Recombinant AAV capsid proteins

Abstract

Provided herein are recombinant adeno-associated virus (AAV) capsid proteins, compositions (e.g., rAAV) comprising the capsid proteins, nucleic acids encoding the capsid proteins, and methods of making and using the capsid proteins.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 366,369, filed on June 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] The contents of the electronically submitted Sequence Listing XML (name: 200722_SL.xml; size: 47,397 bytes; created on June 6, 2023) are incorporated herein by reference in its entirety. Background Art

[0005] Adeno-associated virus (AAV) has unique characteristics that make it attractive as a vector for delivering foreign DNA into cells for gene therapy. For example, infection of cultured cells by AAV is non-cytopathic, and natural infection of humans and other animals is silent. In addition, because the signals directing AAV replication, genome encapsidation, and integration are contained within the inverted terminal repeats (ITRs) of the AAV genome, essentially all of the internal 4.7 kb of the AAV genome (encoding replication and structural capsid proteins, rep-cap) can be replaced by heterologous nucleic acid sequences, such as transgenes for achieving gene therapy.

[0006] Recombinant AAV (rAAV) gene therapy vectors typically consist of a recombinant AAV genome containing a gene therapy transgene contained within an AAV capsid. The AAV capsid is formed by an assembly of 60 AAV capsid proteins, interacts with the cell surface of target cells, and determines the tropism and transduction efficiency of rAAV. The blood-brain barrier (BBB) ​​remains a major obstacle to delivering gene therapy to the central nervous system (CNS) via systemic administration. It would be highly advantageous to have rAAV that can effectively target CNS cells and cross the BBB to target tissues in need of gene therapy.

[0007] Therefore, there is a need in the art for tissue-specific AAV capsids for gene therapy applications. Summary of the invention

[0008] Provided herein are recombinant adeno-associated virus (AAV) capsid proteins, compositions comprising the capsid proteins (e.g., rAAV), nucleic acids encoding the capsid proteins, and methods for making and using the capsid proteins. The recombinant AAV capsid proteins provided herein are engineered to include peptides within the parent AAV capsid proteins. On the one hand, the recombinant AAV capsid proteins provided herein are engineered to include peptides within loop IV and / or loop VIII of the parent AAV capsid proteins. Compared to the parent AAV capsid proteins, these recombinant AAV capsid proteins exhibit significantly increased transduction of (motor) neurons, astrocytes, microglia, and myocytes, as well as increased penetration of the BBB. The recombinant AAV capsid proteins disclosed herein overcome previous shortcomings in the art by providing compositions and methods for delivering viral vectors across the BBB.

[0009] In one aspect, a recombinant adeno-associated virus (AAV) capsid protein comprising a peptide and a parent capsid protein is provided herein, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In one aspect, a recombinant adeno-associated virus (AAV) capsid protein comprising a peptide and a parent capsid protein is provided herein, wherein the peptide is contained within loop IV and / or loop VIII of the parent capsid protein, and wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In an embodiment, the peptide is contained within loop IV of the parent capsid protein. In an embodiment, the peptide is contained within loop VIII of the parent capsid protein.

[0010] In an embodiment, the parent capsid protein is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV or AAV5 capsid protein, or an engineered variant thereof.

[0011] In embodiments, the parent capsid protein comprises an amino acid sequence having at least 95% identity to amino acids 193-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence having at least 99% identity to amino acids 193-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence of amino acids 193-725 of SEQ ID NO:11.

[0012] In embodiments, the parent capsid protein comprises an amino acid sequence that is at least 95% identical to amino acids 137-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence that is at least 99% identical to amino acids 137-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence of amino acids 137-725 of SEQ ID NO: 11.

[0013] In embodiments, the parent capsid protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 10 or 11. In embodiments, the parent capsid protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 10 or 11. In embodiments, the parent capsid protein comprises the amino acid sequence of SEQ ID NO: 10 or 11.

[0014] In embodiments, the peptide is positioned C-terminally adjacent to an amino acid corresponding to amino acid 440, 441, 442, 443, 444, 445, 446, 447, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583 or 584 of the parent capsid protein.

[0015] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 440 of SEQ ID NO: 11 in the parent capsid protein.

[0016] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 441 of SEQ ID NO: 11 in the parent capsid protein.

[0017] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 442 of SEQ ID NO: 11 in the parent capsid protein.

[0018] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 443 of SEQ ID NO: 11 in the parent capsid protein.

[0019] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 444 of SEQ ID NO: 11 in the parent capsid protein.

[0020] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 445 of SEQ ID NO: 11 in the parent capsid protein.

[0021] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 446 of SEQ ID NO: 11 in the parent capsid protein.

[0022] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 447 of SEQ ID NO: 11 in the parent capsid protein.

[0023] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 572 of SEQ ID NO: 11 in the parent capsid protein.

[0024] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 573 of SEQ ID NO: 11 in the parent capsid protein.

[0025] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 574 of SEQ ID NO: 11 in the parent capsid protein.

[0026] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 575 of SEQ ID NO: 11 in the parent capsid protein.

[0027] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 576 of SEQ ID NO: 11 in the parent capsid protein.

[0028] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 577 of SEQ ID NO: 11 in the parent capsid protein.

[0029] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 578 of SEQ ID NO: 11 in the parent capsid protein.

[0030] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 579 of SEQ ID NO: 11 in the parent capsid protein.

[0031] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 580 of SEQ ID NO: 11 in the parent capsid protein.

[0032] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 581 of SEQ ID NO: 11 in the parent capsid protein.

[0033] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 582 of SEQ ID NO: 11 in the parent capsid protein.

[0034] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 583 of SEQ ID NO: 11 in the parent capsid protein.

[0035] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 584 of SEQ ID NO: 11 in the parent capsid protein.

[0036] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0037] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0038] In an embodiment, the recombinant AAV capsid protein comprises: an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12; an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13; an amino acid sequence having 100% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14; an amino acid sequence having 100% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 20; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 21; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 22; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 23.

[0039] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0040] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0041] In an embodiment, the recombinant AAV capsid protein comprises: an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12; an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13; an amino acid sequence having 100% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14; an amino acid sequence having 100% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 20; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 21; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 22; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 23.

[0042] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 6); an amino acid sequence having at least 95% sequence identity with amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 95% sequence identity with amino acids 1 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 95% sequence identity with amino acids 1 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0043] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity with amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity with amino acids 1 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity with amino acids 1 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0044] In an embodiment, the recombinant AAV capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 12-23. In an embodiment, the amino acid sequence of the AAV capsid protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 12-23.

[0045] In one aspect, provided herein is an isolated polynucleotide encoding a recombinant AAV capsid protein disclosed herein.

[0046] In one aspect, provided herein is a vector comprising a polynucleotide disclosed herein.

[0047] In an embodiment, the vector is a plasmid or a viral vector. In an embodiment, the viral vector is a retroviral vector, a herpes virus vector, a baculovirus vector or an adenovirus vector. In an embodiment, the vector is an expression vector.

[0048] In one aspect, provided herein is a recombinant cell comprising a polynucleotide or a vector disclosed herein.

[0049] In one aspect, provided herein is a method for producing an AAV capsid protein, the method comprising culturing a recombinant cell disclosed herein under conditions where a polynucleotide is expressed and the capsid protein is produced.

[0050] In one aspect, provided herein is a recombinant adeno-associated virus (rAAV), comprising: a capsid comprising one or more recombinant capsid proteins disclosed herein; and a rAAV genome.

[0051] In embodiments, the rAAV genome comprises a transgene. In embodiments, the transgene encodes a polypeptide or a non-coding RNA. In embodiments, the transgene encodes an antibody or a fragment thereof. In embodiments, the transgene encodes a scFv, a nanobody, or a VHH.

[0052] In one aspect, a method of delivering a transgene to a cell is provided herein, comprising contacting the cell with a rAAV disclosed herein under conditions of transducing the cell and expressing the transgene. In embodiments, the cell is a myocyte, a microglia, an astrocyte, a neuron, a blood-brain barrier endothelial cell (BBB EC), a pericyte, an oligodendrocyte, an ependymal cell, or a cardiomyocyte. In embodiments, the neuron is a pyramidal neuron, a rosehip neuron, or a Purkinje cell.

[0053] In one aspect, the present invention provides a method of delivering a transgene to the brain, spinal cord or muscle tissue of a subject in need thereof, the method comprising administering a rAAV disclosed herein. In embodiments, the subject suffers from a disease associated with the brain, spinal cord or muscle tissue.

[0054] In one aspect, provided herein is a method of treating a neuromuscular or neurodegenerative disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a rAAV disclosed herein.

[0055] In an embodiment, the neuromuscular disease or disorder is selected from the group consisting of myopathy, hereditary cardiomyopathy, metabolic myopathy, distal myopathy, muscular dystrophy, congenital myopathy, spinal muscular atrophy (SMA), motor neuron disease, congenital myopathy, congenital muscular dystrophy, motor neuron disease, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, myotubular myopathy, centronuclear myopathy, nemaline myopathy, selenoprotein N-related myopathy, Pompe disease, glycogen storage disease type III, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, multiple sclerosis, myositis, polymyositis and dermatomyositis.

[0056] In an embodiment, the neurodegenerative disease or disorder is selected from the group consisting of motor neuron disease (MND), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinocerebellar ataxia (SCA) and Friedreich's ataxia.

[0057] In embodiments, the rAAV is administered to the subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1A is a graph showing GFP expression in myotubes after transduction with recombinant AAV capsid proteins comprising VYPep2, VYPep4, VYPep5, or VYPep11 in loop IV or loop VIII compared to the parental capsid protein without peptide insertion. Figure 1B is a graph showing GFP protein expression in human myotubes after transduction with recombinant AAV capsid proteins comprising VYPep4 or VYPep5 compared to the parental capsid protein without peptide insertion.

[0059] Figure 2A is shown in 1x10 6 Figure 3 Plot of the percentage of GFP-positive human astrocytes after transduction with recombinant AAV capsid proteins containing VYPep4 or VYPep5 in loop IV or loop VIII compared to the parental capsid protein without peptide insert at an MOI of 1.38kDa. Figure 2B is shown in 1x10 4Figure 3 Plot of the percentage of GFP-positive human astrocytes after transduction with recombinant AAV capsid proteins containing VYPep4 or VYPep5 in loop IV or loop VIII compared to the parental capsid protein without peptide insert at an MOI of 1.38kDa.

[0060] Figure 3 is a graph showing the percentage of positive GFP cells on the brain side of the blood-brain barrier after transduction of astrocytes with recombinant AAV capsid protein containing VYPep4 compared to the parental capsid protein without peptide insertion in a blood-brain barrier microfluidic model.

[0061] Figure 4 is shown in 1x10 5 and 1x10 6 Graph of the percentage of GFP-positive non-human primate (NHP) astrocytes after transduction with recombinant AAV capsid protein containing VYPep4 in loop IV compared to the parental capsid protein without peptide insert at an MOI of 1.34 Å.

[0062] Figure 5 is shown in 1x10 5 and 1x10 6 Figure 3 Plot of the percentage of GFP-positive human motor neurons after transduction with recombinant AAV capsid protein containing VYPep4 in loop IV compared to the parental capsid protein without peptide insert at an MOI of 1.34 Å. DETAILED DESCRIPTION

[0063] Provided herein are recombinant adeno-associated virus (AAV) capsid proteins, compositions comprising the capsid proteins (e.g., rAAV), nucleic acids encoding the capsid proteins, and methods for making and using the capsid proteins. The recombinant AAV capsid proteins provided herein are engineered to include peptides within the parent AAV capsid proteins. On the one hand, the recombinant AAV capsid proteins provided herein are engineered to include peptides within loop IV and / or loop VIII of the parent AAV capsid proteins. Compared to the parent AAV capsid proteins, these recombinant AAV capsid proteins exhibit a significant increase in penetration of the blood-brain barrier (BBB).

[0064] definition

[0065] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus.

[0066] As used herein, the term "parent capsid protein" refers to an AAV capsid protein into which a peptide is inserted. The parent capsid protein can be a naturally occurring AAV capsid protein (e.g., belonging to any clade or clone described in the following literature: Bello et al., Sci. Rep. (2014) 4(6644): 1-11. doi: 10.1038 / srep06644 or Gao et al., J Virol. (June 2004); 78(12): 6381–6388. doi: 10.1128 / JVI.78.12.6381-6388.2004) or a non-naturally occurring AAV capsid protein. Suitable non-naturally occurring AAV capsid proteins include, but are not limited to, chimeric capsid proteins and capsid proteins that are engineered to contain one or more amino acid insertions, deletions or substitutions relative to naturally occurring AAV capsid proteins.

[0067] As used herein, the term "recombinant adeno-associated virus" or "rAAV" refers to an AAV comprising a genome lacking functional rep and cap genes.

[0068] As used herein, the term "cap gene" refers to a nucleic acid sequence encoding a capsid protein. For AAV, the capsid protein may be VP1, VP2 or VP3. VP1, VP2 and / or VP3 capsid proteins are assembled into a capsid surrounding the rAAV genome.

[0069] As used herein, the term "rep gene" refers to a nucleic acid sequence encoding non-structural proteins required for replication and production of AAV (eg, rep78, rep68, rep52, and rep40).

[0070] As used herein, the term "loop IV" refers to the amino acids in the AAV capsid protein corresponding to amino acids 434-466 of SEQ ID NO: 10 or amino acids 435-467 of SEQ ID NO: 11, or the corresponding amino acids of a different parent capsid protein.

[0071] As used herein, the term "loop VIII" refers to the amino acids in the AAV capsid protein corresponding to amino acids 563-592 of SEQ ID NO: 10 or amino acids 564-593 of SEQ ID NO: 11, or the corresponding amino acids of a different parent capsid protein.

[0072] As used herein, the term "rAAV genome" refers to a nucleic acid molecule (e.g., DNA and / or RNA) comprising the genomic sequence of rAAV. The skilled artisan will appreciate that where the rAAV genome comprises a transgene (e.g., an antibody heavy chain or light chain encoding sequence operably linked to a transcriptional regulatory element), the rAAV genome may be in a sense or antisense orientation relative to the direction of transcription of the transgene.

[0073] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been separated from one or more nucleic acid molecules present in the natural source of the polynucleotide.

[0074] As used herein, "vector" refers to a nucleic acid molecule that serves as a vehicle for introducing a nucleic acid molecule (eg, a polynucleotide disclosed herein) into a cell.

[0075] As used herein, "expression vector" refers to a vector (eg, a polynucleotide disclosed herein) comprising transcriptional regulatory elements operably linked to a gene of interest, which promotes expression of the gene of interest in a cell and / or a cell-free expression system.

[0076] As used herein, the term "transgene" refers to a non-AAV nucleic acid sequence encoding a polypeptide (e.g., an antibody or scFv) or a non-coding RNA (e.g., miRNA, shRNA, siRNA, antisense RNA, gRNA, antagomir, miRNA sponge, RNA aptamer, RNA ribozyme, or RNA aptamer).

[0077] As used herein, the term "transcriptional regulatory element" or "TRE" refers to a cis-acting nucleotide sequence (e.g., a DNA sequence) that regulates (e.g., controls, increases, or decreases) the transcription of an operably linked nucleotide sequence by RNA polymerase to form an RNA molecule. A TRE may comprise one or more promoter elements and / or enhancer elements. Those skilled in the art will appreciate that promoter and enhancer elements in a gene may be located close to each other, and the term "promoter" may refer to a sequence comprising a promoter element and an enhancer element. Therefore, the term "promoter" does not exclude an enhancer element in a sequence. Promoter and enhancer elements do not need to be derived from the same gene or species, and the sequence of each promoter or enhancer element may be identical or substantially identical to the corresponding endogenous sequence in the genome.

[0078] As used herein, the term "operably linked" is used to describe the connection between a TRE and a polynucleotide sequence to be transcribed (e.g., a transgene disclosed herein). Typically, gene expression is placed under the control of a TRE comprising one or more promoter and / or enhancer elements. A transgene is "operably linked" to a TRE if transcription of the transgene is controlled or influenced by the TRE. The promoter and enhancer elements of the TRE can be in any orientation and / or at any distance from the transgene, as long as the desired transcriptional activity is obtained. In an embodiment, the TRE is located upstream of the transgene.

[0079] As used herein, "percent identity" between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between the aligned sequence pairs by 100 and dividing by the length of the aligned region (including internal gaps). The identity score only calculates perfect matches and does not take into account the similarity between the amino acids to each other. When a sequence is described herein as having a certain percentage of identity with a reference sequence, the percentage identity with the reference sequence is determined across the entire length of the reference sequence.

[0080] As used herein, the term "effective amount" in the context of administering an AAV to a subject refers to the amount of AAV that achieves a desired prophylactic or therapeutic effect.

[0081] AAV capsid protein

[0082] In one aspect, the present invention provides a recombinant adeno-associated virus (AAV) capsid protein, comprising a peptide and a parent AAV capsid protein. The amino acid sequences of exemplary peptides for the recombinant AAV capsid proteins disclosed herein are provided in Table 1 below.

[0083] Table 1. Amino acid sequences of exemplary peptides

[0084]

[0085] In one aspect, the present invention provides a recombinant adeno-associated virus (AAV) capsid protein comprising a peptide and a parent capsid protein, wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6. In one aspect, the present invention provides a recombinant adeno-associated virus (AAV) capsid protein comprising a peptide and a parent capsid protein, wherein the peptide is contained within loop IV and / or loop VIII of the parent capsid protein, and wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0086] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is contained within loop IV of a parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is contained within loop IV of a parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is contained within loop IV of a parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is contained within loop IV of a parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is contained within loop IV of a parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is contained within loop IV of a parent capsid protein.

[0087] In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is contained within the loop VIII of the parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is contained within the loop VIII of the parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is contained within the loop VIII of the parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is contained within the loop VIII of the parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is contained within the loop VIII of the parent capsid protein. In an embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is contained within the loop VIII of the parent capsid protein.

[0088] In an embodiment, the recombinant AAV capsid protein comprises a first peptide within loop IV of a parent capsid protein and a second peptide within loop VIII of a parent capsid protein. In an embodiment, the first peptide and the second peptide are the same peptide. In an embodiment, the first peptide and the second peptide are different peptides. In an embodiment, the first peptide comprises an amino acid sequence of SEQ ID NO: 1, and the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In an embodiment, the first peptide comprises an amino acid sequence of SEQ ID NO: 2, and the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In an embodiment, the first peptide comprises an amino acid sequence of SEQ ID NO: 3, and the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In an embodiment, the first peptide comprises an amino acid sequence of SEQ ID NO: 4, and the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In an embodiment, the first peptide comprises an amino acid sequence of SEQ ID NO: 5, and the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1-6. In an embodiment, the first peptide comprises the amino acid sequence of SEQ ID NO: 6, and the second peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0089] In embodiments, the parent capsid protein is VP1 capsid protein. In embodiments, the parent capsid protein is VP2 capsid protein. In embodiments, the parent capsid protein is VP3 capsid protein.

[0090] In an embodiment, the parent capsid protein is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV, AAV5.2 or AAV5 capsid protein, or an engineered variant thereof.

[0091] In an embodiment, the parent capsid protein is an engineered variant capsid protein comprising amino acid sequences from at least two different AAV capsid proteins. In an embodiment, the parent capsid protein comprises amino acid sequences from AAV2 capsid protein and AAV5 capsid protein. In an embodiment, the parent capsid protein comprises the VP1 amino acid sequence from AAV2 and the VP2 and VP3 amino acid sequences from AAV5.

[0092] In embodiments, the parent capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity to amino acids 193-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence having at least 99% identity to amino acids 193-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence having 100% identity to amino acids 193-725 of SEQ ID NO: 11.

[0093] In embodiments, the parent capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity to amino acids 137-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence having at least 99% identity to amino acids 137-725 of SEQ ID NO: 11. In embodiments, the parent capsid protein comprises an amino acid sequence having 100% identity to amino acids 137-725 of SEQ ID NO: 11.

[0094] In embodiments, the parent capsid protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% identity to the amino acid sequence of SEQ ID NO: 10 or 11. In embodiments, the parent capsid protein comprises an amino acid sequence having at least 99% identity to the amino acid sequence of SEQ ID NO: 10 or 11. In embodiments, the parent capsid protein comprises an amino acid sequence having 100% identity to the amino acid sequence of SEQ ID NO: 10 or 11.

[0095] In embodiments, the peptide is positioned C-terminally adjacent to an amino acid in the parent capsid corresponding to amino acid 440, 441, 442, 443, 444, 445, 446, 447, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, or 584 of SEQ ID NO: 11.

[0096] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 440 of SEQ ID NO: 11 in the parent capsid.

[0097] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 440 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 440 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 440 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 440 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 440 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 440 of SEQ ID NO: 11 in the parent capsid.

[0098] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 441 of SEQ ID NO: 11 in the parent capsid.

[0099] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 441 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 441 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 441 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 441 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 441 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 441 of SEQ ID NO: 11 in the parent capsid.

[0100] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 442 of SEQ ID NO: 11 in the parent capsid.

[0101] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 442 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 442 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 442 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 442 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 442 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 442 of SEQ ID NO: 11 in the parent capsid.

[0102] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 443 of SEQ ID NO: 11 in the parent capsid.

[0103] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 443 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 443 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 443 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 443 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 443 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 443 of SEQ ID NO: 11 in the parent capsid.

[0104] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 444 of SEQ ID NO: 11 in the parent capsid.

[0105] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 444 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 444 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 444 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 444 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 444 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 444 of SEQ ID NO: 11 in the parent capsid.

[0106] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 445 of SEQ ID NO: 11 in the parent capsid.

[0107] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 445 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 445 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 445 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 445 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 445 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 445 of SEQ ID NO: 11 in the parent capsid.

[0108] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 446 of SEQ ID NO: 11 in the parent capsid.

[0109] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 446 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 446 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 446 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 446 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 446 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 446 of SEQ ID NO: 11 in the parent capsid.

[0110] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 447 of SEQ ID NO: 11 in the parent capsid.

[0111] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 447 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 447 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 447 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 447 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 447 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 447 of SEQ ID NO: 11 in the parent capsid.

[0112] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 572 of SEQ ID NO: 11 in the parent capsid.

[0113] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 572 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 572 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 572 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 572 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 572 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 572 of SEQ ID NO: 11 in the parent capsid.

[0114] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 573 of SEQ ID NO: 11 in the parent capsid.

[0115] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 573 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 573 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 573 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 573 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 573 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 573 of SEQ ID NO: 11 in the parent capsid.

[0116] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 574 of SEQ ID NO: 11 in the parent capsid.

[0117] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 574 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 574 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 574 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 574 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 574 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 574 of SEQ ID NO: 11 in the parent capsid.

[0118] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 575 of SEQ ID NO: 11 in the parent capsid.

[0119] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 575 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 575 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 575 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 575 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 575 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 575 of SEQ ID NO: 11 in the parent capsid.

[0120] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 576 of SEQ ID NO: 11 in the parent capsid.

[0121] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 576 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 576 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 576 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 576 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 576 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 576 of SEQ ID NO: 11 in the parent capsid.

[0122] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 577 of SEQ ID NO: 11 in the parent capsid.

[0123] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 577 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 577 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 577 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 577 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 577 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 577 of SEQ ID NO: 11 in the parent capsid.

[0124] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 578 of SEQ ID NO: 11 in the parent capsid.

[0125] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 578 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 578 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 578 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 578 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 578 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 578 of SEQ ID NO: 11 in the parent capsid.

[0126] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 579 of SEQ ID NO: 11 in the parent capsid.

[0127] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 579 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 579 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 579 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 579 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 579 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 579 of SEQ ID NO: 11 in the parent capsid.

[0128] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 580 of SEQ ID NO: 11 in the parent capsid.

[0129] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 580 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 580 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 580 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 580 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 580 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 580 of SEQ ID NO: 11 in the parent capsid.

[0130] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 581 of SEQ ID NO: 11 in the parent capsid.

[0131] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 581 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 581 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 581 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 581 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 581 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 581 of SEQ ID NO: 11 in the parent capsid.

[0132] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 582 of SEQ ID NO: 11 in the parent capsid.

[0133] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 582 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 582 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 582 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 582 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 582 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 582 of SEQ ID NO: 11 in the parent capsid.

[0134] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 583 of SEQ ID NO: 11 in the parent capsid.

[0135] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 583 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 583 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 583 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 583 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 583 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 583 of SEQ ID NO: 11 in the parent capsid.

[0136] In embodiments, the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 584 of SEQ ID NO: 11 in the parent capsid.

[0137] In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 1, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 584 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 2, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 584 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 3, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 584 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 4, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 584 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 5, and the peptide is positioned C-terminally adjacent to the amino acid in the parent capsid corresponding to amino acid 584 of SEQ ID NO: 11. In embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6, and the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 584 of SEQ ID NO: 11 in the parent capsid.

[0138] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3);an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 194 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6). ;

[0139] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 578 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0140] In an embodiment, the recombinant AAV capsid protein comprises: an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12; an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13; an amino acid sequence having 100% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14; an amino acid sequence having 100% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19; an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 20; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 21; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 22; an amino acid sequence that has 100% sequence identity with amino acids 194 to 737 of SEQ ID NO: 23.

[0141] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3);an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 138 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 89 are FGQSSLPRDGPN (SEQ ID NO: 6). ;

[0142] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 89 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0143] In an embodiment, the recombinant AAV capsid protein comprises: an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12; an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13; an amino acid sequence having 100% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14; an amino acid sequence having 100% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19; an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 20; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 21; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 22; an amino acid sequence that has 100% sequence identity with amino acids 138 to 737 of SEQ ID NO: 23.

[0144] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3);an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 1 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 1 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity to amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 1 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to amino acids 1 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 89 are FGQSSLPRDGPN (SEQ ID NO: 6). ;

[0145] In embodiments, the recombinant AAV capsid protein comprises: an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity with amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); an amino acid sequence having at least 99% sequence identity with amino acids 1 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); an amino acid sequence having at least 99% sequence identity with amino acids 1 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 89 are FGQSSLPRDGPN (SEQ ID NO: 6).

[0146] In an embodiment, the recombinant AAV capsid protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-23. In an embodiment, the amino acid sequence of the recombinant AAV capsid protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-23.

[0147] In an embodiment, the peptide is directly connected (i.e., connected by a peptide bond) to the amino acids of the parent AAV capsid protein. In an embodiment, the peptide is connected to the amino acids of the parent AAV capsid protein via an amino acid linker. In an embodiment, the peptide comprises an N-terminal linker. In an embodiment, the peptide comprises a C-terminal linker. In an embodiment, the peptide comprises an N-terminal linker and a C-terminal linker. Suitable linkers include, but are not limited to, a G linker (AG), a GS linker (GS), and a GG linker (GG), TG, GLS, or GQSG.

[0148] In an embodiment, the recombinant AAV capsid protein disclosed herein binds to a protein expressed on the cell membrane of a myocyte, microglia, astrocyte, neuron, blood-brain barrier endothelial cell (BBB EC), pericyte, oligodendrocyte, ependymal cell, pyramidal neuron, rose hip neuron, Purkinje cell or cardiomyocyte. In an embodiment, the recombinant AAV capsid protein disclosed herein binds to a protein selected from the group consisting of α-integrin or β-integrin (e.g., integrin α7 (ITGA7)), αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, αIIbβ3, α8β1), transferrin receptor (Tf1r), insulin receptor (INSR / CD220), ADP-ribosyltransferase 1 (ART1) and cadherin 15 (CDH15).

[0149] The amino acid sequences of exemplary recombinant AAV capsid proteins provided herein are listed in Table 2 below.

[0150] Table 2. Amino acid sequences of exemplary recombinant AAV capsid proteins

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Additional examples of recombinant AAV capsid proteins disclosed herein are listed in Table 3 below.

[0158] Table 3. Amino acid sequences of additional exemplary recombinant AAV capsid proteins

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Polynucleotides, vectors and methods for producing AAV capsid proteins

[0165] In one aspect, provided herein is an isolated polynucleotide encoding a recombinant AAV capsid protein disclosed herein.

[0166] In an embodiment, polynucleotides are optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences and / or elimination of mRNA unstable elements. The method of generating optimized polynucleotides for recombinant expression by introducing codon changes and / or eliminating inhibitory regions in mRNA can be performed by adopting, for example, the optimization methods described in U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132 and 6,794,498, and therefore, all of these U.S. Patents are incorporated herein by reference in their entirety. For example, potential splicing sites and unstable elements (e.g., elements rich in A / T or A / U) in RNA can be mutated without changing the amino acid encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These changes utilize the degeneracy of the genetic code, for example, using alternative codons of the same amino acid. In an embodiment, one or more codons may need to be changed to encode conservative mutations, for example, similar amino acids with chemical structures and properties and / or functions similar to the original amino acids. Such methods can increase expression of the encoded capsid protein relative to expression of a capsid encoded by a non-optimized polynucleotide.

[0167] In one aspect, a vector is provided herein, comprising a polynucleotide disclosed herein. Suitable vectors include, but are not limited to, plasmids, viruses, cosmids, artificial chromosomes, linear DNA, and mRNA. In an embodiment, the vector is a plasmid or a viral vector. In an embodiment, the vector is a retroviral vector, a herpes virus vector, a baculovirus vector, or an adenovirus vector. In an embodiment, the vector is an expression vector.

[0168] The vector (e.g., expression vector) can be introduced into a cell (using any technique known in the art) for propagation of the vector and / or for expression of the AAV capsid protein encoded by the vector. Therefore, in another aspect, the present disclosure provides a recombinant cell comprising a polynucleotide or vector (e.g., expression vector) disclosed herein. And further, in another aspect, the present disclosure provides a method of producing an AAV capsid protein, the method comprising culturing a recombinant cell under conditions where the polynucleotide is expressed and the capsid is produced.

[0169] A variety of host cells and expression vector systems can be used to express the capsid proteins described herein. In these expression systems, the coding sequence of interest can be produced and subsequently purified. These expression systems also represent cells that can express the capsid proteins described herein in situ when transformed or transfected with appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing a capsid protein coding sequence; yeast (e.g., Saccharomyces Pichia) transformed with, for example, a recombinant yeast expression vector containing a capsid protein coding sequence; insect cell systems infected with, for example, a recombinant viral expression vector (e.g., baculovirus) containing a capsid protein coding sequence; plant cell systems (e.g., green algae, such as Chlamydomonas reinhardtii) infected with, for example, a recombinant viral expression vector (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, for example, a recombinant plasmid expression vector (e.g., Ti plasmid) containing a capsid protein coding sequence; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, NIH) containing, for example, a recombinant expression construct. 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells), the recombinant expression construct contains a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or contains a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In an embodiment, the cell used to express the capsid protein described herein is a human cell, such as a human cell line. In an embodiment, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In an embodiment, bacterial cells (such as Escherichia coli) or eukaryotic cells (e.g., mammalian cells) are used for the expression of capsid proteins. For example, mammalian cells (such as CHO or HEK293 cells) combined with a vector (such as the major intermediate early gene promoter element from human cytomegalovirus) are effective expression systems for the capsid proteins disclosed herein. In an embodiment, insect cells (e.g., Sf9 cells) are used for the expression of capsid proteins.

[0170] For example, in insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The capsid protein coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0171] In mammalian host cells, many virus-based expression systems can be used. In the case of using adenovirus as an expression vector, the capsid protein coding sequence of interest can be connected to an adenovirus transcription / translation control complex, such as a late promoter and a tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., E1 region or E3 region) will produce a recombinant virus that is viable and capable of expressing capsid protein molecules in infected hosts (see, e.g., Logan J & Shenk T (1984) PNAS 81 (12): 3655-9, which is incorporated herein by reference in its entirety). The efficient translation of the inserted capsid protein coding sequence may also require specific start signals. These signals include the ATG start codon and adjacent sequences. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can have a variety of sources, including natural and synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancer elements, transcription terminators, etc. (See, e.g., Bitter G et al., (1987) Methods Enzymol. 153: 516-544, which is incorporated herein by reference in its entirety). Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT20, T-47D, NSO, CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells.

[0172] For long-term, high-yield production of recombinant proteins, stable expressing cells can be generated. For example, cell lines that stably express the capsid proteins described herein can be engineered.

[0173] In an embodiment, instead of using an expression vector containing a viral replication start point, a polynucleotide (e.g., DNA or RNA) and a selectable marker controlled by an appropriate transcriptional regulatory element (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) can be used to transform a host cell. After the polynucleotide is introduced, the engineered cell can be allowed to grow in an enriched medium for 1-2 days, and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, and allows cells to stably integrate the plasmid into their chromosome and grow to form a focus, which can then be cloned and amplified into a cell line. The method can be advantageously used to engineer a cell line expressing a capsid protein described herein or a fragment thereof.

[0174] A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes in tk cells, hgprt cells, or aprt cells, respectively, all of which are incorporated herein by reference in their entirety. In addition, antimetabolite resistance can be used as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78: 1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3: 87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan RC (1993) Science 260: 926-932; Morgan RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; and Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5): 211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are incorporated herein by reference in their entirety.Methods generally known in the field of recombinant DNA technology can be routinely applied to select desired recombinant clones, and such methods are described, for example, in Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, all of which are incorporated herein by reference in their entirety.

[0175] AAV composition

[0176] In one aspect, provided herein is a rAAV composition comprising a capsid comprising a recombinant AAV capsid protein disclosed herein.

[0177] In one aspect, provided herein is an rAAV comprising: a capsid comprising one or more recombinant capsid proteins disclosed herein; and an rAAV genome.

[0178] In embodiments, the rAAV genome comprises a transgene. In embodiments, the transgene encodes a polypeptide. In embodiments, the transgene encodes an antibody or fragment thereof. In embodiments, the transgene encodes a scFv.

[0179] The rAAV disclosed herein generally comprises a recombinant genome (e.g., a rAAV genome) packaged in a capsid. The rAAV genome can be of any type capable of being packaged in an AAV capsid disclosed herein. For example, in an embodiment, the rAAV genome is a single-stranded DNA genome. In an embodiment, the rAAV genome is a self-complementary genome, such as described in US7790154, which is hereby incorporated by reference in its entirety.

[0180] In embodiments, the rAAV genome comprises a transgene. In embodiments, the transgene encodes a therapeutic protein. In embodiments, the transgene encodes an antibody or fragment thereof (e.g., a Fab, scFv, or full-length antibody). In embodiments, the transgene encodes a scFv, a nanobody, or a VHH. In embodiments, the transgene encodes a non-coding RNA.

[0181] In an embodiment, the transgene encodes a reporter gene sequence that produces a detectable signal when expressed. Such reporter gene sequences include, but are not limited to, DNA sequences encoding: β-lactamase; β-galactosidase (LacZ); alkaline phosphatase; thymidine kinase; green fluorescent protein (GFP); red fluorescent protein (RFP); chloramphenicol acetyltransferase (CAT); luciferase; membrane-bound proteins, including, for example, CD2, CD4, CD8; influenza hemagglutinin protein and other proteins well known in the art, for which high-affinity antibodies are present or can be produced by conventional means; and fusion proteins comprising membrane-bound proteins, which are appropriately fused to antigen tag domains from hemagglutinin or Myc, etc.

[0182] In an embodiment, the rAAV genome comprises a TRE operably linked to a transgene to control the expression of an RNA or polypeptide encoded by the transgene. In an embodiment, the TRE comprises a constitutive promoter. In an embodiment, the TRE is active in any mammalian cell (e.g., any human cell). In an embodiment, the TRE is active in a wide range of human cells. Such TREs may comprise constitutive promoters and / or enhancer elements, including any of those described herein, and any of those elements known to those skilled in the art. In an embodiment, the TRE comprises an inducible promoter. In an embodiment, the TRE is a tissue-specific TRE, that is, it is active in a specific tissue and / or organ. The tissue-specific TRE comprises one or more tissue-specific promoters and / or enhancer elements, and optionally one or more constitutive promoters and / or enhancer elements. Tissue-specific promoters and / or enhancer elements can be separated from genes specifically expressed in tissues by methods well known in the art.

[0183] Suitable promoters include, for example, the cytomegalovirus promoter (CMV) (Stinski et al. (1985) Journal of Virology 55(2): 431-441), the CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al., (1989) Gene 79(2): 269-277), a hybrid form of the CBA promoter (CBh) (Gray et al., (2011) Hum Gene Ther. 22(9): 1143-53), the CB SB (Jacobson et al., (2006) Molecular Therapy 13(6): 1074-1084), human elongation factor 1α promoter (EF1α) (Kim et al., (1990) Gene 91(2): 217-223), human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al., (1984) Gene32(3): 409-417), mitochondrial heavy chain promoter (Lodeiro et al., (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al., (1990) FEBS Letters 261: 101-105).

[0184] In one aspect, provided herein is a pharmaceutical composition comprising a rAAV disclosed herein and a pharmaceutically acceptable excipient, adjuvant, diluent, carrier or vector, or a combination thereof. A "pharmaceutically acceptable carrier" includes any material that, when combined with the active ingredient of the composition, allows the ingredient to retain biological activity and does not cause a disruptive physiological response (such as an unintended immune response). Pharmaceutically acceptable carriers include water, phosphate-buffered saline, emulsions (such as oil / water emulsions), and wetting agents. Compositions containing such carriers are formulated by well-known conventional methods, such as those described in Remington's Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton Pa. 18042, USA; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HC Ansel et al., 7th edition, Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., 3rd edition Amer. Pharmaceutical Assoc.

[0185] How to use

[0186] In one aspect, a method of transducing a cell is provided herein, comprising contacting the cell with a rAAV disclosed herein under conditions of transducing the cell and expressing a transgene. In embodiments, the cell is a myocyte, a microglia, an astrocyte, a neuron, a blood-brain barrier endothelial cell (BBB EC), a pericyte, an oligodendrocyte, an ependymal cell, or a cardiomyocyte. In embodiments, the neuron is a pyramidal neuron, a rosehip neuron, or a Purkinje cell.

[0187] In an embodiment, a method for expressing a transgene in a cell is provided herein, the method generally comprising contacting the cell with such a rAAV under conditions where the cell is transduced and the transgene is expressed. As described herein, the transgene may encode a polypeptide, such as an antibody or a fragment thereof (e.g., scFv). Thus, in an embodiment, a method for producing a polypeptide, such as an antibody or a fragment thereof (e.g., scFv), in a cell is provided herein, the method generally comprising contacting the cell with such a rAAV under conditions where the cell is transduced and the polypeptide, such as an antibody or a fragment thereof (e.g., scFv), is produced.

[0188] On the one hand, there is provided herein a method for delivering a transgene to a cell, the method comprising contacting the cell with a rAAV disclosed herein under conditions of transducing the cell and expressing the transgene. The rAAV disclosed herein can be used for in vitro, in vivo and ex vivo transduction of cells. Cells suitable for being transduced by rAAV disclosed herein include, but are not limited to, blood cells, liver cells, heart cells, joint tissue cells, muscle cells, brain cells, spinal cord cells, kidney cells or lung cells. In an embodiment, the cell is a myocyte, a microglia, an astrocyte, a neuron, a blood-brain barrier endothelial cell (BBB EC), a pericyte, an oligodendrocyte, an ependymal cell or a cardiomyocyte. In an embodiment, the neuron is a pyramidal neuron, a rose hip neuron or a Purkinje cell. In an embodiment, the rAAV provided herein is used for in vivo transduction of cells in a subject suffering from a disease associated with muscle, brain or spinal cord tissue.

[0189] In one aspect, provided herein is a method of delivering a transgene to muscle, brain, or spinal cord tissue of a subject in need thereof, the method comprising administering to the subject a disclosed rAAV.

[0190] In one aspect, provided herein is a method of treating a neuromuscular or neurodegenerative disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a rAAV disclosed herein. In an embodiment, the neuromuscular disease is selected from the group consisting of myopathy, hereditary cardiomyopathy, metabolic myopathy, distal myopathy, muscular dystrophy, congenital myopathy, spinal muscular atrophy (SMA), motor neuron disease, congenital myopathy, congenital muscular dystrophy, motor neuron disease, Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, myotonic dystrophy, myotubular myopathy, centronuclear myopathy, nemaline myopathy, selenoprotein N-related myopathy, Pompe disease, glycogen storage disease type III, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), Charcot-Marie-Tooth disease, multiple sclerosis, myositis, polymyositis, and dermatomyositis.

[0191] In an embodiment, the neurodegenerative disease or disorder is selected from the group consisting of motor neuron disease (MND), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinocerebellar ataxia (SCA) and Friedreich's ataxia.

[0192] In embodiments, the rAAV disclosed herein can be administered to a subject (e.g., a human subject) by all routes suitable for rAAV, including but not limited to intravenous, intraperitoneal, subcutaneous, intramuscular, intrathecal, or intradermal.

[0193] In one aspect, provided herein is a rAAV as disclosed herein for use in medicine. In one aspect, provided herein is a rAAV as disclosed herein for use as therapy. In one aspect, provided herein is a rAAV as disclosed herein for use as a medicament.

[0194] Adeno-associated virus packaging system

[0195] In one aspect, the present invention provides a packaging system for the recombinant preparation of a recombinant adeno-associated virus (rAAV) disclosed herein. Such a packaging system generally comprises: a first nucleotide sequence encoding one or more AAV Rep proteins; a second nucleotide sequence encoding a capsid protein of any recombinant AAV capsid disclosed herein; and a third nucleotide sequence comprising any AAV genome sequence disclosed herein, wherein the packaging system is operable in a cell to encapsulate the AAV genome in a capsid to form rAAV.

[0196] In an embodiment, the packaging system comprises a first vector and a second vector, the first vector comprising a first nucleotide sequence encoding one or more AAV Rep proteins and a second nucleotide sequence encoding an AAV capsid protein, and the second vector comprising a third nucleotide sequence comprising an rAAV genome. As used in the context of the packaging systems described herein, "vector" refers to a nucleic acid molecule (e.g., a plasmid, a virus, a cosmid, an artificial chromosome, etc.) that serves as a vehicle for introducing nucleic acid into a cell.

[0197] Any AAV Rep protein can be used in the packaging system disclosed herein. In an embodiment of the packaging system, the Rep nucleotide sequence encodes an AAV2 Rep protein. Suitable AAV2 Rep proteins may include, but are not limited to, Rep 78 / 68 or Rep68 / 52.

[0198] In an embodiment, the packaging system further comprises a fourth nucleotide sequence comprising one or more helper virus genes. In an embodiment, the fourth nucleotide sequence comprises adenovirus E2, E4 and VA genes. In an embodiment, the packaging system further comprises a third vector (e.g., a helper virus vector) comprising the fourth nucleotide sequence. The third vector can be an independent third vector, integrated with the first vector, or integrated with the second vector.

[0199] In embodiments of the packaging system, the helper virus is selected from the group consisting of adenovirus, herpes virus (including herpes simplex virus (HSV)), poxvirus (such as vaccinia virus), cytomegalovirus (CMV) and baculovirus. In embodiments where the helper virus is an adenovirus, the adenovirus genome comprises one or more adenovirus RNA genes selected from the group consisting of E1, E2, E4 and VA. In embodiments, the adenovirus genome comprises one or more adenovirus RNA genes selected from the group consisting of E2, E4 and VA. In embodiments where the helper virus is HSV, the HSV genome comprises one or more HSV genes selected from the group consisting of UL5 / 8 / 52, ICPO, ICP4, ICP22 and UL30 / UL42.

[0200] In embodiments of the packaging system, the first vector, the second vector, and / or the third vector are contained within one or more plasmids. In embodiments, the first vector and the third vector are contained within a first plasmid. In embodiments, the second vector and the third vector are contained within a second plasmid.

[0201] In an embodiment of the packaging system, the first vector, the second vector and / or the third vector are contained in one or more recombinant helper viruses. In an embodiment, the first vector and the third vector are contained in a recombinant helper virus. In an embodiment, the second vector and the third vector are contained in a recombinant helper virus.

[0202] In one aspect, a method for recombinant production of rAAV as described herein is provided herein, wherein the method comprises transfecting or transducing cells with a packaging system as described herein under conditions operable to encapsulate the rAAV genome in a capsid to form an rAAV as described herein. Exemplary methods for recombinant production of rAAV include: transient transfection (e.g., with one or more transfection plasmids containing a first vector, a second vector, and an optional third vector as described herein); viral infection (e.g., with one or more recombinant helper viruses containing a first vector, a second vector, and an optional third vector as described herein, such as adenovirus, poxvirus (such as vaccinia virus), herpes virus (including HSV, cytomegalovirus, or baculovirus); and transfection or infection of a stable production cell line (e.g., with a stable production cell containing a Rep nucleotide sequence encoding one or more AAVRep proteins and / or a Cap nucleotide sequence encoding one or more capsid proteins as described herein, such as a mammalian cell or an insect cell, and with a transfer genome as described herein delivered in the form of a plasmid or a recombinant helper virus).

[0203] Therefore, the present disclosure provides a packaging system for the preparation of rAAV, wherein the packaging system comprises: a first nucleotide sequence encoding one or more AAV Rep proteins; a second nucleotide sequence encoding the AAV capsid protein disclosed herein; a third nucleotide sequence comprising the rAAV genome sequence disclosed herein; and optionally a fourth nucleotide sequence comprising one or more helper virus genes (e.g., adenovirus E2, E4 and VA genes).

[0204] Examples

[0205] Example 1: Production of modified capsids

[0206] Recombinant AAV capsids that can efficiently cross the BBB were developed by inserting peptides within the amino acid sequence of the parent AAV capsid protein.

[0207] a. AAV5.2 capsid

[0208] A chimeric AAV capsid (AAV5.2, SEQ ID NO: 11) was used as the parent capsid protein to generate recombinant AAV capsid protein. The AAV capsid is encoded by three viral proteins (VPs) VP1, VP2, and VP3, which are incorporated into the 60-subunit capsid at a ratio of 1:1:10. The AAV5.2 capsid is an engineered variant of the AAV2 and AAV5 capsids, which includes the VP1 sequence from AAV2 and the VP2 / 3 sequence from AAV5.

[0209] b. Capsid Design

[0210] The VP1 and VP2 unique sequences are located on the interior of the capsid and contain a phospholipase (PLA2) domain (in VP1) and a nuclear localization signal (VP1 and VP2). The VP3 sequence (contained in all three VPs) is the primary protein sequence that makes up the surface topology of the AAV capsid, which in turn determines tropism and antigenicity. The VP3 topology contains a core eight-stranded (βB to βI) barrel motif and large loop insertions between the β strands that form the capsid surface. Conformational variations in these nine surface loops (VR-I to VR-IX) define the unique surface features of different AAV serotypes. Therefore, the peptide inserts described here were inserted into these outward-facing loops. Specifically, loops IV and VIII were selected for peptide insertion sites.

[0211] Position 443, located at the tip of loop IV on AAV5 (SEQ ID NO: 10) or position 444 on the AAV5.2 (SEQ ID NO: 11) capsid, was selected as an insertion site in loop IV, and the peptide was inserted with the C-terminus next to these amino acid positions. Position 576, located at the tip of loop VIII on AAV5 (SEQ ID NO: 10) or position 577 on the AAV5.2 (SEQ ID NO: 11) capsid, was selected as an insertion site in loop VIII, and the peptide was inserted with the C-terminus next to these amino acid positions. Insertion of the peptide C-terminally next to these positions should have minimal impact on capsid integrity, as the remaining VP residues interact to remain intact at the bottom of the capsid.

[0212] Six different peptides (SEQ ID NO: 1-6; see Table 1) were used to engineer recombinant AAV capsid proteins. Specifically, polynucleotides encoding capsid proteins comprising parent AAV5.2 capsid proteins with peptide insertions from Table 1 were synthesized and inserted into plasmid vectors. Each AAV capsid protein was engineered to contain only one peptide insertion, which was inserted C-terminally adjacent to position 444 in loop IV or position 577 in loop VIII numbered according to AAV5.2 (SEQ ID NO: 11). The amino acid sequences of the resulting recombinant AAV capsid proteins (SEQ ID NO: 12-23) are disclosed in Table 2.

[0213] Example 2: Transduction of human myotubes

[0214] The transduction efficiency of recombinant AAV capsids was further analyzed by transducing human myotubes with the capsids.

[0215] Human skeletal muscle cells (CC-2580, Lonza) were plated in 24-well plates at a cell density of 50,000 cells per well. When the cells reached 85-90% confluence, myoblast differentiation was induced by replacing the myoblast proliferation medium with myoblast (low serum) differentiation medium. Myotube differentiation was performed for 7 days with partial replacement of differentiation medium every 2 days. AAV transduction of myotubes was performed at 1x10 6 MOI was performed, mixed with 50uL of PBS at the beginning of differentiation, and the negative control was treated with 50uL of PBS only. One day after transduction, the medium was changed. To analyze the transduction efficacy, GFP+ cells were monitored for a long time using the EVOS FLoid imaging system (Catalog No. 447116). One week after transduction, the transduced myotubes were imaged at 20 times magnification using the EVOS FLoid imaging system (Catalog No. 447116), and then harvested and frozen for further quantitative analysis.

[0216] Shown in Figure 1A The results in show that several recombinant AAV capsids showed successful transduction of human myotubes, and that some capsids showed increased transduction of myotubes when the peptide was inserted in loop IV compared to loop VIII (VYPep4 and VYPep5). Figure 1B The results in show that recombinant AAV capsids containing VYPep4 or VYPep5 at position 444 of loop IV or position 577 of loop VIII successfully transduced myotubes.

[0217] Example 3: Transduction of human astrocytes and motor neurons

[0218] The transduction efficiency of recombinant AAV capsids was further analyzed by transducing human astrocytes and motor neurons with the capsids.AAV5.2 capsids containing VYPep4, VYPep5, or VYPep11 in loop IV or loop VIII were analyzed in the following assays.

[0219] Human astrocytes and motor neurons were plated in 24-well plates at a cell density of 50,000 cells per well. AAV transduction of astrocytes and motor neurons was performed at a density of 1x10 4 or 1x10 6MOI was performed, mixed with 50uL of PBS, and the negative control was treated with 50uL of PBS only. One day after transduction, the medium was changed. To analyze the transduction efficacy, GFP+ cells were monitored for a long time using the EVOS FLoid imaging system (Catalog No. 447116). One week after transduction, the transduced astrocytes and motor neurons were imaged at 20 times magnification using the EVOS FLoid imaging system (Catalog No. 447116), and then harvested and frozen for further quantitative analysis.

[0220] GFP expression from astrocytes was quantified and expressed as the percentage of GFP-positive cells to the total number of cells. Figure 2A and Figure 2B The results in Figure 2 show that several recombinant AAV capsids were expressed at 1x10 per cell. 4 ( Figure 2B ) or 1x10 6 ( Figure 2A ) successfully transduced human astrocytes and demonstrated that some capsids showed increased transduction of astrocytes when the peptide was inserted in loop IV compared to loop VIII.

[0221] Example 4: Transduction of astrocytes across the blood-brain barrier (BBB)

[0222] The ability of recombinant AAV capsids to penetrate the blood-brain barrier (BBB) ​​was analyzed using a BBB microfluidics model.

[0223] The BBB model used to test the capsids contains human cell lines of brain endothelial cells (blood side) and astrocytes (brain side) in a three-channel microfluidic platform containing 40 chips (OrganoPlate® 3-Channel 40) in a 384-well plate format. In each chip, perfused blood vessels of brain endothelial cells are grown against an extracellular matrix gel that is patterned by surface tension technology. Astrocytes are added on the other side of the gel to complete the BBB-on-a-chip model. The barrier function of this model is studied using a fluorescent barrier integrity assay. 6On day 10 after endothelial cells were seeded at an MOI of 1:1, different AAV capsids, VYAAV-5.2-GFP, AAV5.2-VYPep4-loop IV-GFP, or AAV5.2-VYPep4-loop VIII-GFP, were added to the chip on the blood side. The medium was replaced with fresh human brain microvascular endothelial cell medium and astrocyte medium on the blood and brain sides, respectively. On day 6 after AAV transduction, the medium was replaced with PBS containing Hoechst 33342, 1:10,000 (Cat. No. 62249, Thermo Fisher Scientific), and the OrganoPlate® was imaged using ImageXpress PICO at 4X magnification.

[0224] Figure 3 The results in show that recombinant AAV capsids successfully penetrate the BBB and transduce astrocytes on the brain side of the BBB.

[0225] Example 5: Transduction of non-human primate astrocytes

[0226] The transduction efficiency of recombinant AAV capsids containing VYPep4 in loop IV was further analyzed by transducing non-human primate (NHP) primary astrocytes with the recombinant capsids.

[0227] NHP primary astrocytes were plated in 24-well plates at a cell density of 40,000 cells per well. Four days after seeding, 1×10 5 or 1x10 6 AAV transduction of NHP astrocytes was performed at an MOI of 4.4 %. Four days after transduction, the transduced astrocytes were stained with Hoechst and imaged at 4× magnification using an ImageXpress PICO microscope.

[0228] GFP expression from NHP astrocytes was quantified using PICO software and expressed as the percentage of GFP-positive cells to the total number of cells. Figure 4 The results in show that transduction of NHP astrocytes was increased when the peptide was inserted in loop IV compared to AAV5.2 and AAV9 without the peptide inserted.

[0229] Example 6: Transduction of human motor neurons

[0230] The transduction efficiency of recombinant AAV capsids containing VYPep4 in loop IV was further analyzed by transducing human motor neurons with the recombinant capsids.

[0231] iPSC-derived motor neurons were plated in 96-well plates at a cell density of 32,000 cells per well. 5 or 1x10 6 AAV transduction was performed at an MOI of 1. ...

[0232] GFP expression from motor neurons was quantified using PICO software and expressed as the percentage of GFP-positive cells to the total number of cells. Figure 5 The results in show that transduction of motor neurons was increased when the peptide was inserted in loop IV compared to AAV5.2 and AAV9 without the peptide inserted.

[0233] ***

[0234] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to the modifications described, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0235] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

1. A recombinant adeno-associated virus (AAV) capsid protein, comprising a peptide and a parent capsid protein, wherein the peptide is contained within loop IV and / or loop VIII of the parent capsid protein, and wherein the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 6.

2. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein is a clade A, clade B, clade C, clade D, clade E, clade F, clade G, clade H, clade I, AAVgo.1, AAV3, AAV4, AAV10, AAV11, AAV12, rh.32, rh32.33, rh.33, rh.34, BAAV or AAV5 capsid protein, or an engineered variant thereof.

3. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence that is at least 95% identical to amino acids 193 to 725 of SEQ ID NO:

11.

4. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence that is at least 99% identical to amino acids 193 to 725 of SEQ ID NO:

11.

5. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence of amino acids 193 to 725 of SEQ ID NO:

11.

6. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence that is at least 95% identical to amino acids 137 to 725 of SEQ ID NO:

11.

7. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence that is at least 99% identical to amino acids 137 to 725 of SEQ ID NO:

11.

8. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence of amino acids 137 to 725 of SEQ ID NO:

11.

9. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 10 or 11.

10. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 10 or 11.

11. The recombinant AAV capsid protein of claim 1, wherein the parent capsid protein comprises the amino acids of SEQ ID NO: 10 or 11.

12. The recombinant AAV capsid protein of any one of claims 1 to 11, wherein the peptide is positioned C-terminally proximal to an amino acid in the parent capsid corresponding to amino acids 440, 441, 442, 443, 444, 445, 446, 447, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, or 584 of SEQ ID NO:

11.

13. A recombinant AAV capsid protein according to claim 12, wherein the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 440 of SEQ ID NO: 11 in the parent capsid.

14. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 441 of SEQ ID NO: 11 in the parent capsid.

15. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 442 of SEQ ID NO: 11 in the parent capsid.

16. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 443 of SEQ ID NO: 11 in the parent capsid.

17. A recombinant AAV capsid protein according to claim 12, wherein the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 444 of SEQ ID NO: 11 in the parent capsid.

18. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 445 of SEQ ID NO: 11 in the parent capsid.

19. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 446 of SEQ ID NO: 11 in the parent capsid.

20. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 447 of SEQ ID NO: 11 in the parent capsid.

21. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 572 of SEQ ID NO: 11 in the parent capsid.

22. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 573 of SEQ ID NO: 11 in the parent capsid.

23. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 574 of SEQ ID NO: 11 in the parent capsid.

24. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 575 of SEQ ID NO: 11 in the parent capsid.

25. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 576 of SEQ ID NO: 11 in the parent capsid.

26. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 577 of SEQ ID NO: 11 in the parent capsid.

27. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 578 of SEQ ID NO: 11 in the parent capsid.

28. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 579 of SEQ ID NO: 11 in the parent capsid.

29. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 580 of SEQ ID NO: 11 in the parent capsid.

30. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 581 of SEQ ID NO: 11 in the parent capsid.

31. A recombinant AAV capsid protein according to claim 12, wherein the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 582 of SEQ ID NO: 11 in the parent capsid.

32. The recombinant AAV capsid protein of claim 12, wherein the peptide is positioned C-terminally proximal to the amino acid corresponding to amino acid 583 of SEQ ID NO: 11 in the parent capsid.

33. A recombinant AAV capsid protein according to claim 12, wherein the peptide is positioned C-terminally adjacent to the amino acid corresponding to amino acid 584 of SEQ ID NO: 11 in the parent capsid.

34. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); (b) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); (c) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); (d) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); (e) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); (f) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); (g) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); (h) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); (i) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); (j) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); (k) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); or (l) an amino acid sequence having at least 95% sequence identity to amino acids 194 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

35. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); (b) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); (c) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); (d) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); (e) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); (f) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); (g) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); (h) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); (i) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); (j) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); (k) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); or (l) an amino acid sequence having at least 99% sequence identity to amino acids 194 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

36. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 12; (b) an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 13; (c) an amino acid sequence having 100% sequence identity to amino acids 194 to 734 of SEQ ID NO: 14; (d) an amino acid sequence having 100% sequence identity to amino acids 194 to 734 of SEQ ID NO: 15; (e) an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 16; (f) an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 17; (g) an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 18; (h) an amino acid sequence having 100% sequence identity to amino acids 194 to 732 of SEQ ID NO: 19; (i) an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 20; (j) an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 21; (k) an amino acid sequence having 100% sequence identity to amino acids 194 to 737 of SEQ ID NO: 22; or (l) an amino acid sequence having 100% sequence identity with amino acids 194 to 737 of SEQ ID NO:

23.

37. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); (b) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); (c) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); (d) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); (e) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); (f) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); (g) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); (h) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); (i) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); (j) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); (k) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); or (l) an amino acid sequence having at least 95% sequence identity to amino acids 138 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

38. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); (b) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); (c) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); (d) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); (e) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); (f) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); (g) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); (h) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); (i) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); (j) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); (k) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); or (l) an amino acid sequence having at least 99% sequence identity to amino acids 138 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

39. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 12; (b) an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 13; (c) an amino acid sequence having 100% sequence identity to amino acids 138 to 734 of SEQ ID NO: 14; (d) an amino acid sequence having 100% sequence identity to amino acids 138 to 734 of SEQ ID NO: 15; (e) an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 16; (f) an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 17; (g) an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 18; (h) an amino acid sequence having 100% sequence identity to amino acids 138 to 732 of SEQ ID NO: 19; (i) an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 20; (j) an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 21; (k) an amino acid sequence having 100% sequence identity to amino acids 138 to 737 of SEQ ID NO: 22; or (l) an amino acid sequence having 100% sequence identity with amino acids 138 to 737 of SEQ ID NO:

23.

40. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); (b) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); (c) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); (d) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); (e) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); (f) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); (g) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); (h) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); (i) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); (j) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); (k) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); or (l) an amino acid sequence having at least 95% sequence identity to amino acids 1 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

41. The recombinant AAV capsid protein of claim 1, comprising: (a) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 12, wherein amino acids 445 to 456 are GGHKAKGPRKLG (SEQ ID NO: 1); (b) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 13, wherein amino acids 578 to 589 are GGHKAKGPRKLG (SEQ ID NO: 1); (c) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 734 of SEQ ID NO: 14, wherein amino acids 445 to 453 are GGHAIYPRH (SEQ ID NO: 2); (d) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 734 of SEQ ID NO: 15, wherein amino acids 578 to 586 are GGHAIYPRH (SEQ ID NO: 2); (e) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 16, wherein amino acids 445 to 451 are RTIGPSV (SEQ ID NO: 3); (f) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 17, wherein amino acids 578 to 584 are RTIGPSV (SEQ ID NO: 3); (g) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 18, wherein amino acids 445 to 451 are LSSRLDA (SEQ ID NO: 4); (h) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 732 of SEQ ID NO: 19, wherein amino acids 578 to 584 are LSSRLDA (SEQ ID NO: 4); (i) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 20, wherein amino acids 445 to 456 are THRPPMWSPVWP (SEQ ID NO: 5); (j) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 21, wherein amino acids 578 to 589 are THRPPMWSPVWP (SEQ ID NO: 5); (k) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 22, wherein amino acids 445 to 456 are FGQSSLPRDGPN (SEQ ID NO: 6); or (l) an amino acid sequence having at least 99% sequence identity to amino acids 1 to 737 of SEQ ID NO: 23, wherein amino acids 578 to 589 are FGQSSLPRDGPN (SEQ ID NO: 6).

42. The recombinant AAV capsid protein of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 23.

43. The recombinant AAV capsid protein of claim 1, wherein the amino acid sequence of the recombinant AAV capsid protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 23.

44. An isolated polynucleotide encoding the recombinant AAV capsid protein according to any one of claims 1 to 43.

45. A vector comprising the polynucleotide according to claim 44.

46. ​​The vector of claim 45, which is a plasmid or a viral vector.

47. The vector of claim 46, wherein the viral vector is a retroviral vector, a herpes virus vector, a baculovirus vector or an adenovirus vector.

48. The vector according to any one of claims 45 to 47, which is an expression vector.

49. A recombinant cell comprising the polynucleotide of claim 44 or the vector of any one of claims 45 to 47.

50. A method for producing a recombinant AAV capsid protein, the method comprising culturing the recombinant cell according to claim 49 under conditions where the polynucleotide is expressed and the capsid protein is produced.

51. A recombinant adeno-associated virus (rAAV), comprising: (a) a capsid comprising one or more of the recombinant AAV capsid proteins of any one of claims 1 to 43; and (b) rAAV genome.

52. The rAAV of claim 51, wherein the rAAV genome comprises a transgene.

53. The rAAV of claim 52, wherein the transgene encodes a polypeptide or a non-coding RNA.

54. The rAAV of claim 52 or 53, wherein the transgene encodes an antibody or fragment thereof.

55. The rAAV of claim 54, wherein the transgene encodes a scFv, a nanobody, or a VHH.

56. A method of delivering a transgene to a cell, the method comprising contacting the cell with the rAAV according to any one of claims 51 to 55 under conditions which transduce the cell and express the transgene.

57. The method of claim 56, wherein the cell is a myocyte, a microglia, an astrocyte, a neuron, a blood-brain barrier endothelial cell (BBB EC), a pericyte, an oligodendrocyte, an ependymal cell, or a cardiomyocyte.

58. The method of claim 57, wherein the neuron is a pyramidal neuron, a rosehip neuron, or a Purkinje cell.

59. A method of delivering a transgene to muscle tissue of a subject in need thereof, the method comprising administering to the subject the rAAV of any one of claims 51 to 55.

60. The method of claim 59, wherein the subject suffers from a disease associated with muscle tissue.

61. A method of treating a neuromuscular or neurodegenerative disease or condition in a subject in need thereof, the method comprising administering to the subject an effective amount of the rAAV according to any one of claims 51 to 55.

62. The method of claim 61, wherein the neuromuscular disease or disorder is selected from the group consisting of a myopathy, a hereditary cardiomyopathy, a metabolic myopathy, a distal myopathy, a muscular dystrophy, a congenital myopathy, a spinal muscular atrophy (SMA), a motor neuron disease, a congenital myopathy, a congenital muscular dystrophy, a motor neuron disease, a Duchenne muscular dystrophy, a Becker muscular dystrophy, a limb-girdle muscular dystrophy, a myotonic dystrophy, a myotubular myopathy, a centronuclear myopathy, a nemaline myopathy, a selenoprotein N-related myopathy, a Pompe disease, a glycogen storage disease type III, a spinal muscular atrophy, an amyotrophic lateral sclerosis (ALS), a Charcot-Marie-Tooth disease, multiple sclerosis, a myositis, a polymyositis, and a dermatomyositis.

63. The method of claim 61, wherein the neurodegenerative disease or disorder is selected from the group consisting of motor neuron disease (MND), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinocerebellar ataxia (SCA), and Friedreich's ataxia.

64. The method of any one of claims 59 to 63, wherein the rAAV is administered to the subject intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, or intradermally.

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