T453 and T454 of VR-IV. The inserted peptide is in bold.

[0250] 6.22. Example 22: Evaluation of modified capsids in vitro and in vivo AAV capsid sequences were modified by either peptide insertion or directed mutagenesis and pooled to obtain barcoded libraries packaged with a GFP expression cassette. The modified vectors were then evaluated in in vitro assays and for in vivo biodistribution in mice using next generation sequencing (NGS) and quantitative PCR. AAV.hDyn was identified as a high brain transduction vector from this pool and further evaluated in individual delivery studies in mice to characterize its transduction profile. Immunohistochemistry analysis of brain sections was also performed to understand the cellular tropism of this vector.

[0251] 6.22.1 Example 22A - In vitro studies of transduction across the blood-brain barrier The ability of the modified capsids to cross the blood-brain barrier was tested in an in vitro Transwell assay using hCMEC / D3 BBB cells (SCC066, Millipore-Sigma) (see Figures 20A-20B). More specifically, the assay was essentially adapted from Sade, H. et al. (2014 PLoS ONE 9(4):e96340) A human Blood-Brain Barrier transcytosis assay reveals Antibody Transcytosis influenced by pH-dependent Receptor Binding, April 2014, Vol.9, Issue 4; and Zhang, X., Blood-brain barrier shuttle peptides enhance AAV transduction in the brain after systemic administration, 2018 Biomaterials 176:71-83. Briefly, 5×10 4 hCMEC / D3 cells / cm 2 were seeded onto collagen-coated Transwell inserts in 12-well plates. Each insert contained 500 μL of medium, and the lower chamber contained 1 mL of medium. Medium was changed every other day. The supernatant was removed 10 days after seeding (time point zero (0)). At this time point, 1 × 10 9 Cells were transduced by adding GC vector to the medium in the upper insert chamber. 10 μL samples of the lower chamber supernatant were removed for testing at intervals of 0.5, 3, 6, and 23 hours after transduction. Each condition (vector) was tested in duplicate and titers were measured in triplicate via qPCR against PolyA.

[0252] Figures 20A-20B show an in vitro Transwell assay (20A) for AAV.hDyn (AAV9 having TLAAPFK (SEQ ID NO: 2) between amino acid residues 588-589) crossing the blood-brain barrier (BBB) ​​cell layer, and the results (Figure 20B) showing that AAV.hDyn (indicated by an inverted triangle in the figure) crosses the BBB cell layer of the assay faster than AAV9 (squares) and faster and to a greater extent than AAV2 (circles). The in vitro assay developed predicts improved trafficking across the BBB, and similar assays may also be used to predict targeting to other organs.

[0253] 6.22.2 Example 22B - Transduction and Biodistribution of Modified Capsids 6.22.2.1 Materials and methods Capsid modifications were made to widely used AAV capsids (including AAV8, AAV9, and AAVrh.10) by inserting various peptide sequences after position S454 in VR-IV (Table 7) or after position Q588 in the VR-VIII surface-exposed loop of the AAV capsid, as well as by inserting inserts after the start codon of VP2 starting at amino acid 137 (AAV4, AAV4-4, and AAV5) or amino acid 138 (AAV1, AAV2, AAV3, AAV3-3, AAV6, AAV7, AAV8, AAV9, AAV9e, rh.10, rh.20, rh.39, rh.74, and hu.37) (FIG. 8) (see also Table 10 for selected capsid sequences). Selected single to multiple amino acid mutations were also used to modify the capsid. See also Yost et al., Structure-guided engineering of surface exposed loops on AAV Capsids. 2019. ASGCT Annual Meeting; and Wu et al., 2000 J. Virology (supra). It was confirmed that packaging efficiency was not adversely affected after any of these capsid modifications on a small scale.

[0254] rAAVs with certain modified capsids were tested for transduction in Lec2 cells in vitro as described above in Example 5. The modified AAVs tested for transduction in Lec2 cells are as follows: eB 588 Ad, eB 588 Hep, eB 588 p79, eB 588 Rab, AAV9 588 Ad, AAV9 588 Hep, AAV9 588 p79, AAV9 588 Rab, eB VP2 Ad, eB VP2 Hep, eB VP2 p79, eB VP2 Rab, AAV9 VP2 Ad, AAV9 VP2 Hep, AAV9 VP2 p79, AAV9 VP2Rab, compared to AAV9. See Table 7B below for AAV capsid identity.

[0255] To test biodistribution, the modified AAVs were packaged with an eGFP transgene cassette containing a specific barcode corresponding to each individual capsid. To improve the efficiency of screening, the novel barcoded vectors were pooled and injected into mice.

[0256] To analyze the biodistribution of genetically altered AAV vectors, various vectors encoding GFP were prepared and formulated in 1x PBS + 0.0001% pluronic acid. All vectors were generated with a cis plasmid containing a 10 bp barcode to enable next generation sequencing (NGS) library (pool) preparation. The three vector pools (Study 1, Study 2 and Study 3 vectors) were injected intravenously into a cohort of five female C57Bl / 6 mice according to Tables 7A-C. Dosing volumes were 10 mL / kg (0.2 mL / 20 g mouse), respectively.

[0257] Mice were randomized into treatment groups based on their body weight on day 1, and their age at the start date was 8-12 weeks. On day 15 post-dosing, animals were euthanized and peripheral tissues were collected, including brain, kidney, liver, sciatic nerve, lung, heart, and muscle tissue. The same protocol was followed in studies where selected capsids from the pool were injected individually.

[0258] Genomic DNA (gDNA) was isolated from tissue samples using the DNeasy Blood and Tissue kit (69506) from Qiagen. The barcode region of each vector was amplified with primers containing overlap for NGS and unique dual index (UDI) and multiplex sequencing strategies as recommended by the manufacturer (Illumina). The relative abundance of each barcoded AAV vector was determined for samples collected from mice using an Illumina MiSeq using the reagents Nano and Micro Kit v2 (MS-103-1001 / 1002). Thus, each vector sample in Tables 7A-C below was barcoded as described above to allow identification and sorting of each read prior to final data analysis. Data was normalized based on the composition of AAV in the originally injected pool and quantified using total genome copy number obtained from qPCR analysis using primer-probe combinations specific for the barcoded sample. [Table 7A] [Table 7B] [Table 7C]

[0259] In studies where selected capsids from the pool were injected individually, qPCR was used to determine the number of vector genomes per μg of tissue genomic DNA. qPCR was performed on a QuantStudio 5 (Life Technologies, Inc.) using a primer-probe combination specific for eGFP according to the standard curve method (Figure 22).

[0260] From studies in which individual vectors were injected into mice for characterization, formalin-fixed mouse brains were sectioned at 40 μm thickness with a vibrating blade microtome (VT1000S, Leica) and free-floating sections were probed with an antibody against GFP to examine the cellular distribution of the delivered vectors.

[0261] More specifically, fixed brains from AAV.hDyn-injected mice were sectioned using a Vibratome (Leica, VT-1000) and GFP expression was assessed using an anti-GFP antibody (AB3080, Millipore Sigma), Vectastain ABC kit (PK-6100, Vector Labs) and DAB Peroxidase kit (SK-4100, Vector Labs). There was a wide distribution of GFP-expressing cells throughout the brain in AAV.hDyn-injected mice, including distribution in the cortex, striatum, and hippocampus of the brain. Figures 23A-23C show images from these regions, with scale bars at 400 um (discussed below).

[0262] 6.22.2.2 Results The results are shown in Figure 21, Figures 22A-22H, and Figures 23A-23C.

[0263] Data from Lec2 cell transduction assays are not shown. AAV9 588 Hep (AAV9 with peptide TILSRSTQTG (SEQ ID NO: 22) (DLC-AS2 in Table 1b) inserted after position 588) showed significantly higher transduction (4-fold) than wild-type AAV9, and AAV9 VP2 Ad (AAV9 with peptide SITLVKSTQTV (SEQ ID NO: 21) (DLC-AS1 in Table 1b) inserted after position 138), AAV9 VP2 Hep (AAV9 with peptide TILSRSTQTG (SEQ ID NO: 22) (DLC-AS2 in Table 1b) inserted after position 138), and AAV9 VP2 Rab (AAV9 with peptide RSSEEDKSTQTT (SEQ ID NO: 26) (DLC-AS4 in Table 1b) inserted after position 138) showed slightly higher transduction of Lec2 cells compared to AAV9. Other AAVs assayed demonstrated lower levels of transduction than AAV9.

[0264] Figure 21 shows the results of next generation sequencing (NGS) analysis of brain gDNA, revealing the relative abundance (percent composition) of capsid pools delivered to mouse brain after intravenous injection. Data was normalized based on the composition of AAV in the initially injected pool and quantified using total genome copy numbers obtained from qPCR analysis using a primer-probe combination specific for the eGFP sequence. Data shown are from three different experiments. Dotted lines indicate which vectors were pooled together. Parental AAV9 was used as a standard and included in each pool. The "BC" identifiers are as shown in Tables 7A, 7B and 7C above.

[0265] Figures 22A-22H show the in vivo transduction profile of AAV.hDyn in female C57Bl / 6 mice, showing copy numbers / microgram gDNA in brain (Figure 22A), liver (Figure 22B), heart (Figure 22C), lung (Figure 22D), kidney (Figure 22E), skeletal muscle (Figure 22F), sciatic nerve (Figure 22G), and ovaries (Figure 22H) in naive mice or mice injected with either AAV9 or AAV.hDyn, with AAV.hDyn demonstrating increased brain biodistribution compared to AAV9. AAV vector genome copies per μg of brain genomic DNA was at least 1 log higher in mice administered AAV.hDyn compared to the parental AAV9 vector.

[0266] Figures 23A-23C show images from the regions analyzed in the immunohistochemistry analysis described above; scale bar is 400 μm. Figures 23A-23C show the distribution of GFP from AAV.hDyn throughout the brain, and images of immunohistochemistry staining of brain sections from the striatum (Figure 23A), hippocampus (Figure 23B), and cortex (Figure 23C) revealed global transduction of the brain with the engineered vector.

[0267] 6.22.2.3 Conclusion AAV capsid modifications performed either by insertions or by specific amino acid mutations in the surface-exposed loops of VR-IV and VR-VIII did not affect their packaging efficiency and were able to yield similar titers in the production system described herein.

[0268] Intravenous administration of AAV.hDyn to mice resulted in higher relative abundance of viral genome and greater brain cell transduction than other modified AAV vectors tested and AAV9.

[0269] 6.23. Example 23 - Homing peptide kidney 1C shows enhanced transduction following systemic delivery. The AAV capsid sequence was modified with either peptide insert and pooled to obtain a barcoded library packaged with a GFP expression cassette. The biodistribution profile of the modified AAV9 vectors was then evaluated in vivo in mice using next generation sequencing (NGS) and quantitative PCR. Recombinant AAV9 vectors containing peptide inserts of amino acid sequence CLPVASC (SEQ ID NO: 12) (kidney 1C) or ASSLNIA (SEQ ID NO: 14) (muscle 1) between S454 and G455 of VR-IV showed increased transduction efficiency in kidney compared to liver (Figure 24).

[0270] 6.23.1 Materials and methods Capsid modifications were performed on AAV9 by inserting various homing peptide sequences after position S454 in the VR-IV surface-exposed loop of the AAV capsid. It was confirmed that packaging efficiency was not adversely affected after any of these capsid modifications on a small scale. The peptide sequences are shown in Table 8 below. All modified AAVs were packaged with an eGFP transgene cassette containing a specific barcode corresponding to each individual capsid. These novel barcoded vectors were pooled to improve the efficiency of screening (as described above in Example 22B; see Study 3, Table 7C).

[0271] Genetically altered AAV vectors were injected intravenously into mice as described above in Example 22B for the altered vectors in Study 3. Data were normalized based on the composition of AAV in the original injected pool and quantified using total genome copy number obtained from qPCR analysis using primer-probe combinations specific for the eGFP sequence to more closely examine kidney versus liver tissue targeting.

[0272] 6.23.2 Results and Conclusions Figure 24 shows the kidney-to-liver ratio of in vivo transduction of AAV9 S454 vectors with different homing peptide inserts (Table 8) in female C57Bl / 6 mice. Kidney-to-liver transduction relative to total kidney transduction of the pool for modified capsids was used for calculation. AAV9 S454 Kidney 1 and AAV9 S454 Kidney 2 (Kidney 1C) show increased kidney biodistribution compared to parental AAV9. The parental AAV9 vector shows increased liver transduction compared to kidney with a ratio of about 0.25, whereas insertion of kidney homing peptide 1C (and also muscle 1) results in an increase of this ratio to about 1.0. AAV vector genome copies per μg of kidney gDNA were at least 5-fold higher in mice administered AAV9 S454 Kidney 1 or AAV9 S454 Muscle 1 compared to all other AAV9 S454 vectors (see Figure 24). [Table 8]

[0273] AAV capsid modifications performed by insertion of different homing peptides in the surface-exposed loop VR-IV did not affect their packaging efficiency and were able to yield similar titers in the production system described here.

[0274] Intravenous administration of AAV9 S454 Kidney 1 and AAV9 S454 Kidney 1C to mice resulted in higher relative abundance of viral genomes and greater kidney cell transduction than other modified and parental AAV9 vectors tested. Intravenous administration of AAV9 S454 Kidney 1 or AAV9 S454 Muscle 1 vectors to mice also resulted in lower liver cell transduction.

[0275] 6.24. Example 24 - Construction of rAAV capsid containing TLAVPFK (SEQ ID NO:27) FIG. 25 shows the amino acid sequence for a recombinant AAV9 vector capsid containing a peptide insert of amino acid sequence TLAVPFK (SEQ ID NO:27) between S454 and G455 of VR-IV.

[0276] 6.25. Example 25 - Biodistribution of rAAV Vector Pools in Cynomolgus Monkeys Administration, in vivo and postmortem observations, and biodistribution of pools of recombinant AAV with modified capsids and GFP transgenes are evaluated after a single intravenous, intracerebroventricular, or intravitreal injection in cynomolgus monkeys (Table 9). The pools contain multiple capsids, each of which contains a unique barcode identification that allows identification using next generation sequencing (NGS) analysis after administration to cynomolgus monkeys. Cynomolgus monkeys are selected as a test system due to their established utility and acceptability as a model for AAV biodistribution studies in large animal species and for further translation to humans. All animals in this study are naive with respect to prior treatment. The pools may contain at least the following recombinant AAV with modified capsids listed in Table 9: [Table 9]

[0277] 6.25.1. Research design Nine female cynomolgus monkey animals will be used. Animals deemed suitable for the experiment based on clinical sign data and prescreening antibody titers will be divided into study groups by weight using computer-generated random numbers. Three different routes of administration will be used and relevant tissues will be collected to evaluate the biodistribution associated with the different routes (measured by NGS and PCR). Three animals will be implanted with a catheter in the left lateral ventricle for intracerebroventricular (ICV) dose administration (Group 1), three animals will receive a single intravenous infusion (Group 2), and three animals will receive a single intravitreal injection (Group 3). Two animals will serve as replacement animals and will be implanted as needed. Animals in Group 1 will undergo MRI scans to determine coordinates for proper ICV catheter placement.

[0278] IV infusion is administered at a rate of 3 mL / min, followed by 0.2 mL of vehicle to flush the dose from the IV catheter. Three animals receive a single dose of pooled recombinant AAV in a volume of 4 mL / kg intravenously. Total dose (vg) and dose volume (mL / kg) are recorded in the raw data. Based on literature review and previous studies in non-human primates, a dose of 1 × 10 13 It has been determined that an IV dose of GC / kg body weight is required to have the desired distribution in peripheral tissues, including the CNS and skeletal muscle, from systemic delivery.

[0279] ICV implanted animals received a single bolus dose of AAV-NAV-GFPbc in a volume of 1 mL (slow infusion, approximately 0.1 mL / min), followed by 0.1 mL of vehicle to flush the dose from the catheter system. The ICV dose was based on distribution data from prior non-human primate studies to support the ongoing clinical program.

[0280] Intravitreal (IVT) injections are administered bilaterally as bolus injections in a dose volume of 50 μL.

[0281] 6.25.2. Observation and testing Clinical signs are recorded at least daily, beginning approximately 2 weeks prior to the initiation of dosing and continuing throughout the duration of the study. Animals are observed for signs of clinical effects, illness, and / or mortality. At the discretion of the study director and / or technician, additional observations may be recorded based on the condition of the animals.

[0282] Ophthalmic examinations are performed on animals in Group 3 prior to dose administration, and on days 2, 8, 15, and 22. All animals are sedated with ketamine hydrochloride IM for ophthalmic examinations performed after day 1. For examinations on day 1, animals are sedated with injectable anesthesia (see section 15.3.3). Eyes are dilated with 1% tropicamide prior to examination. Examinations include slit-lamp biomicroscopy and indirect ophthalmoscopy. Applanation tonometry is also performed on animals in Group 3 prior to dosing, immediately (approximately 10-15 minutes) after dose administration, and on days 2 and 22.

[0283] Collect a blood sample (approximately 3 mL) from a peripheral vein for neutralizing antibody analysis approximately 2-3 weeks prior to dose administration.

[0284] 6.25.3. Biological Analysis Sample Collection From animals in Group 2 (IV) only, collect whole blood samples (approximately 0.5 mL) from a peripheral vein for biological analysis (AAV capsid clearance) prior to dose administration, 3 hours (± 10 min), 6 hours (± 10 min), and 24 hours (± 0.5 h) after dose administration. Collect samples using a syringe and needle and dispense two K 2 Transfer to EDTA tube and record time.

[0285] Collect blood samples (approximately 5 mL) from a peripheral vein from fasted animals for PBMC analysis prior to dose administration (day 1), days 8 and 15, and prior to necropsy (day 22). Obtain samples using lithium heparin tubes and record the time.

[0286] Blood samples are collected from a peripheral vein for biological analysis prior to dose administration (day 1, 2 mL) and at necropsy (day 22, 5 mL). Samples are collected in coagulation tubes and the time is recorded. Tubes are kept at room temperature until adequately coagulated, then centrifuged at approximately 2400 rpm for 15 minutes at room temperature. Serum is harvested, placed in labeled vials (necropsy samples are divided into 1 mL aliquots), frozen in liquid nitrogen, and stored at -60°C or colder.

[0287] Collect CSF (approximately 1.5 mL) from cisternal spinal puncture from animals in group 1 only prior to dose administration. Collect CSF (approximately 2 mL) from cisternal spinal puncture from all animals (groups 1-3) immediately prior to necropsy. An attempt will be made to collect CSF, but due to unsuccessful spinal punctures, samples may not be collected at all intervals from the animal(s). Once collected, store samples on ice until processing.

[0288] Autopsy Gross necropsies are performed on animals found dead or moribundly sacrificed at least 21 days after treatment (day 22) and at scheduled necropsies. All animals except those found dead are sedated with 8 mg / kg ketamine HCl IM, maintained on an isoflurane / oxygen mix, and provided with an intravenous bolus of sodium heparin (200 IU / kg). Animals are perfused with 0.001% sodium nitrite in saline via the left ventricle. Animals found dead are necropsied but not perfused.

[0289] The following tissues will be obtained from all animals (including those found dead): bone marrow, brain, cecum, colon, dorsal nerve root and ganglion, duodenum, esophagus, eyes including optic nerve, gross lesions, heart, ileum, jejunum, kidneys, knee joints, liver, lungs including bronchi, lymph nodes, ovaries, pancreas, sciatic nerve, skeletal muscle, spinal cord, spleen, thyroid, trachea, and vagus nerve.

[0290] 6.25.5. Biological analysis Whole blood collected from animals in group 2 (IV) will be evaluated by qPCR and next generation sequencing (NGS).

[0291] PBMC samples collected from all animals will be evaluated by flow cytometry and enzyme-linked immunosorbent spot (ELISpot) as appropriate.

[0292] The presence of circulating neutralizing antibodies and free vector in serum and / or CSF will be assessed by ELISA and cell-based assays, as appropriate.

[0293] Vector copy number and transcript number in tissues will be examined by quantitative PCR and NGS methods.

[0294] 6.26. Capsid Amino Acid Sequence Table 10 provides the amino acid sequences of selected modified capsid proteins described and / or used in the studies described herein. Heterologous peptides and amino acid substitutions are shown in grey shading. [Table 10] TIFF0007619958000021.tif181139TIFF0007619958000022.tif190140TIFF0007619958000023.tif186141 TIFF0007619958000024.tif187140TIFF0007619958000025.tif183141TIFF0007619958000026.tif135141

[0295] 7. Equivalents Although the present invention has been described in detail with reference to specific embodiments thereof, it is understood that functionally equivalent variants are within the scope of the present invention. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed within the scope of the following claims.

[0296] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0297] The discussion herein provides a better understanding of the nature of the problems facing the art and should not be construed as an admission of prior art in any way, nor should any reference herein be construed as an admission that such reference constitutes "prior art" to the instant application.

[0298] All references, including patent applications and publications, cited in this specification are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are provided by way of example only, and the present invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. Various embodiments of the present invention are presented below. 1. A recombinant adeno-associated virus (rAAV) capsid protein comprising a peptide insert of at least 4 and at most 12 contiguous amino acids from a heterologous protein that is not an AAV protein, said peptide insert immediately following an amino acid residue corresponding to one of amino acids 138 or 451-461 of the AAV9 capsid protein of FIG. 8, wherein said peptide insert is surface-exposed when said capsid protein is packaged as an AAV particle. 2. A recombinant adeno-associated virus (rAAV) capsid protein, comprising: (i) a neural tissue homing protein or domain, provided that the peptide insert does not contain the sequence TLAVPFK (SEQ ID NO:27); (ii) axonemal or cytoplasmic dynein homing domains; (iii) bone-homing domain; (iv) kidney-homing domain; (v) muscle homing domain; (vi) an endothelial cell homing domain; (vii) an integrin receptor binding domain; (viii) a transferrin receptor binding domain, with the proviso that the peptide insert does not contain the sequence RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20); (ix) a tumor cell targeting domain; or (x) a retinal cell homing protein or domain, with the proviso that the peptide insert does not contain the sequence LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16). and a peptide insertion of at least 4 and up to 12 consecutive amino acids from a heterologous protein or domain selected from the group consisting of: The recombinant adeno-associated virus (rAAV) capsid protein, wherein the peptide insert is surface-exposed when the capsid protein is packaged as an AAV particle. 3. The rAAV capsid protein according to 1 or 2 above, wherein the capsid protein is from at least one AAV type selected from AAV type 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), serotype rh8 (AAVrh8), serotype 9 (AAV9), serotype 9e (AAV9e), serotype rh10 (AAVrh10), serotype rh20 (AAVrh20), serotype hu.37 (AVhu.37), serotype rh39 (AAVrh39), and serotype rh74 (AAVrh74). 4. The peptide insert is in the sequence shown in FIG. (a) 450 to 459 of the AAV1 capsid amino acid sequence (SEQ ID NO: 110); (b) 449 to 458 of the AAV2 capsid amino acid sequence (SEQ ID NO: 111); (c) 449 to 459 of the AAV3 capsid amino acid sequence (SEQ ID NO: 112); (d) 443 to 453 of the AAV4 capsid amino acid sequence (SEQ ID NO: 113); (e) 442 to 445 of the AAV5 capsid amino acid sequence (SEQ ID NO: 114); (f) 450 to 459 of the AAV6 capsid amino acid sequence (SEQ ID NO: 115); (g) 451 to 461 of the AAV7 capsid amino acid sequence (SEQ ID NO: 116); (h) 451 to 461 of the AAV8 capsid amino acid sequence (SEQ ID NO: 117); (i) 451 to 461 of the AAV9 capsid amino acid sequence (SEQ ID NO: 118); (j) 452 to 461 of the AAV9e capsid amino acid sequence (SEQ ID NO: 119); (k) 452 to 461 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 120); (l) 452 to 461 of the AAVrh20 capsid amino acid sequence (SEQ ID NO: 121); (m) 452 to 461 of the AAVhu.37 capsid amino acid sequence (SEQ ID NO: 122); (n) 452 to 461 of the AAVrh74 capsid amino acid sequence (SEQ ID NO: 123 or SEQ ID NO: 154); or (o) 452 to 461 of the AAVrh39 capsid amino acid sequence (SEQ ID NO: 124) The rAAV capsid protein according to any one of 1 to 3 above, wherein the rAAV capsid protein is present immediately after one of the amino acid residues in the above. 5. The rAAV capsid protein of claim 4, wherein the peptide insertion is present after an amino acid residue corresponding to one of amino acids I451, N452, G453, S454, G455, Q456, N457, Q458, Q459, T460, or L461 of the AAV9 capsid. 6. The rAAV capsid protein according to any one of 1, 3 to 5 above, wherein the heterologous protein is a homing domain, a neutralizing antibody epitope, or a purification tag. 7. The homing domain comprises: (i) Neural tissue-homing domain; (ii) axonemal or cytoplasmic dynein homing domains; (iii) bone-homing domain; (iv) kidney-homing domain; (v) muscle homing domain; (vi) an endothelial cell homing domain; (vii) an integrin receptor binding domain; (viii) a transferrin receptor binding domain; (ix) a tumor cell targeting domain; or (x) Retinal cell-homing domain 7. The rAAV capsid protein according to claim 6, 8. The rAAV capsid protein according to claim 7, wherein the peptide insert comprises or consists of at least four consecutive amino acids, or seven consecutive amino acids, of a dynein peptide of the amino acid sequence SITLVKSTQTV (SEQ ID NO:21), TILSRSTQTG (SEQ ID NO:22), VVMVGEKPITITQHSVETEG (SEQ ID NO:25), RSSEEDKSTQTT (SEQ ID NO:26), KMQVPFQ (SEQ ID NO:1), LKLPPIV (SEQ ID NO:5), PFIKPFE (SEQ ID NO:6), TLSLPWK (SEQ ID NO:7), QQAAPSF (SEQ ID NO:3), RYNAPFK (SEQ ID NO:4), TLAVPFK (SEQ ID NO:27), or TLAAPFK (SEQ ID NO:2). 9. The rAAV capsid protein of claim 7, wherein the peptide insertion from the transferrin receptor binding domain is at least four consecutive amino acids of the amino acid sequence RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20), or seven amino acids thereof. 10. The rAAV capsid protein according to claim 7, wherein the peptide insert from the retinal cell homing domain is at least four consecutive amino acids of the amino acid sequence TLAAPFK (sequence number 2), LGETTRP (sequence number 15) or LALGETTRP (sequence number 16), or seven amino acids thereof. 11. The rAAV capsid protein described in claim 10, wherein the AAV capsid protein is an AAV8 capsid protein or an AAV9 capsid protein. 12. The rAAV capsid protein according to claim 7, wherein the peptide insertion from the purification tag is at least four consecutive amino acids of the hemagglutinin (HA) epitope of amino acid sequence YPYDVPDYA (sequence number 86) or a FLAG tag of amino acid sequence DYKDDDDK (sequence number 52), or seven consecutive amino acids thereof. 13. The rAAV capsid protein according to 2 or 3 above, wherein the neural tissue homing protein or the retinal cell homing protein is a human axonemal dynein (HAD) heavy chain tail. 14. The rAAV capsid protein according to claim 13, wherein the peptide insert comprises at least 4 and at most 15 consecutive amino acids from the dimerization domain of the HAD heavy chain tail. 15. The peptide insert is (as shown in Figure 7): (a) (aa1 to 1542 of DYH1_HUMAN UniProtKB-Q9P2D7) (SEQ ID NO: 97); (b) (aa1 to 1764 of DYH2_HUMAN UniProtKB-Q9P225) (SEQ ID NO: 98); (c) (aa1 to 1390 of DYH3_HUMAN UniProtKB-Q8TD57) (SEQ ID NO: 99); (d) (aa1 to 1941 of DYH5_HUMAN UniProtKB-Q8TE73) (SEQ ID NO: 100); (e) (aa1 to 1433 of DYH6_HUMAN UniProtKB-Q9C0G6) (SEQ ID NO: 101); (f) (aa1 to 1289 of DYH7_HUMAN UniProtKB-Q8WXX0) (SEQ ID NO: 102); (g) (aa1 to 1807 of DYH8_HUMAN UniProtKB-Q96JB1) (SEQ ID NO: 103); (h) (aa1 to 1831 of DYH9_HUMAN UniProtKB-Q9NYC9) (SEQ ID NO: 104); (i) (aa1 to 1793 of DYH10_HUMAN UniProtKB-Q8IVF4) (SEQ ID NO: 105); (j) (aa1 to 1854 of DYH11_HUMAN UniProtKB-Q96DT5) (SEQ ID NO: 106); (k) (aa1 to 1214 of DYH12_HUMAN UniProtKB-Q6ZR08) (SEQ ID NO: 107); (l) (aa1 to 200 of DYH14_HUMAN UniProtKB-Q0VDD8) (SEQ ID NO: 108); or (m) (aa1 to 1794 of DYH17_HUMAN UniProtKB-Q9UFH2) (SEQ ID NO: 109) 15. The rAAV capsid protein according to claim 14, comprising at least 4 and at most 15 consecutive amino acids from the group consisting of: 16. The rAAV capsid protein according to claim 15, wherein the peptide insert comprises at least 4 and at most 15 consecutive amino acids from residues 1-200 of any one of the dynein heavy chain sequences (Figure 7). 17. The rAAV capsid protein of claim 15, wherein the peptide insert comprises seven consecutive amino acids from any one of the dynein heavy chain sequences of Figure 7. 18. The rAAV capsid protein of claim 16, wherein the peptide insert comprises seven consecutive amino acids from residues 1 to 200 of any one of the dynein heavy chain sequences (Figure 7). 19. The peptide insert comprises the peptide: (a) KMQVPFQ (SEQ ID NO: 1); (b) TLAAPFK (SEQ ID NO:2); (c) QQAAPSF (SEQ ID NO: 3); (d) RYNAPFK (SEQ ID NO: 4); (e) LKLPPIV (SEQ ID NO:5); (f) PFIKPFE (SEQ ID NO: 6); or (g) TLSLPWK (SEQ ID NO: 7) 4. The rAAV capsid protein according to claim 2 or 3, comprising at least four consecutive amino acids of one of the following: 20. The peptide insert comprises the peptide: (a) KMQVPFQ (SEQ ID NO: 1); (b) TLAAPFK (SEQ ID NO:2); (c) QQAAPSF (SEQ ID NO: 3); (d) RYNAPFK (SEQ ID NO: 4); (e) LKLPPIV (SEQ ID NO:5); (f) PFIKPFE (SEQ ID NO: 6); or (g) TLSLPWK (SEQ ID NO: 7) rAAV capsid protein according to claim 2 or 3, which consists of one of the following: 21. The rAAV capsid protein according to claim 20, wherein the peptide insert is the amino acid sequence TLAAPFK (sequence number 2). 22. The rAAV capsid protein according to claim 2 or 3, wherein the neural tissue homing protein is a mouse axonemal dynein (MAD) heavy chain tail. 23. The rAAV capsid protein according to claim 2 or 3, wherein the neural tissue homing domain is an EPO (erythropoietin) domain that binds to a natural repair receptor and is not erythropoietin, or a conformational analog of said domain. 24. The rAAV capsid protein according to claim 23, wherein the peptide insert is at least 4 and at most 11 consecutive amino acids from QEQLERALNSS (sequence number 8). 25. The rAAV capsid protein described in claim 24, wherein the peptide insert has the amino acid sequence QEQLERALNSS (sequence number 8). 26. The rAAV capsid protein described in 2 or 3 above, wherein the neural tissue homing protein is a brain homing domain having an SRL (serine-arginine-lysine) motif. 27. The rAAV capsid protein according to claim 26, wherein the peptide insert from the brain-homing domain has at least four consecutive amino acids of the amino acid sequence LSSRLDA (sequence number 10) or CLSSRLDAC (sequence number 11), or is the amino acid sequence LSSRLDA (sequence number 10) or CLSSRLDAC (sequence number 11). 28. The rAAV capsid protein according to 2 or 3 above, wherein the axonemal or cytoplasmic dynein homing domain is a dynein light chain homing domain. 29. The rAAV capsid protein according to claim 28, wherein the peptide insert from the dynein light chain homing domain is at least four and at most 12 consecutive amino acids of one of SITLVKSTQTV (sequence number 21), TILSRSTQTG (sequence number 22), VVMVGEKPITITQHSVETEG (sequence number 25), or RSSEEDKSTQTT (sequence number 26). 30. The rAAV capsid protein according to 2 or 3 above, wherein the bone homing protein is a hydroxyapatite (HA) binding domain. 31. The rAAV capsid protein described in claim 30, wherein the peptide insert from the hydroxyapatite (HA) binding domain is at least 6 amino acid residues of the sequence DDDDDDDD (sequence number 9). 32. The rAAV capsid protein according to claim 2 or 3, wherein the kidney homing domain comprises the amino acid sequence CLPVASC (sequence number 12). 33. The rAAV capsid protein according to claim 32, wherein the peptide insert from the kidney homing domain peptide is the amino acid sequence LPVAS (sequence number 13) or CLPVASC (sequence number 12). 34. The rAAV capsid protein according to claim 2 or 3, wherein the peptide insert from the muscle-homing domain comprises or consists of the amino acid sequence ASSLNIA (sequence number 14). 35. The rAAV capsid protein according to claim 2 or 3, wherein the peptide insert comprises or consists of the amino acid sequence QAVRTSL (sequence number 23) or QAVRTSH (sequence number 24). 36. The rAAV capsid protein according to claim 2 or 3, wherein the peptide insert from the endothelial cell homing domain comprises or consists of the amino acid sequence SIGYPLP (sequence number 28). 37. The rAAV capsid protein according to claim 2 or 3, wherein the peptide insert from the integrin binding domain has the amino acid sequence CDCRGDCFC (sequence number 29). 38. The rAAV capsid protein according to claim 2 or 3, wherein the transferrin receptor binding domain is a transferrin domain, or a conformational analogue or iron mimetic thereof. 39. The rAAV capsid protein according to claim 38, wherein the peptide insertion from the transferrin domain is at least 4 consecutive amino acids from the sequence HAIYPRH (sequence number 17) or THRPPMWSPVWP (sequence number 18), or 7 consecutive amino acids thereof. 40. The rAAV capsid protein according to claim 39, wherein the peptide insert comprises or consists of the amino acid sequence HAIYPRH (sequence number 17) or THRPPMWSPVWP (sequence number 18). 41. The rAAV capsid protein according to claim 2 or 3, wherein the peptide insert from the tumor cell targeting domain comprises or consists of the amino acid sequence NGRAHA (sequence number 30). 42. The rAAV capsid protein according to claim 2 or 3, wherein the peptide insert is at least four consecutive amino acids from one of TLAAPFK (sequence number 2), TLAVPFK (sequence number 27), RTIGPSV (sequence number 19), CRTIGPSVC (sequence number 20), LGETTRP (sequence number 15), and LALGETTRP (sequence number 16), or seven consecutive amino acids thereof. 43. The peptide insert comprises the amino acid residues (shown in FIG. 8): (a) 138; 262-272; 450-459; or 585-593 of the AAV1 capsid amino acid sequence (SEQ ID NO: 110); (b) 138; 262-272; 449-458; or 584-592 of the AAV2 capsid amino acid sequence (SEQ ID NO:111); (c) 138; 262-272; 449-459; or 585-593 of the AAV3 capsid amino acid sequence (SEQ ID NO: 112); (d) 137; 256 to 262; 443 to 453; or 583 to 591 of the AAV4 capsid amino acid sequence (SEQ ID NO: 113); (e) 137; 252 to 262; 442 to 445; or 574 to 582 of the AAV5 capsid amino acid sequence (SEQ ID NO: 114); (f) 138; 262-272; 450-459; 585-593 of the AAV6 capsid amino acid sequence (SEQ ID NO: 115); (g) 138; 263-273; 451-461; 586-594 of the AAV7 capsid amino acid sequence (SEQ ID NO: 116); (h) 138; 263-274; 452-461; 587-595 of the AAV8 capsid amino acid sequence (SEQ ID NO: 117); (i) 138; 262-273; 452-461; 585-593 of the AAV9 capsid amino acid sequence (SEQ ID NO: 118); (j) 138; 262-273; 452-461; 585-593 of the AAV9e capsid amino acid sequence (SEQ ID NO: 119); (k) 138; 263-274; 452-461; 587-595 of the AAVrh10 capsid amino acid sequence (SEQ ID NO: 120); (l) 138; 263-274; 452-461; 587-595 of the AAVrh20 capsid amino acid sequence (SEQ ID NO: 121); (m) 138; 263-274; 452-461; 587-595 of the AAVhu37 capsid amino acid sequence (SEQ ID NO: 122); (n) 138; 263 to 274; 452 to 461; 587 to 595 of the AAVrh74 capsid amino acid sequence (SEQ ID NO: 123 or SEQ ID NO: 154); or (o) 138; 263-274; 452-461; 587-595 of the AAVrh39 capsid amino acid sequence (SEQ ID NO: 124) 43. The rAAV capsid protein according to any one of 2 or 13 to 42 above, which is present immediately after one of the above. 44. The rAAV capsid protein according to claim 43, comprising the amino acid sequence TLAAPFK (sequence number 2) inserted between amino acid residues 588 and 589 of the AAV9 capsid or immediately following the amino acid residue corresponding to amino acid 138 of the AAV9 capsid (see FIG. 8). 45. The rAAV capsid protein according to claim 43, comprising the amino acid sequence TLAAPFK (sequence number 2) inserted after one of I451 to L461, S268 or Q588 of the AAV9 capsid (Figure 8). 46. ​​The rAAV capsid protein according to claim 43, comprising the amino acid sequence QEQLERALNSS (sequence number 8) between amino acid residues 588 to 589 of the AAV9 capsid (see FIG. 8). 47. The rAAV capsid protein according to claim 43, comprising the amino acid sequence QEQLERALNSS (sequence number 8) inserted at one or more positions selected from I451 to L461, or S268 of the AAV9 capsid (Figure 8). 48. The rAAV capsid protein described in claim 43, wherein the peptide insert comprises the amino acid sequence TLAVPFK (sequence number 27) immediately following one of amino acid residues 262 to 273 of the AAV9 capsid protein. 49. The rAAV capsid protein described in claim 43, wherein the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) between amino acid residues 269 and 270 of the AAV8 capsid protein. 50. The rAAV capsid protein described in claim 49, wherein the peptide insert has the amino acid sequence LALGETTRP (sequence number 16). 51. The rAAV capsid protein described in claim 43, wherein the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) between amino acid residues 590 and 591 of the AAV8 capsid protein. 52. The rAAV capsid protein described in 51 above, wherein the peptide insert has the amino acid sequence LALGETTRP (sequence number 16). 53. The rAAV capsid protein described in claim 43, wherein the peptide insert comprises the amino acid sequence LGETTRP (sequence number 15) immediately following one of amino acid residues 453 and 454 of the AAV8 capsid protein. 54. The rAAV capsid protein described in 53 above, wherein the peptide insert comprises the amino acid sequence LALGETTRP (sequence number 16). 55. The rAAV capsid protein according to any one of 2 or 13 to 43 above, wherein the capsid is AAV9 and the peptide insert is present between amino acid residues 454 to 455 of AAV9 (Figure 8). 56. An rAAV capsid protein according to any one of 2 or 13 to 43 above, wherein the peptide insertion is located immediately after an acid residue corresponding to one of I451 to L461, S268 or Q588 of the AAV9 capsid (Figure 8). 57. The rAAV capsid protein according to claim 43, wherein the peptide insertion is located immediately after one of amino acids 451 to 461 of the AAV9 capsid protein. 58. An rAAV capsid protein according to any one of 2 or 13 to 43 above, wherein the peptide insertion is present in the AAV capsid eighth variable region (VR-VIII). 59. The rAAV capsid protein of any of the preceding claims, wherein the capsid protein is not the AAV2 capsid protein. 60. A recombinant AAV capsid protein comprising one or more amino acid substitutions relative to a wild-type or unmodified capsid protein, wherein the rAAV capsid protein is an AAV8 capsid protein having an A269S amino acid substitution, or an AAV9 capsid protein having S263G / S269R / A273T substitutions, or a W503R or Q474A substitution, or a corresponding substitution in a capsid protein of another AAV type capsid. 61. The rAAV capsid protein of embodiment 60, further comprising 498-NNN / AAA-500 for the AAV8 capsid protein or 496-NNN / AAA-498 for the AAV9 capsid protein, or a corresponding substitution in the capsid protein of another AAV type capsid. 62. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to any of the preceding paragraphs, or encoding an amino acid sequence sharing at least 80% identity therewith. 63. A nucleic acid according to claim 62, encoding an rAAV capsid protein according to any of the preceding claims. 64. A packaging cell capable of expressing the nucleic acid according to 62 or 63 above to produce an AAV vector comprising the capsid protein encoded by said nucleotide sequence. 65. An rAAV vector comprising a capsid protein described in any one of 1 to 61 above. 66. The rAAV vector described in 65 above, further comprising a transgene. 67. A pharmaceutical composition comprising the rAAV vector described in 65 or 66 above and a pharma- ceutical acceptable carrier. 68. A method for delivering a transgene to a cell, the method comprising contacting the cell with an rAAV vector according to 65 or 66 above; or an rAAV vector according to 65 or 66 above for use in delivering a transgene to a cell, the cell being contacted with the vector. 69. A method for delivering a transgene to a target tissue of a subject in need thereof, the method comprising administering to the subject an rAAV vector as described in 65 or 66 above, wherein the peptide insert is a homing peptide; or an rAAV vector as described in 65 or 66 above for use in delivering a transgene to a target tissue of a subject in need thereof, the vector being administered to the subject. 70. The method or use of an rAAV vector according to claim 69, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, intravitreally, via lumbar puncture or via the cisterna magna. 71. The target tissue is (i) neural tissue, and the vector comprises the peptide insert from the neural tissue homing domain; (ii) bone, wherein the vector comprises a peptide insert from the bone-homing domain; (iii) kidney, wherein the vector comprises a peptide insert from the kidney-homing domain; (iv) muscle, wherein the vector comprises a peptide insert from the muscle-homing domain; (v) an endothelial cell, the vector comprising a peptide insert from the endothelial cell homing domain; (vi) an integrin receptor, and the vector comprises a peptide insert from the integrin receptor binding domain; (vii) a transferrin receptor on a tumor cell, the vector comprising a peptide insert from the transferrin receptor binding domain; (viii) a tumor cell, and the vector comprises a peptide insert from the tumor cell targeting domain; or (ix) a retinal cell, the vector comprising a peptide insert from the retinal cell homing domain. 71. A rAAV vector for use according to claim 70, comprising: 72. The method or rAAV vector for use according to claim 71, wherein the target tissue is a retinal cell and the peptide insert comprises the amino acid sequence TLAAPFK (sequence number 2), LGETTRP (sequence number 15) or LALGETTRP (sequence number 16). 73. The method or rAAV vector for use according to claim 72, wherein the target tissue is a retinal cell and the peptide insert comprises the amino acid sequence TLAAPFK (sequence number 2).

Claims

1. a. a rAAV serotype 8 (AAV8) capsid protein comprising SEQ ID NO: 117, or b. a rAAV serotype 9 (AAV9) capsid protein comprising SEQ ID NO: 118, or c. A recombinant adeno-associated virus (rAAV) capsid protein comprising an amino acid sequence at least 90% identical to SEQ ID NO:117 or SEQ ID NO:118, wherein said AAV capsid protein comprises a peptide insertion of at least 5 and at most 10 contiguous amino acids from a heterologous protein that is not an AAV protein, said peptide insertion immediately follows an amino acid residue corresponding to one of amino acids 451-461 of the AAV9 capsid protein of SEQ ID NO:118, and wherein said peptide insertion is surface-exposed when said capsid protein is packaged into an AAV particle.

2. The rAAV capsid protein of claim 1, wherein the peptide insertion is located immediately after the amino acid residue corresponding to amino acid S454 of the AAV9 capsid.

3. The rAAV capsid protein of claim 1 or 2, wherein the heterologous protein is a homing domain.

4. The homing domain is (i) a neural tissue-homing domain; (ii) an axonemal or cytoplasmic dynein homing domain; (iii) a bone-homing domain; (iv) a kidney-homing domain; (v) muscle-homing domain; (vi) an endothelial cell homing domain; (vii) an integrin receptor binding domain; (viii) a transferrin receptor binding domain; (ix) a tumor cell targeting domain; or (x) retinal cell-homing domain The rAAV capsid protein of claim 3,

5. 5. The rAAV capsid protein of claim 4, wherein the peptide insert comprises or consists of at least 5 consecutive amino acids of a dynein peptide of amino acid sequence SITLVKSTQTV (SEQ ID NO:21), TILSRSTQTG (SEQ ID NO:22), VVMVGEKPITITQHSVETEG (SEQ ID NO:25), RSSEEDKSTQTT (SEQ ID NO:26), KMQVPFQ (SEQ ID NO:1), LKLPPIV (SEQ ID NO:5), PFIKPFE (SEQ ID NO:6), TLSLPWK (SEQ ID NO:7), QQAAPSF (SEQ ID NO:3), RYNAPFK (SEQ ID NO:4), TLAVPFK (SEQ ID NO:27), or TLAAPFK (SEQ ID NO:2), or 7 consecutive amino acids thereof.

6. 5. The rAAV capsid protein of claim 4, wherein the peptide insertion from the transferrin receptor binding domain is at least 5 consecutive amino acids of the amino acid sequence HAIYPRH (SEQ ID NO: 17), THRPPMWSPVWP (SEQ ID NO: 18), RTIGPSV (SEQ ID NO: 19) or CRTIGPSVC (SEQ ID NO: 20), or 7 amino acids thereof.

7. 5. The rAAV capsid protein of claim 4, wherein the peptide insert from the retinal cell homing domain is at least 5 consecutive amino acids of the amino acid sequence TLAAPFK (SEQ ID NO: 2), LGETTRP (SEQ ID NO: 15) or LALGETTRP (SEQ ID NO: 16), or 7 amino acids thereof.

8. The peptide insert is: (a) (aa 1-1542 of DYH1_HUMAN UniProtKB-Q9P2D7) (SEQ ID NO: 97); (b) (aa 1-1764 of DYH2_HUMAN UniProtKB-Q9P225) (SEQ ID NO: 98); (c) (aa 1-1390 of DYH3_HUMAN UniProtKB-Q8TD57) (SEQ ID NO: 99); (d) (aa 1-1941 of DYH5_HUMAN UniProtKB-Q8TE73) (SEQ ID NO: 100); (e) (aa 1-1433 of DYH6_HUMAN UniProtKB-Q9C0G6) (SEQ ID NO: 101); (f) (aa 1-1289 of DYH7_HUMAN UniProtKB-Q8WXX0) (SEQ ID NO: 102); (g) (aa 1-1807 of DYH8_HUMAN UniProtKB-Q96JB1) (SEQ ID NO: 103); (h) (aa1-1831 of DYH9_HUMAN UniProtKB-Q9NYC9) (SEQ ID NO: 104); (i) (aa 1-1793 of DYH10_HUMAN UniProtKB-Q8IVF4) (SEQ ID NO: 105); (j) (aa1-1854 of DYH11_HUMAN UniProtKB-Q96DT5) (SEQ ID NO: 106); (k) (aa1-1214 of DYH12_HUMAN UniProtKB-Q6ZR08) (SEQ ID NO: 107); (l) (aa 1-200 of DYH14_HUMAN UniProtKB-Q0VDD8) (SEQ ID NO: 108); or (m) (aa1-1794 of DYH17_HUMAN UniProtKB-Q9UFH2) (SEQ ID NO: 109) 8. The rAAV capsid protein of any one of claims 1 to 7, comprising at least 5 and at most 10 consecutive amino acids from the group consisting of:

9. 9. The rAAV capsid protein of claim 8, wherein the peptide insert comprises at least 5 and up to 10 contiguous amino acids from residues 1-200 of any one of the dynein heavy chain sequences (a)-(m).

10. 10. The rAAV capsid protein of claim 9, wherein the peptide insert comprises seven contiguous amino acids from residues 1-200 of any one of the dynein heavy chain sequences (a)-(m).

11. The rAAV capsid protein of claim 7, wherein the peptide insert is the amino acid sequence TLAAPFK (SEQ ID NO:2).

12. 5. The rAAV capsid protein of claim 4, wherein the neural tissue homing domain is an erythropoietin (EPO) domain that binds to a natural repair receptor and is not erythropoietin, or a conformational analog of said domain.

13. The rAAV capsid protein of claim 12, wherein the peptide insertion is at least 5 and at most 10 consecutive amino acids from QEQLERALNSS (SEQ ID NO: 8).

14. The rAAV capsid protein of claim 4, wherein the neural tissue homing protein is a brain homing domain having an SRL (serine-arginine-lysine) motif.

15. 15. The rAAV capsid protein of claim 14, wherein the peptide insert from the brain-homing domain has at least five consecutive amino acids of the amino acid sequence LSSRLDA (SEQ ID NO: 10) or CLSSRLDAC (SEQ ID NO: 11), or is the amino acid sequence LSSRLDA (SEQ ID NO: 10) or CLSSRLDAC (SEQ ID NO: 11).

16. 5. The rAAV capsid protein of claim 4, wherein the bone-homing protein is a hydroxyapatite (HA) binding domain.

17. 17. The rAAV capsid protein of claim 16, wherein the peptide insert from the hydroxyapatite (HA) binding domain is at least six amino acid residues of the sequence DDDDDDDDD (SEQ ID NO:9).

18. The rAAV capsid protein of claim 4, wherein the kidney-homing domain comprises the amino acid sequence CLPVASC (SEQ ID NO: 12).

19. 19. The rAAV capsid protein of claim 18, wherein the peptide insert from the kidney-homing domain is the amino acid sequence LPVAS (SEQ ID NO: 13) or CLPVASC (SEQ ID NO: 12).

20. 5. The rAAV capsid protein of claim 4, wherein the peptide insert from the muscle-homing domain comprises or consists of the amino acid sequence ASSLNIA (SEQ ID NO: 14).

21. 5. The rAAV capsid protein of claim 4, wherein the peptide insert comprises or consists of the amino acid sequence QAVRTSL (SEQ ID NO:23) or QAVRTSH (SEQ ID NO:24).

22. 5. The rAAV capsid protein of claim 4, wherein the peptide insert from the endothelial cell homing domain comprises or consists of the amino acid sequence SIGYPLP (SEQ ID NO:28).

23. 5. The rAAV capsid protein of claim 4, wherein the peptide insert from the integrin binding domain has the amino acid sequence CDCRGDCFC (SEQ ID NO:29).

24. 5. The rAAV capsid protein of claim 4, wherein the peptide insert from the tumor cell targeting domain comprises or consists of the amino acid sequence NGRAHA (SEQ ID NO: 30).

25. 4. The rAAV capsid protein of claim 3, wherein the peptide insert comprises the amino acid sequence LGETTRP (SEQ ID NO: 15) immediately following one of amino acid residues 453 and 454 of the AAV8 capsid protein.

26. 26. The rAAV capsid protein of claim 25, wherein the peptide insert comprises the amino acid sequence LALGETTRP (SEQ ID NO: 16).

27. A nucleic acid comprising a nucleotide sequence encoding an rAAV capsid protein according to any one of claims 1 to 26.

28. A packaging cell capable of expressing the nucleic acid of claim 27 to produce an AAV vector comprising the capsid protein encoded by the nucleotide sequence.

29. 27. An rAAV vector comprising the rAAV capsid protein of any one of claims 1 to 26.

30. 30. The rAAV vector of claim 29, further comprising a transgene.

31. 31. A pharmaceutical composition comprising the rAAV vector of claim 29 or 30 and a pharma- ceutically acceptable carrier.

32. 31. The rAAV vector of claim 29 or 30 for use in delivering a transgene to a cell, wherein the cell is contacted with the vector.

33. 31. A pharmaceutical composition comprising the rAAV vector of claim 29 or 30 for use in delivering an introduced gene to a target tissue of a subject in need thereof, wherein the vector is administered to the subject.

34. The pharmaceutical composition of claim 33, wherein the rAAV vector is administered systemically, intravenously, intrathecally, intranasally, intraperitoneally, intravitreally, via lumbar puncture or via the cisterna magna.

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