Tropism-modified recombinant viral particles and uses thereof for targeted introduction of genetic material into human cells

Abstract

To provide viral vector systems that maintain the integrity of a modified viral structure while remaining adaptable for targeted transfer of nucleic acids of interest to a variety of target cells.SOLUTION: Described herein is a viral retargeting strategy that solves the problems inherent in previous retargeting strategies by utilizing a first member and a second cognate member of a specific binding pair, where the first member and the second cognate member specifically interact to form a chemical, preferably covalent, bond. The first member, when displayed on a capsid protein, acts as a scaffold for any targeting ligand fused to the second cognate member, but upon binding of the first member and the second cognate member, an isopeptide bond forms and recombinant viral particles act as a one-component targeting vector.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing Reference Submitted as a text file via EFS Web The sequence listing set forth in file 10359WO01_ST25.txt is 183 kilobytes, was created on June 27, 2018, and is incorporated herein by reference.

[0002] The disclosure herein generally relates to tropism-modified recombinant viral particles and compositions comprising them that are useful for the targeted transfer of genetic material into cells. [Background technology]

[0003] The delivery of genes to specific target cells has become one of the most important technologies in modern medicine for the potential treatment of various chronic and genetic diseases. Currently, the lack of an ideal gene delivery vehicle has limited progress in the clinical application of gene therapy. To achieve therapeutic success, gene delivery vehicles must be able to transduce target cells while avoiding transduction of non-target cells. Specifically, when the natural tropism of a virus does not match the desired therapeutic target tissue or cell type, there is a need for recombinant viral particles in which the natural tropism has been deleted or attenuated, and the desired tropism has been successfully engineered. (Buchholz et al.)

[0004] In recent years, most progress in vector development has been achieved using non-enveloped viruses (e.g., viruses that contain a capsid formed by viral capsid proteins without an envelope (e.g., lipid bilayer)), such as adeno-associated virus (AAV) and adenovirus (Ad), as well as enveloped viruses (e.g., viruses whose capsid is surrounded by a lipid bilayer), such as retroviruses, lentiviruses, and herpes simplex viruses. Because AAV is only modestly immunogenic and can transduce a wide range of species and tissues in vivo without any evidence of toxicity, AAV-based vectors have been the focus of much research.

[0005] AAV is a small, non-enveloped, single-stranded DNA virus. The AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. The two ITRs are the only cis-receptors essential for AAV replication and encapsidation. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins primarily function to regulate transcription and replication of the AAV genome. The Cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). More than 80% of the total protein in AAV virions includes VP3, which is found in a relative abundance of approximately 1:1:10 in mature virions. In vivo, the three proteins spontaneously assemble into virion-like structures, e.g., viral capsids. Thus, viral encapsidation in infected cells appears to proceed independently of viral DNA synthesis (as verified by Kotin et al. (1994) Hum. Gene Ther. 5:793).

[0006] Among all known AAV serotypes, AAV2 is perhaps the best characterized serotype, since its infectious clone was first produced. (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA, 79:2077-2081) Subsequently, the complete sequences of several AAV serotypes have also been determined (see, for example, Rutledge et al. (1998) J. Virol., 72:309-319; Gao et al. (2005) Curr. Gen. Ther. 5(3)285-97; Chiorini et al. (1997) J. Virol., 71:6823-6833; S. Muramatsu et al., (1996) Virol., 221:208-217). In general, all AAVs share more than 80% nucleotide sequence identity.

[0007] AAV is a promising vector for human gene therapy because, unlike other viral vectors, AAV has not been shown to be associated with any known human diseases and is generally not considered pathogenic (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Furthermore, AAV safely transduces postmitotic tissues with relatively low immunogenicity, and although the virus may occasionally integrate into host chromosomes, it very frequently does so only at the safe harbor locus on human chromosome 19 when Rep proteins are supplied in trans. The AAV genome rapidly circularizes and concatenates in infected cells, exists in a stable episomal state within the infected cells, and provides long-term, stable expression of the payload.

[0008] Some viruses, including AAV, infect cells via a virus / ligand:cell / receptor interaction, ultimately resulting in endocytosis of the virus by the infected cell. This ligand:receptor interaction has been the focus of much of viral vector research, and can be manipulated, for example, to redirect the natural tropism of the virus from cells that are naturally permissive for infection by wild-type viruses to non-native target cells via receptors expressed by the target cells.

[0009] In theory, retargeting a vector toward any cell surface protein or marker should result in infection, since most cell surface receptors are involved in endocytic pathways, either constitutively (e.g., due to recycling) or ligand-induced (e.g., receptor-mediated). These receptors aggregate in clathrin-coated pits, enter cells via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and then recycle to the cell surface to either be sorted intracellularly or degraded in lysosomes. Thus, platforms for retargeting viral vectors often aim to eliminate the viral vector's natural tropism and redirect it toward a receptor or marker expressed solely or primarily by the target cell. Many advances in targeted gene therapy using viral vectors can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of the viral vector, resulting in pseudotyping, expansion, and / or retargeting of the viral vector's natural tropism. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[0010] The most common approach is recombinant genetic modification of viral capsid proteins, thus forming the surface of the viral capsid. Meanwhile, in indirect recombinant approaches, viral capsids are modified with heterologous "scaffolds" and then linked to adapters. The adapters bind to both the scaffolds and target cells. In direct recombinant targeting approaches, targeting ligands are directly inserted into or linked to viral capsids, i.e., the viral capsid protein is modified to express heterologous ligands. Then, the ligands redirect, for example, bind, to receptors or markers that are preferentially or only expressed on target cells.

[0011] Each approach has its advantages and disadvantages. The ability to genetically modify viruses requires maintaining the structure of the capsid and placing targeting ligands or scaffolds in locations within the capsid protein that can tolerate or properly present the targeting ligands or scaffolds. For example, the targeting ligands or scaffolds introduced into viral proteins must meet size constraints without interfering with the structure of the modified capsid, so that direct ligands or scaffolds may not be able to precisely limit the spectrum of natural molecules that can be used as targeting ligands or scaffolds. Furthermore, the use of targeting ligands inserted directly into viral capsids is not modular and must be re-engineered for every target. While scaffold platforms have the advantage of the flexibility and modular nature of the adapters used, the virus particle and the scaffold on the adapter interact ionically and remain two separate entities, and the inherent instability of their interaction may limit their practical use in vivo. With such two-component systems, optimal transduction efficiency may be difficult to achieve. Clearly, there remains a need for a viral vector system that maintains the structural integrity of modified viruses while remaining adaptable for targeted delivery of nucleic acids of interest to a variety of target cells. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Kotin et al. (1994) Hum. Gene Ther. 5:793 [Non-Patent Document 2] Samulski et al. (1982) Proc. Natl. Acad. Sci. USA, 79:2077-2081 [Non-Patent Document 3] Rutledge et al. (1998) J. Virol., 72:309-319 [[ID=十六]] [[ID=十七]]

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[0013] [[ID=四十八]] Described herein is a viral retargeting strategy that overcomes the problems inherent in previous retargeting strategies by utilizing a first and second cognate member of a specific binding pair, where the first and second cognate members interact to form a specific chemical bond, preferably a covalent bond. The first member, when displayed on the capsid protein, serves as a scaffold for any targeting ligand fused to the second cognate member, while upon binding of the first and second cognate members, the isopeptide bond forms, and the recombinant viral particle functions as a single-component targeting vector.

[0014] The present specification provides recombinant viral particles (e.g., recombinant viral capsid proteins, recombinant viral capsids containing recombinant viral capsid proteins, and / or recombinant viral vectors containing recombinant viral capsids encapsulating target nucleotides) that have been genetically modified to display a heterologous amino acid sequence comprising a first member of a specific binding pair, such as a peptide tag, wherein the amino acid sequence is less than 50 amino acids in length, and the recombinant viral capsid / particle protein reduces or eliminates its natural tropism. The tropism of the recombinant viral capsid protein / capsid / vector can be restored and / or reoriented upon the formation of an isopeptide bond with a second cognate member of the specific binding pair, and this second member is fused with a targeting ligand that specifically binds to target cells. Such binding results in the recombinant viral capsid protein / capsid / vector displaying the targeting ligand. When a targeting ligand is presented in limited amounts by a recombinant viral capsid / vector via a linker and / or on the surface of the viral capsid, surprisingly, transduction efficiency and specificity are enhanced. Provided herein are such viral particles, compositions comprising them, and methods for making and using them.

[0015] Thus, described herein is a recombinant viral capsid protein comprising a peptide tag operably linked (e.g., covalently linked) to the capsid protein, where the viral capsid protein is derived from a capsid gene of a virus that infects eukaryotic cells, and the peptide tag is a first member of a specific binding pair that forms an isopeptide bond with a second cognate member of the specific binding pair. In some embodiments, the recombinant capsid protein described herein (which may be derived from a capsid gene of a virus that infects eukaryotic cells, e.g., is a genetically engineered capsid protein of a virus that infects eukaryotic cells) comprises a first member of a specific binding pair (i.e., peptide tag) operably linked to the capsid protein and further comprises a second cognate member of the specific binding pair, where the first and second members of the specific binding pair are linked by a covalent (isopeptide) bond, e.g., the capsid protein comprises a first member operably linked to the capsid protein and further comprises a second cognate member of the specific binding pair covalently bonded to the first member. In some embodiments, the recombinant capsid proteins described herein (which may be derived from a capsid gene of a virus that infects eukaryotic cells, e.g., are genetically modified capsid proteins of a virus that infects eukaryotic cells) comprise a first member of a specific binding pair (i.e., a peptide tag) operably linked to the capsid protein, and further comprise a second cognate member of the specific binding pair fused to a targeting ligand, wherein the first and second members of the specific binding pair are linked by a covalent (isopeptide) bond, e.g., the capsid protein comprises a first member of a specific binding pair operably linked to the capsid protein, and further comprises a second cognate member of the specific binding pair covalently linked to the first member, wherein the second cognate member of the specific binding pair is operably linked to a targeting ligand that specifically binds to a cell surface marker (e.g., a cell surface oligosaccharide, a cell surface receptor, and / or a cell surface marker, etc.) on a target cell.Also described herein are viral capsids comprising recombinant viral capsid proteins, and viral vectors comprising a nucleotide of interest encapsulated by the viral capsids described herein. Also described herein are compositions comprising the recombinant viral particles (e.g., recombinant viral capsid proteins, recombinant viral capsids, and / or recombinant viral vectors), methods of using them for targeted delivery of a nucleotide of interest, and methods of making them.

[0016] In some embodiments, the peptide tag (first member of the specific binding pair) is operably linked to (translated in frame, chemically attached, and / or displayed by) the capsid protein via a first or second linker, e.g., an amino acid spacer that is at least one amino acid in length. In some embodiments, the peptide tag (first member) is flanked by first and / or second linkers, e.g., first and / or second amino acid spacers, each of which is at least one amino acid in length.

[0017] In some embodiments, the first and / or second linkers are not identical. In some embodiments, the first and / or second linkers are each independently one or two amino acids in length. In some embodiments, the first and / or second linkers are each independently one, two, or three amino acids in length. In some embodiments, the first and / or second linkers are each independently one, two, three, or four amino acids in length. In some embodiments, the first and / or second linkers are each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and / or second linkers are each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and / or second linkers are each independently one, two, three, four, or five amino acids in length. In some embodiments, the first and / or second linker is independently 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the first and / or second linker is independently 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length. In some embodiments, the first and / or second linker is independently 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In some embodiments, the first and second linker is independently 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length.In some embodiments, the first and second linkers are each independently 1 amino acid long, 2 amino acids long, 3 amino acids long, 4 amino acids long, 5 amino acids long, 6 amino acids long, 7 amino acids long, 8 amino acids long, or 9 amino acids long, or 10 amino acids long or more amino acids long.

[0018] In some embodiments, the first and second linkers are identical in sequence and / or length, each being one amino acid long. In some embodiments, the first and second linkers are identical in length, each being one amino acid long. In some embodiments, the first and second linkers are identical in length, each being two amino acids long. In some embodiments, the first and second linkers are identical in length, each being three amino acids long. In some embodiments, the first and second linkers are identical in length, each being four amino acids long, e.g., the linkers are GLSG (SEQ ID NO: 40). In some embodiments, the first and second linkers are identical in length, each being five amino acids long. In some embodiments, the first and second linkers are identical in length, each being six amino acids long, e.g., the first and second linkers each comprise the sequence GLSGSG (SEQ ID NO: 41). In some embodiments, the first and second linkers are identical in length, each being seven amino acids long. In some embodiments, the first and second linkers are identical in length, each 8 amino acids long, e.g., the first and second linkers each comprise the sequence GLSGLSGS (SEQ ID NO: 42). In some embodiments, the first and second linkers are identical in length, each 9 amino acids long. In some embodiments, the first and second linkers are identical in length, each 10 amino acids long, e.g., the first and second linkers each comprise the sequence GLSGLSGLSG (SEQ ID NO: 43) or GLSGGSGLSG (SEQ ID NO: 44). In some embodiments, the first and second linkers are identical in length, each greater than 10 amino acids long.

[0019] Generally, peptide tags and optionally one or more linkers described herein, e.g., peptide tags alone or in combination with one or more linkers, are about 5 amino acids to about 50 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are at least 5 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 6 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 7 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 8 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 9 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 10 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 11 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 12 amino acids in length. In some embodiments, peptide tags alone or in combination with one or more linkers are 13 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 14 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 15 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 16 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 17 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 18 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 19 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 20 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 21 amino acids in length.In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 22 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 23 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 24 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 25 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 26 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 27 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 28 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 29 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 30 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 31 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 32 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 33 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 34 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 35 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 36 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 37 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 38 amino acids in length. In some embodiments, the peptide tag, alone or in combination with one or more linkers, is 39 amino acids in length.In some embodiments, the peptide tag alone or in combination with one or more linkers is 40 amino acids long. In some embodiments, the peptide tag alone or in combination with one or more linkers is longer than 40 amino acids long. In some embodiments, the peptide tag alone or in combination with one or more linkers is 50 amino acids or less long.

[0020] Generally, the recombinant viral capsid proteins described herein can be derived from the capsid gene of a non-enveloped virus, e.g., a cap gene that has been modified to express a genetically engineered capsid protein of a non-enveloped virus that infects human cells or a serotype of a non-enveloped virus that commonly infects human cells, e.g., adenovirus, adeno-associated virus, etc. In some embodiments, the recombinant viral capsid proteins described herein are derived from AAV capsid genes encoding AAV VP1, VP2, and / or VP3 capsid proteins (or portions of VP1, VP2, and / or VP3 capsid proteins) and are encoded by cap genes that have been modified to encode genetically modified adeno-associated virus (AAV) VP1, VP2, and / or VP3 capsid proteins, e.g., genetically modified capsid proteins of an AAV serotype that infects humans, selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant viral capsid proteins are derived from AAV2 or AAV9 capsid genes encoding AAV2 VP1, VP2, and / or VP3 capsid proteins, or AAV9 VP1, VP2, and / or VP3 capsid proteins, respectively, for example, encoded by AAV2 or AAV9 cap genes modified to encode genetically modified AAV2 VP1, VP2, and / or VP3 capsid proteins, or genetically modified AAV9 VP1, VP2, and / or VP3 capsid proteins, respectively. In some embodiments, the recombinant viral capsid proteins are derived from AAV6 capsid genes, for example, encoded by AAV6 cap genes modified to encode genetically modified AAV6 VP1, VP2, and / or VP3 capsid proteins, the wild-type amino acid sequences of which are represented as SEQ ID NO:51.In some embodiments, the recombinant viral capsid proteins are derived from an AAV2 capsid gene, e.g., encoded by an AAV2 cap gene modified to encode genetically modified AAV2 VP1, VP2, and / or VP3 capsid proteins, the wild-type amino acid sequences of which are each represented as SEQ ID NO: 9. In some embodiments, the recombinant viral capsid proteins are derived from an AAV9 capsid gene, e.g., encoded by an AAV9 cap gene modified to encode genetically modified AAV9 VP1, VP2, and / or VP3 capsid proteins, the wild-type amino acid sequences of which are each represented as SEQ ID NO: 31.

[0021] In some embodiments, the recombinant viral capsid protein is derived from (encoded by) a chimeric AAV capsid gene, the chimeric capsid gene comprising multiple nucleic acid sequences, each of which encodes a portion of a capsid protein of a different AAV serotype, and the multiple nucleic acid sequences together encode the chimeric AAV capsid protein. In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV2 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV6 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV9 capsid gene.

[0022] Generally, the recombinant viral capsid proteins described herein are modified to include a peptide tag (e.g., a first member of a protein:protein binding pair) operably linked to (e.g., inserted into and / or displayed by) the recombinant capsid protein, optionally via a linker, such that the peptide tag (first member of the protein:protein binding pair) and optional linker themselves reduce and / or abolish the native tropism of the recombinant capsid protein or capsid comprising it, compared to a reference capsid protein or capsid comprising the reference capsid protein lacking the peptide tag (first member of the protein:protein binding pair) and optional linker, respectively. In some embodiments, the peptide tag (first member of the protein:protein binding pair) is operably linked to (e.g., inserted into and / or displayed by), optionally via a linker, a region of the capsid protein responsible for the native tropism of the wild-type reference capsid protein, e.g., a region of the capsid protein responsible for cellular targeting. In some embodiments, the peptide tag (first member of a protein:protein binding pair) and optional linker are operably linked to (e.g., inserted into and / or displayed by) the knob domain of an Ad fiber protein, optionally via a linker. In some embodiments, the peptide tag (first member of a protein:protein binding pair) is operably linked to (e.g., inserted into and / or displayed by) the HI loop of an Ad fiber protein, optionally via a linker. In some embodiments, the peptide tag (first member of a protein:protein binding pair) is operably linked to (e.g., inserted into and / or displayed by) an exposed variable loop in an AAV capsid protein, optionally via a linker.In some embodiments, a peptide tag (first member of a protein:protein binding pair) is operably linked to (e.g., inserted into and / or displayed by) an exposed variable loop of an AAV2 capsid protein, optionally via a linker. In some embodiments, a peptide tag (first member of a protein:protein binding pair) is operably linked to (e.g., inserted into and / or displayed by) an exposed variable loop of an AAV9 capsid protein, optionally via a linker.

[0023] In some embodiments, (i) the viral capsid protein is derived from an AAV2 capsid gene encoding AAV2 VP1, VP2, and / or VP3 capsid proteins, and the peptide tag is operably linked, optionally via a linker, to (e.g., inserted into and / or displayed by) amino acid at position 1453 or 1587 of the AAV2 VP1 capsid protein (or the corresponding position of a VP2 and / or VP3 capsid protein encoded by the same capsid gene, or the corresponding amino acid at the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9); or (ii) the viral capsid protein is derived from an AAV6 capsid gene, and the peptide tag (first member of a protein:protein binding pair) is operably linked, optionally via a linker, to (e.g., inserted into and / or displayed by) the AAV6 capsid gene encoding AAV6 or (iii) the viral capsid protein is derived from an AAV9 capsid gene encoding AAV9 VP1, VP2, and / or VP3 capsid proteins, and the peptide tag is operably linked to (e.g., inserted into and / or displayed by) an amino acid at position 1585 of the VP1 capsid protein (or the corresponding position of a VP2 and / or VP3 capsid protein encoded by the same capsid gene, or the corresponding amino acid of a VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9), optionally via a linker, at position 1453 or 1589 of the AAV9 capsid protein. It is operably linked to (e.g., inserted into and / or displayed by) amino acids of the VP1 capsid (or corresponding positions of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or corresponding amino acids of the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8).

[0024] In some embodiments, the peptide tag (first member of the protein:protein binding pair) is an amino acid selected from the group consisting of: 453 of AAV2 capsid protein VP1, 587 of AAV2 capsid protein VP1, 585 of AAV6 capsid protein VP1, 453 of AAV9 capsid protein VP1, 589 of AAV9 capsid protein VP1 (or corresponding positions in VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or VP1, VP2, and / or VP3 capsids of different AAVs that infect humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). The AAV1 capsid protein is operably linked to a corresponding amino acid of the AAV2 capsid protein VP1, for example, fused to the C-terminus of an amino acid selected from the group consisting of 453 of the AAV2 capsid protein VP1, 587 of the AAV2 capsid protein VP1, 585 of the AAV6 capsid protein VP1, 453 of the AAV9 capsid protein VP1, and 589 of the AAV9 capsid protein VP1 (or the corresponding positions of the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids of the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). In some embodiments, the peptide tag (first member of the protein:protein binding pair) and optional linker are inserted immediately after (e.g., fused to the C-terminus of) amino acid position 453 of the AAV2 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).In some embodiments, the peptide tag (first member of the protein:protein binding pair) and optional linker are inserted immediately after (e.g., fused to the C-terminus of) amino acid position 587 of the AAV2 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). In some embodiments, the peptide tag (first member of the protein:protein binding pair) and optional linker are inserted immediately after (e.g., fused to the C-terminus of) amino acid position 585 of the AAV6 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9). In some embodiments, the peptide tag (first member of the protein:protein binding pair) and optional linker are inserted immediately after (e.g., fused to the C-terminus of) amino acid position 453 of the AAV9 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8).In some embodiments, the peptide tag (first member of the protein:protein binding pair) and optional linker are inserted immediately after (e.g., fused to the C-terminus of) amino acid position 589 of the AAV9 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8). In some embodiments, the peptide tag (first member of a protein:protein binding pair) and optional linker are inserted and / or displayed between positions 587 and 588 of the AAV2 VP1 capsid protein (or at corresponding positions in the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or at corresponding amino acids in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8).

[0025] In some embodiments, the recombinant capsid proteins described herein comprise a (second, different) mutation, which may be in addition to the peptide tag (first member of a protein:protein binding pair) and optional linker. In some embodiments, the (second, different) mutation comprises an insertion of a heterologous peptide into the capsid protein, a substitution of one or more amino acids of the capsid protein with one or more heterologous amino acids, a deletion of one or more amino acids of the capsid protein, or a combination thereof. For example, in some embodiments, the recombinant viral capsid proteins described herein are derived from an AAV2 capsid gene (e.g., are genetically modified AAV2 VP1, VP2, and / or VP3 capsid proteins), include a peptide tag (the first member of a protein:protein binding pair) and an optional linker, and may further include a mutation, e.g., an R585A and / or an R588A mutation in the AAV2 VP1 capsid protein (or a corresponding mutation in the VP2 and / or VP3 capsid proteins encoded from the same AAV2 capsid gene). In some embodiments, the recombinant viral capsid proteins are derived from an AAV2 capsid gene (e.g., are genetically modified AAV2 VP1, VP2, and / or VP3 capsid proteins), include a peptide tag (the first member of a protein:protein binding pair) and an optional linker, and may further include a mutation, e.g., an R585A and / or an R588A mutation in the AAV2 VP1 capsid protein (or a corresponding mutation in the VP2 and / or VP3 capsid proteins encoded from the same AAV2 capsid gene). It comprises a peptide tag (a first member of a protein:protein binding pair) and an optional linker inserted immediately after (e.g., fused to the C-terminus of) amino acid 453 of the VP1 protein (or the amino acid at the corresponding position in the AAV2 VP2 and / or VP3 capsid protein encoded by the AAV2 capsid gene), and further comprises a mutation selected from the group consisting of R585A and / or R588A (or the corresponding mutation in the VP2 and / or VP3 capsid protein encoded by the same AAV2 capsid gene). In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, e.g., a genetically modified AAV2 VP1, VP2, and / or VP3 capsid protein, and comprises a peptide tag (a first member of a protein:protein binding pair) and optional linker inserted immediately after (e.g., fused to the C-terminus of) amino acid 587 of the AAV2 VP1 capsid protein (or the amino acid at the corresponding position in the AAV2 VP2 and / or VP3 capsid protein encoded by the same AAV2 capsid gene), and further comprises a mutation selected from the group consisting of R585A, R588A, and / or the corresponding mutation in the VP2 and / or VP3 capsid protein encoded by the same AAV2 capsid gene.

[0026] In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, e.g., a genetically modified AAV9 VP1, VP2, and / or VP3 capsid protein, that includes a peptide tag (a first member of a protein:protein binding pair) and optional linker inserted immediately after (e.g., fused to the C-terminus of) amino acid 453 of the AAV9 VP1 protein (or the amino acid at the corresponding position in the AAV9 VP2 or VP3 capsid protein encoded from the AAV9 capsid gene), and further includes a W503A mutation (or the corresponding mutation in the VP2 and / or VP3 capsid protein encoded from the same AAV2 capsid gene). In some embodiments, the recombinant viral capsid protein is derived from an AAV9 capsid gene, e.g., a genetically modified AAV9 VP1, VP2, and / or VP3 capsid protein, that includes a peptide tag (a first member of a protein:protein binding pair) and optional linker inserted immediately after (e.g., fused to the C-terminus of) amino acid 589 of the AAV9 VP1 protein (or the amino acid at the corresponding position in the AAV9 VP2 and / or VP3 capsid protein encoded by the same AAV9 capsid gene), and further includes a W503A mutation (or the corresponding mutation in the VP2 and / or VP3 capsid protein encoded by the same AAV2 capsid gene).

[0027] In some embodiments, the protein:protein binding pair may be selected from the group consisting of SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, Isopeptag:Pyrin-C, and SnoopTag:SnoopCatcher. In some embodiments, the peptide tag (first member) is SpyTag (or a biologically active portion thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion thereof). In some embodiments, the peptide tag (first member) is SpyTag (or a biologically active portion thereof) and the protein (second cognate member) is KTag (or a biologically active portion thereof). In some embodiments, the peptide tag (first member) is KTag (or a biologically active portion thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion thereof). In some embodiments, the peptide tag (first member) is SnoopTag (or a biologically active portion thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion thereof). In some embodiments, the peptide tag (first member) is IsopepTag (or a biologically active portion thereof) and the protein (second cognate member) is Pirin-C (or a biologically active portion thereof). In some embodiments, the peptide tag (first member) is SpyTag002 (or a biologically active portion thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion thereof).

[0028] In some embodiments, the recombinant viral capsid protein comprises a SpyTag. In some embodiments, the recombinant viral capsid, the recombinant viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid or viral vector comprises an amino acid sequence represented by any of the SEQ ID NOs listed in Table 1 as the amino acid sequence of the recombinant viral capsid protein. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented by SEQ ID NO: 13. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented by SEQ ID NO: 15. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented by SEQ ID NO: 17. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 19. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 21. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 23. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 25. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 27.In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 29. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 35. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 37. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO: 39.

[0029] In some embodiments, the recombinant viral capsid proteins described herein comprise a first member of a specific binding pair (e.g., a peptide tag) covalently linked to a second cognate protein member of the specific binding pair. In some embodiments, the recombinant viral capsid proteins described herein comprise a peptide tag (first member) covalently linked to an adapter polypeptide comprising a cognate protein (second member) operably linked to a targeting ligand. In some embodiments, the targeting ligand is operably linked to, e.g., fused to, the protein (second member), optionally via a linker. Generally, the targeting ligand can be a binding moiety, e.g., a natural ligand, an antibody, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or portion thereof. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a cell surface protein on a target cell and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a cell surface protein on a target cell and an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a cell surface protein on a target cell and an IgG heavy chain constant domain, the IgG heavy chain constant domain being operably linked, e.g., via a linker, to a protein (e.g., a second member of a protein:protein binding pair) that forms an isopeptide covalent bond with the peptide tag. In some embodiments, the recombinant capsid protein described herein comprises a SpyTag operably linked to a viral capsid protein and covalently linked to the SpyTag, an adaptor polypeptide comprising SpyCatcher linked to a targeting ligand comprising an antibody variable domain, and an IgG heavy chain domain, the SpyCatcher and the IgG heavy chain domain being linked via an amino acid linker, e.g., GSGESG (SEQ ID NO: 48).In some embodiments, the adapter polypeptide comprises the sequence represented as SEQ ID NO: 46, which comprises a portion of a human IgG4 heavy chain, the IgG4 portion having the sequence represented as SEQ ID NO: 49 linked to SpyCatcher (SEQ ID NO: 3) via a linker (SEQ ID NO: 48).

[0030] In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a SpyTag covalently linked to a SpyCatcher fused to a targeting ligand. In some embodiments, the recombinant viral capsid, the recombinant viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises an amino acid sequence represented by any SEQ ID NO: listed in Table 1 encoding a recombinant viral capsid protein and a polypeptide adapter comprising the amino acid sequence represented by SEQ ID NO: 46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises an amino acid sequence represented by SEQ ID NO: 13 and a polypeptide adapter comprising the amino acid sequence represented by SEQ ID NO: 46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises an amino acid sequence represented by SEQ ID NO: 15 and a polypeptide adapter comprising the amino acid sequence represented by SEQ ID NO: 46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO: 17 and the amino acid sequence represented as SEQ ID NO: 46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO: 19 and the amino acid sequence represented as SEQ ID NO: 46. In some embodiments, the recombinant viral capsid comprising the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO: 21 and the amino acid sequence represented as SEQ ID NO: 46.In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO:23 and the amino acid sequence represented as SEQ ID NO:46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO:25 and the amino acid sequence represented as SEQ ID NO:46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO:27 and the amino acid sequence represented as SEQ ID NO:46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO:29 and the amino acid sequence represented as SEQ ID NO:46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO: 35, and the amino acid sequence represented as SEQ ID NO: 46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO: 37, and the amino acid sequence represented as SEQ ID NO: 46. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises a polypeptide adapter comprising the amino acid sequence represented as SEQ ID NO: 39, and the amino acid sequence represented as SEQ ID NO: 46.

[0031] Generally, the targeting ligand specifically binds to a cell surface molecule, e.g., an oligosaccharide, receptor, cell surface marker, etc., expressed on the surface of a mammalian (e.g., human) eukaryotic cell, e.g., a target cell. In some embodiments, the targeting ligand binds to a (human) liver cell, a (human) brain cell, a (human) T cell, a (human) kidney cell, a (human) intestinal cell, a (human) lung cell, a (human) cancer cell, or a (human) cell infected with a heterologous pathogen.

[0032] In some embodiments, the targeting ligand binds to a receptor expressed by (human) hepatocytes, e.g., an asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the targeting ligand binds to a molecule expressed by (human) neuronal cells, e.g., GABA, transferrin, etc. In some embodiments, the targeting ligand binds to a molecule expressed by (human) T cells, e.g., CD3, e.g., CD3ε. In some embodiments, the targeting ligand binds to CD63. In some embodiments, the targeting ligand binds to a molecule expressed by (human) hematopoietic stem cells, e.g., CD34. In some embodiments, the targeting ligand binds to a molecule expressed by (human) kidney cells. In some embodiments, the targeting ligand binds to a molecule expressed by (human) muscle cells, e.g., integrins. In some embodiments, the targeting ligand is a molecule expressed by (human) cancer cells, e.g., a tumor-associated antigen, e.g., adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin- A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100 / Pme117, GPNMB, HAUS3, hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13R alpha 2, intestinal carboxylesterase,K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, Malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS- 9, P polypeptide, p53, PAP, PAX5, PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, cecernin1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigens (in lymphomas and other solid tumors), ErbB receptors, Melan A [MART1], gp 100, tyrosinase, TRP-1 / gp 75, and TRP-2 (in melanoma), MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma), HPV EG and E7 proteins (in cervical cancer), mucin [MUC-1] (in breast, pancreatic, colon, and prostate cancer),The targeting ligand binds to common tumor-specific antigens such as prostate-specific antigen [PSA] (in prostate cancer), carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1, CAGE-2, CAGE-8, CAGE-3 TO 7, LAGE-1, NY-ESO-1 / LAGE-2, NA-88, GnTV, and TRP2-INT2. In some embodiments, the targeting ligand binds to E6 and / or E7. In some embodiments, the targeting ligand binds to Her2. In some embodiments, the targeting ligand binds to CD63. In some embodiments, the targeting ligand binds to human glucagon receptor (hGCGR). In some embodiments, the targeting ligand binds to human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

[0033] Generally, viral capsids comprising the recombinant viral capsid proteins described herein are unable to infect target cells in the absence of a targeting ligand, e.g., a second member operably linked to the targeting ligand. Generally, in the absence of an appropriate targeting ligand, viral capsids comprising the recombinant viral capsid proteins described herein have reduced or abolished natural tropism, e.g., reduced or abolished ability to target or bind to naturally permissive reference cells for transduction, compared to the transduction of a reference viral capsid, e.g., a capsid comprising a reference viral capsid protein, e.g., a wild-type control viral capsid protein, or a viral capsid protein identical to the reference viral capsid protein but lacking either or both of the targeting ligand and, optionally, the protein:protein binding pair. In some embodiments, the transduction efficiency of a recombinant viral capsid protein comprising a SpyTag is reduced or abolished compared to the control wild-type viral capsid protein.

[0034] In some embodiments, in the absence of a suitable targeting ligand, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibit a decrease in transduction efficiency of at least 10% compared to a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibit a decrease in transduction efficiency of at least 20% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibit a decrease in transduction efficiency of at least 30% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits a decrease in transduction efficiency of at least 40% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits a decrease in transduction efficiency of at least 50% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits a decrease in transduction efficiency of at least 60% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of an appropriate targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits at least a 70% reduction in transduction efficiency compared to a control wild-type viral capsid, e.g., listed in Table 1.In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits a reduction in transduction efficiency of at least 75% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits a reduction in transduction efficiency of at least 80% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits a reduction in transduction efficiency of at least 85% compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits at least a 90% decrease in transduction efficiency compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits at least a 95% decrease in transduction efficiency compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of a suitable targeting ligand, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, exhibits at least a 99% decrease in transduction efficiency compared to a control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, in the absence of an appropriate targeting ligand, transduction of regulatory cells by viral capsids comprising the recombinant viral capsid proteins described herein is abrogated, e.g., undetectable, e.g., via a method that measures expression of a nucleotide of interest, such as, for example, a reporter assay.

[0035] Conversely, viral capsids comprising a recombinant viral capsid protein comprising a peptide tag covalently linked to a suitable adapter polypeptide, e.g., a cognate protein covalently linked to a targeting ligand, are capable of infecting target cells and have a partially or fully restored ability to target and bind to reference cells naturally permissive for transduction, e.g., compared to transduction of a reference viral capsid, e.g., a capsid comprising a reference viral capsid protein, e.g., a wild-type control capsid protein. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide, exhibit a transduction efficiency that is at least 10% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide, exhibit a transduction efficiency that is at least 20% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 30% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 40% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 50% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1.In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 60% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 70% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 75% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 80% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 85% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 90% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1.In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 95% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is at least 99% of the transduction efficiency of a suitable control wild-type viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is the same as the transduction efficiency of a suitable control wild-type viral capsid covalently linked to, e.g., Table 1.

[0036] Similarly, a recombinant viral capsid protein comprising a peptide tag covalently linked to a suitable adapter polypeptide, e.g., a viral capsid comprising a cognate protein operably linked to a targeting ligand, can, e.g., infect target cells and have an enhanced ability to target and bind to a reference cell that is naturally permissive for transduction, compared to transduction of a reference viral capsid that is identical to the recombinant viral capsid protein except for, e.g., comprising, the reference capsid protein, e.g., lacking one or both of the protein:protein binding pairs. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide, exhibits a transduction efficiency that is 10% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibits a transduction efficiency that is 20% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibits a transduction efficiency that is 30% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibits a transduction efficiency that is 40% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide, exhibits a transduction efficiency that is 50% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1.In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibits a transduction efficiency that is 60% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibits a transduction efficiency that is 70% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibits a transduction efficiency that is 75% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is 80% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is 85% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, viral capsids comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1. In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide, exhibits a transduction efficiency that is greater than 95% than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1.In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein, e.g., listed in Table 1, covalently linked to a suitable adapter polypeptide, exhibits a transduction efficiency that is greater than 99% than the transduction efficiency of a suitable control reference viral capsid, e.g., listed in Table 1.

[0037] In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein is a mosaic capsid, e.g., comprising at least two sets of VP1, VP2, and / or VP3 proteins, each set encoded by a different cap gene, comprising a recombinant viral capsid protein comprising, e.g., a peptide tag and a specific ratio of a reference capsid protein that does not contain a peptide tag. In some embodiments, the reference capsid protein is a wild-type reference capsid protein in that it comprises the amino acid sequence of a wild-type capsid protein having the same serotype as the recombinant viral capsid protein. In some embodiments, the reference capsid protein is a control reference capsid protein in that it comprises the amino acid sequence of a recombinant viral capsid protein, except that the control reference capsid protein lacks the peptide tag. In some embodiments, the reference capsid protein is a mutant wild-type reference protein in that it has the same serotype as the recombinant viral capsid protein, but contains substantially the same amino acid sequence as the wild-type capsid protein, accounting for amino acids in the wild-type capsid protein for mutations (e.g., amino acid sequence deletions, amino acid sequence insertions, chimerization, etc.) that reduce the tropism of the wild-type capsid protein. In some embodiments, the compositions described herein include the recombinant viral capsid protein and the reference capsid protein in a ratio ranging from 1:1 to 1:15, or the methods described herein combine the recombinant viral capsid protein and the reference capsid protein in a ratio ranging from 1:1 to 1:15. In some embodiments, the ratio is 1:2. In some embodiments, the ratio is 1:3. In some embodiments, the ratio is 1:4. In some embodiments, the ratio is 1:5. In some embodiments, the ratio is 1:6. In some embodiments, the ratio is 1:7. In some embodiments, the ratio is 1:8. In some embodiments, the ratio is 1:9. In some embodiments, the ratio is 1:10. In some embodiments, the ratio is 1:11. In some embodiments, the ratio is 1:12.In some embodiments, the ratio is 1:13. In some embodiments, the ratio is 1:14. In some embodiments, the ratio is 1:15.

[0038] In some embodiments, the compositions described herein comprise a recombinant viral capsid protein listed in Table 1 and its appropriate reference capsid protein (or a combination of reference capsid proteins) also listed in Table 1 in a ratio ranging from 1:1 to 1:15 (recombinant capsid protein:reference capsid protein(s)), or the methods described herein combine a recombinant viral capsid protein listed in Table 1 and its appropriate reference capsid protein (or a combination of reference capsid proteins) also listed in Table 1 in a ratio ranging from 1:1 to 1:15 (recombinant capsid protein:reference capsid protein(s)). In some embodiments, the ratio is 1:2. In some embodiments, the ratio is 1:3. In some embodiments, the ratio is 1:4. In some embodiments, the ratio is 1:5. In some embodiments, the ratio is 1:6. In some embodiments, the ratio is 1:7. In some embodiments, the ratio is 1:8. In some embodiments, the ratio is 1:9. In some embodiments, the ratio is 1:10. In some embodiments, the ratio is 1:11. In some embodiments, the ratio is 1:12. In some embodiments, the ratio is 1:13. In some embodiments, the ratio is 1:14. In some embodiments, the ratio is 1:15.

[0039] Table 1 provides sequence identification numbers (SEQ ID NOs) expressed as amino acid sequences of (1) exemplary, non-limiting recombinant viral capsid proteins comprising the peptide tags described herein, (2) exemplary, non-limiting examples of corresponding control (C) wild-type viral capsid proteins that can optionally be used as references to determine the reduction or abolition of transduction efficiency of recombinant capsid proteins comprising shared protein tags in the absence of a targeting vector, and (3) exemplary, non-limiting examples of corresponding reference viral capsid proteins for generating mosaic capsids and / or use as references to determine the restoration of transduction efficiency of recombinant capsid proteins comprising protein:protein binding groups and targeting ligands. [Table 1-1] [Table 1-2]

[0040] Generally, the recombinant viral vectors described herein comprise a viral capsid comprising a recombinant viral capsid protein described herein, including a mosaic viral capsid, wherein the viral capsid encloses a nucleotide of interest. In some embodiments, the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide of interest is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CAGG promoter. In some embodiments, the promoter is a ubiquitin C (UbC) promoter.

[0041] Generally, the nucleotides of interest can be one or more genes, which can encode a detectable marker, e.g., a reporter, or a therapeutic polypeptide. In some embodiments, the nucleotides of interest are reporter genes. In some embodiments, the nucleotides of interest are reporter genes encoding a detectable marker selected from the group consisting of green fluorescent protein, luciferase, β-galactosidase, and the like. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the nucleotides of interest are selected from the group consisting of suicide genes, nucleotides encoding antibodies or fragments thereof, nucleotides encoding CRISPR / Cas systems or portion(s) thereof, nucleotides encoding antisense RNAs, nucleotides encoding siRNAs, secreted enzymes, genes encoding therapeutic proteins, and the like. In one embodiment, the nucleotides of interest encode proteins, e.g., comprising at least two domains that provide two distinct functions.

[0042] The compositions described herein generally include a viral vector comprising a recombinant viral capsid protein described herein, e.g., a capsid comprising a recombinant viral capsid protein (e.g., including a mosaic capsid), where the capsid encapsulates a nucleotide of interest. In some embodiments, the compositions described herein include (1) a viral vector having a capsid comprising a recombinant viral capsid protein described herein, and (2) a pharmaceutically acceptable carrier.

[0043] Also described herein are methods for making and using recombinant viral capsid proteins, viral vectors comprising the same, compositions, etc. In some embodiments, methods including redirecting viruses, e.g., adenoviruses, adeno-associated viruses, etc., delivering diagnostic / therapeutic cargo to target cells, etc., include contacting a target cell (which may be in vitro or in vivo, e.g., in a human) with a recombinant viral vector comprising a recombinant viral capsid protein described herein, wherein the viral capsid or viral vector comprises a targeting ligand that specifically binds to a protein expressed on the surface of the target cell. Such methods may include, as a first step, producing a recombinant viral vector, e.g., culturing packaging cells under conditions sufficient for the production of the viral vector, wherein the packaging cells comprise a plasmid encoding a capsid protein comprising a peptide tag (first member) in the absence or presence of a plasmid encoding a reference capsid protein, incubating the recombinant capsid protein with a second cognate member operably linked to a targeting ligand, etc. In some embodiments, the target cell is a (human) hepatocyte, and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to an asialoglycoprotein receptor, e.g., (h)ASGR1. In some embodiments, the target cell is a (human) neuronal cell, and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to GABA, transferrin receptor, etc. In some embodiments, the target cell is a (human) T cell, and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to CD3, e.g., CD3ε. In some embodiments, the target cell is a (human) hematopoietic stem cell, and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to CD34. In some embodiments, the target cell is a (human) kidney cell. In some embodiments, the target cell is a (human) muscle cell, and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to an integrin.In some embodiments, the target cell is a (human) cancer cell and the (mosaic) recombinant viral vector comprises a targeting ligand that specifically binds to a tumor-associated antigen, e.g., E6 and E7, Her2, etc. In some embodiments, the targeting ligand binds to the human glucagon receptor (hGCGR).

[0044] Also described herein are methods for inactivating viral capsids and / or producing viral vectors, generally comprising: (a) inserting a nucleic acid encoding a heterologous protein into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein containing a peptide tag (in the absence or presence of a plasmid encoding a reference capsid protein); and / or (b) culturing packaging cells under conditions sufficient for producing a viral vector, wherein the packaging cells contain the nucleotide sequence. In some embodiments, the packaging cells further contain a helper plasmid and / or a transfer plasmid containing the nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementary adeno-associated viral vector from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral vector from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the supernatant containing the viral vector with a nuclease, e.g., DNase I, in the presence of MgCl2, (c) concentrating the viral vector, (d) purifying the viral vector, and (e) any combination of (a)-(d). Also provided herein are viral vectors made according to the methods described herein, and packaging cells useful for producing the viral vectors described herein, e.g., packaging cells containing plasmids encoding recombinant capsid proteins.

[0045] The patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0046] [Figure 1] Scatter plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by either "uninfected" HER2-positive (+) 293 hErbB2 or HER2-negative (-) 293 parental cells, or cells infected with either "AAV2 N587-SpyTag" particles or "AAV2 N587-SpyTag + C6.5-SpyC" particles. The capsids of both viruses contain the following mutations: R585A, delR588, and insertion of the SpyTag peptide (SEQ ID NO: 1) immediately after residue N587 (SEQ ID NO: 13). C6.5-SpyC particles were conjugated with an anti-HER2 scFv (C6.5) fused to SpyCatcher (SEQ ID NO: 3) via SpyTag. The viruses express GFP as a marker of transformation. [Figure 2] Scatter plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by either "uninfected" HER2-positive (+) 293 hErbB2 or HER2-negative (-) 293 parental cells, or cells infected with either "AAV2 N587-SpyTag" particles or "AAV2 N587-SpyTag + SpyC-anti-HER2" particles. The capsids of both viruses contain the following mutations: R585A, delR588, and insertion of the SpyTag peptide (SEQ ID NO: 1) immediately after residue N587 (SEQ ID NO: 13). SpyC-anti-HER2 particles were conjugated with an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) via SpyTag. The virus expresses GFP as a marker of transformation. [Figure 3]Scatter plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by HER2-positive (+) 293 hErbB2 or HER2-negative (-) 293 parental cells infected with "AAV2 wild-type" particles or cells infected with "AAV2 G453-SpyTag+SpyC-anti-HER2" particles. "AAV2 wild-type" capsids do not have the mutation or modification (SEQ ID NO: 9), and "AAV2 G453-SpyTag" capsids are mosaic viral particles composed of a 1:7 ratio between the "SpyTag" capsid protein inserted immediately after residue G453, adjacent to a 10-amino acid linker (SEQ ID NO: 29), and "AAV2 HBM" capsids without SpyTag but containing the mutations R585A and R588A (SEQ ID NO: 11). AAV2 G453-SpyTag mosaic particles were conjugated with an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) via SpyTag. The virus expresses GFP as a marker of transformation. [Figure 4A] Western blots are provided using the B1 antibody, which recognizes a linear epitope shared by the AAV2 VP1, VP2, and VP3 capsid proteins, to analyze the reaction between anti-HER2 scFv fused to SpyCatcher "SpyC-anti-Her2 scFv" and a panel of AAV2 particles composed of capsids with the following mutations: R585A, delR588, and insertion of the SpyTag peptide immediately after residue N587 adjacent to amino acid linkers of various lengths (Linker 1, Linker 2, Linker 4, Linker 6, Linker 8, and Linker 10) (SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25). [Figure 4B]The percentage of HER2+ cells expressing GFP (gray, y-axis) versus the percentage of HER2− cells expressing GFP (black, y-axis) is shown 5 days after infection with AAV2 viral particles composed of capsids with the following mutations: R585A, delR588, and N587-SpyTag flanked by amino acid linkers of the indicated lengths (Linker 1, Linker 2, Linker 4, Linker 6, Linker 8, and Linker 10) (SEQ ID NOs: 13, 15, 17, 19, 21, 23, and 25, respectively) (x-axis). AAV2 N587-SpyTag particles were conjugated with an anti-HER2 scFv fused to SpyCatcher (SEQ ID NO: 3). [Figure 5A] Western blots are provided analyzing the reaction between an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher "SpyC-anti-Her2 antibody" using the B1 antibody, which recognizes a linear epitope shared by the AAV2 VP1, VP2, and VP3 capsid proteins, and a panel of AAV2 viral particles composed of capsids with the following mutations: R585A, delR588, and insertion of the SpyTag peptide immediately after residue N587 adjacent to amino acid linkers of various lengths (no linker, linker 1, linker 2, linker 4, linker 6, linker 8, and linker 10) (SEQ ID NOs: 13, 15, 17, 19, 21, 23, and 25, respectively). [Figure 5B] The percentage of HER2+ cells expressing GFP (gray, y-axis) versus the percentage of HER2− cells expressing GFP (black, y-axis) is shown 5 days after infection with wild-type (wt) AAV2 particles or AAV2 viral particles composed of capsids with the following mutations: R585A, delR588, and N587-SpyTag flanked by amino acid linkers of the indicated lengths (no linker, linker 1, linker 2, linker 4, linker 6, linker 8, and linker 10) (SEQ ID NOs: 13, 15, 17, 19, 21, 23, and 25, respectively) (x-axis). AAV2 N587 SpyTag particles were conjugated with an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3). [Figure 6A] Using the B1 antibody, which recognizes a linear epitope shared by the AAV2 VP1, VP2, and VP3 capsid proteins, we provide a Western blot analyzing the reaction between anti-HER2 scFv C6.5 fused to "SpyC-anti-HER2 scFv" of SpyCatcher (SEQ ID NO: 3) and a panel of mosaic AAV2 viral particles, where the AAV2 viral particles are composed of a mixture of "SpyTag" capsid protein containing the mutations R585A, delR588, and N587-linker10-SpyTag (SEQ ID NO: 25) and "HBM" capsid protein containing the mutations R585A and R588A but without SpyTag (SEQ ID NO: 11). The "SpyTag" and "HBM" capsid proteins were mixed in various ratios (1:0, 1:1, and 1:3). [Figure 6B] Figure 1 shows the percentage of HER2+ 293 hErbB2 cells (gray bars) and HER2- 293 parental cells (black bars) expressing GFP (y-axis) 5 days after infection with mosaic AAV2 viral particles (x-axis) composed of a mixture of "Linker10" capsid protein containing mutations R585A, delR588, and N587-Linker10-SpyTag (SEQ ID NO: 25) and "HBM" capsid protein containing mutations R585A and R588A but without SpyTag (SEQ ID NO: 11). "Linker10" and "HBM" capsid proteins were mixed in various ratios (1:0, 3:1, 1:1, and 1:3) and conjugated to an anti-HER2 scFv fused to SpyCatcher (SEQ ID NO: 3). [Figure 7A]Using the B1 antibody, which recognizes a linear epitope shared by the AAV2 VP1, VP2, and VP3 capsid proteins, a Western blot is provided analyzing the reaction between an anti-HER2 antibody (HERCEPTIN®) fused to "SpyC-anti-HER2 antibody" of SpyCatcher (SEQ ID NO: 3) and a panel of mosaic AAV2 viral particles, where the AAV2 viral particles are composed of a mixture of "SpyTag" capsid protein containing the mutations R585A, delR588, and N587-linker10-SpyTag (SEQ ID NO: 25) and "HBM" capsid protein containing the mutations R585A and R588A but without SpyTag (SEQ ID NO: 11). The "SpyTag" and "HBM" capsid proteins were mixed at various ratios (1:0, 3:1, 1:1, and 1:3). [Figure 7B] Figure 1 shows the percentage of HER2+ 293 hErbB2 cells (gray bars) and HER2- 293 parental cells (black bars) expressing GFP (y-axis) 5 days after infection with mosaic AAV2 viral particles (x-axis) composed of a mixture of "Linker10" capsid protein containing the mutations R585A, delR588, and N587-Linker10-SpyTag (SEQ ID NO: 25) and "HBM" capsid protein containing the mutations R585A and R588A but without the SpyTag (SEQ ID NO: 11). "Linker10" and "HBM" capsid proteins were mixed in various ratios (1:0, 3:1, 1:1, and 1:3) and conjugated to an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3). [Figure 8A]A Western blot is provided analyzing the reaction between an anti-HER2 antibody (HERCEPTIN®) fused to "SpyC-anti-HER2 antibody" of SpyCatcher (SEQ ID NO: 3) and a panel of mosaic AAV2 viral particles, using the B1 antibody, which recognizes a linear epitope shared by the AAV2 VP1, VP2, and VP3 capsid proteins, where the AAV2 viral particles are composed of a mixture of "SpyTag" capsid protein and "HBM" capsid protein containing the mutations R585A and R588A but without SpyTag (SEQ ID NO: 11). The "SpyTag" capsid proteins include "G453 SpyTag," which contains the mutations R585A, R588A, and an insertion of the SpyTag peptide immediately after residue G453 (SEQ ID NO: 27), and "G453 Linker 10 SpyTag," which contains the mutations R585A, R588A, and an insertion of the SpyTag peptide immediately after residue G453, flanked on either side by 10 linker amino acids (SEQ ID NO: 29). The indicated "G453 SpyTag" and "G453 Linker 10 SpyTag" capsids were mixed with "HBM" capsids in various ratios (1:0, 1:3, and 1:7). [Figure 8B]The percentage of HER2+ 293 hErbB2 cells (gray bars) and HER2- 293 parental cells (black bars) expressing GFP (y-axis) 5 days after infection with wild-type "wt" or mosaic AAV2 viral particles (x-axis) composed of a mixture between a "G453 SpyTag" capsid protein containing mutations R585A, R588A, and an insertion of the SpyTag peptide immediately after residue G453 (SEQ ID NO: 27), or a "G453 linker 10 SpyTag" capsid protein containing mutations R585A, R588A, and an insertion of the SpyTag peptide immediately after residue G453 and flanked on either side by 10 linker amino acids (SEQ ID NO: 29), and an "HBM" capsid protein containing mutations R585A and R588A but no SpyTag (SEQ ID NO: 11). "G453 SpyTag" or "G453 Linker 10 SpyTag" and "HBM" capsids were mixed in various ratios (1:0 "pure", 1:3, and 1:7) and conjugated to an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3). [Figure 9-1]Scatter plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescent protein (GFP) expression by cells positive (+) for ASGR1 expression after infection with "AAV2 wt" particles, "AAV2 SpyTag no antibody" particles, or "AAV2 SpyTag anti-ASGR1" particles. The "AAV2 wt" capsid is wild-type with no mutations or modifications (SEQ ID NO: 9), while the "AAV2 SpyTag" capsid contains the following mutations: R585A, delR588, and insertion of the SpyTag peptide immediately after residue N587 (SEQ ID NO: 13). The "AAV2 SpyTag anti-ASGR1" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to ASGR1. Also shown are scatter plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescent protein (GFP) expression by cells positive (+) for CD63 expression following infection with "AAV2 wt" particles, "AAV2 SpyTag no antibody" particles, or "AAV2 SpyTag anti-CD63" particles. The "AAV2 wt" capsid is wild-type with no mutations or modifications (SEQ ID NO: 9), while the "AAV2 SpyTag" capsid contains the following mutations: R585A, delR588, and insertion of the SpyTag peptide immediately after residue N587 (SEQ ID NO: 13). The "AAV2 SpyTag anti-CD63" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to CD63. The virus expresses GFP as a marker of transformation. Also shown are scatter plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescent protein (GFP) expression by cells positive (+) for PTPRN expression after infection with "AAV9 wt" particles, "AAV2 SpyTag irrelevant antibody" particles, or "AAV2 SpyTag anti-PTPRN" particles.The "AAV9 wt" capsid is a wild-type (SEQ ID NO: 31) with no mutations or modifications. The "AAV2 SpyTag" capsid is a mosaic virus particle composed of a 1:7 ratio between the "SpyTag" capsid protein, in which SpyTag is inserted immediately after residue G453 adjacent to a 10-amino acid linker (SEQ ID NO: 29), and a capsid without SpyTag but containing a Myc tag amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 53), which reduces native receptor binding. The "AAV2 SpyTag irrelevant antibody" particle was conjugated to a SpyCatcher fusion antibody that does not bind to PTPRN. The "AAV2 SpyTag anti-PTPRN" particle was conjugated to a SpyCatcher fusion antibody that specifically binds to PTPRN. The virus expresses GFP as a marker of transformation. Also shown are the results of a luciferase assay evaluating firefly luciferase expression by cells positive for hENTPD3 after infection with "AAV2 wt" particles, "AAV2 + irrelevant mAb" particles, "AAV2 + anti-ENTPD3" particles, and "AAV2 + anti-hCD20" particles. The "AAV2 wt" capsid is wild-type with no mutations or modifications (SEQ ID NO: 9), while the "AAV2 + antibody" capsid contains the following mutations: R585A, delR588, and insertion of a SpyTag peptide immediately after residue N587 (SEQ ID NO: 13). The "AAV2 + irrelevant mAb" particles were conjugated to a SpyCatcher fusion antibody that does not bind to hENTPD3. The "AAV2 + anti-hCD20" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to hCD20, which is not expressed on hENTPD3+ cells and serves as an additional negative control. The "AAV2 + anti-ENTPD3" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to hENTPD3. The virus expresses firefly luciferase as a marker of transduction. Also shown are the results of a luciferase assay assessing firefly luciferase expression by cells positive for hCD20 after infection with "AAV2 wt" particles, "AAV2 + irrelevant mAb" particles, "AAV2 + anti-ENTPD3" particles, and "AAV2 + anti-hCD20" particles.The "AAV2 wt" capsid is wild-type with no mutations or modifications (SEQ ID NO: 9), while the "AAV2+ antibody" capsid contains the following mutations: R585A, delR588, and insertion of a SpyTag peptide immediately after residue N587 (SEQ ID NO: 13). "AAV2+ irrelevant mAb" particles were conjugated to a SpyCatcher fusion antibody that does not bind to hENTPD3. "AAV2+ anti-ENTPD3" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to hENTPD3, which is not expressed on hCD20+ cells and serves as an additional negative control. "AAV2+ anti-hCD20" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to hCD20. The virus expresses firefly luciferase as a marker of transduction. [Figure 9-2] Same as above. [Figure 10A] Western blots are provided analyzing the reaction between an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) "SpyC-Herceptin" and a panel of AAV9 viral particles using the B1 antibody, which recognizes a linear epitope shared by the AAV9 VP1, VP2, and VP3 capsid proteins. These AAV9 viral particles consist of capsids containing the mutation W503A, which reduces receptor binding, and either no linker or an insertion of the SpyTag peptide immediately after residues A589 or G453 adjacent to the 10-amino acid linker, or mosaic AAV9 viral particles composed of a 1:7 ratio between a "SpyTag" capsid protein containing the mutation W503A and either A589-linker10-SpyTag or G453-linker10-SpyTag, where the "W503A" capsid protein has the mutation W503A but no SpyTag. [Figure 10B]The percentages of HER2+ 293 hErbB2 cells (gray bars) and HER2- 293 parental cells (black bars) (y-axis) expressing GFP 5 days after infection with AAV9 viral particles conjugated to an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (x-axis) are shown. These AAV9 viral particles consist of capsids containing the mutation W503A, which reduces receptor binding, and either no linker or an insertion of the SpyTag peptide immediately after residue A589 (or G453) adjacent to the 10-amino acid linker, or mosaic AAV9 viral particles composed of a 1:7 ratio between a "SpyTag" capsid protein containing the mutation W503A and either A589-linker10-SpyTag or G453-linker10-SpyTag, where the "W503A" capsid protein has the mutation W503A but no SpyTag. The virus expresses GFP as a marker of transformation. [Figure 11]Immunofluorescence microscopy images of liver samples taken from C57BL / 6 mice transgenicly modified to express human ASGR1 on hepatocytes are shown. Samples were collected 10 days after intravenous injection with phosphate-buffered saline (PBS) or with 2.5 x 10 viral genomes (vg) / animal of SpyTag-tagged AAV2 particles carrying the CAGG eGFP nucleotide of interest and modified with either (1) SpyCatcher-anti-human CD3 antibody (AAV SpyT-anti-hCD3 CAGG eGFP) or (2) SpyCatcher-anti-human ASGR1 antibody (AAV SpyT-anti-hASGR1 CAGG eGFP). Mice were sacrificed and transgenic perfused with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). Each image represents one mouse. The "AAV2 with SpyTag" capsid contains the following mutations: R585A, delR588, and insertion of the SpyTag peptide immediately after residue N587 (SEQ ID NO: 13). The virus expresses eGFP as a marker of transduction. [Figure 12]Fluorescence images are provided of individual mice that do not express human ASGR1 on hepatocytes (control) and genetically modified mice that express human ASGR1 on hepatocytes (ASGR1-humanized mice) 14 days after intravenous injection with phosphate-buffered saline (PBS) or with 3.0 x 1011 viral genomes (vg) / animal of wild-type (wt) AAV2 particles or SpyTag-tagged AAV2 particles carrying the firefly luciferase nucleotide of interest and modified with (1) SpyCatcher-anti-human CD63 antibody or (2) SpyCatcher-anti-human ASGR1 antibody. These "AAV2" viral particles are mosaic viral particles composed at a 1:7 ratio between "SpyTag" capsid proteins, in which SpyTag was inserted immediately after residue G453 adjacent to a 10-amino acid linker (SEQ ID NO: 29), and capsids without SpyTag but containing a Myc tag amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 53), which reduces native receptor binding. The virus expresses firefly luciferase as a marker of transduction. Mice were anesthetized using isoflurane, injected with luciferin substrate, and imaged 10 minutes later using an IVIS Spectrum in vivo imaging system (PerkinElmer). [Figure 13]Immunohistochemistry images of liver and pancreas samples taken from C57BL / 6 mice 4 weeks after intravenous injection with phosphate-buffered saline (PBS), 1.0 x 10 viral genomes (vg) / animal of wild-type (wt) AAV9 particles, or 1.0 x 10 viral genomes (vg) / animal of SpyTag-tagged AAV2 particles carrying the CMV eGFP nucleotide of interest and modified with either (1) SpyCatcher-anti-human ASGR1 antibody (AAV2 SpyTag + irrelevant mAb) or (2) SpyCatcher-anti-human ENTPD3 antibody (AAV2 SpyTag + anti-ENTPD3). Mice were sacrificed, and livers and pancreases were harvested and fixed in 10% neutral-buffered formalin. The organs were then embedded on slides, cryosectioned, and stained with anti-GFP antibody. Each image represents one mouse. These "AAV2" viral particles are mosaic viral particles composed at a 1:7 ratio between "SpyTag" capsid proteins, in which SpyTag was inserted immediately after residue G453 adjacent to a 10-amino acid linker (SEQ ID NO: 29), and capsids without SpyTag but containing a Myc tag amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 53), which reduces native receptor binding. The virus expresses eGFP as a marker of transduction. ENTPD3 has been reported to be expressed in pancreatic islet cells and tongue, among other cell types, but not in the liver. SpyTag-tagged AAV2 particles were shed from the liver, and no eGFP expression was observed in the livers of mice injected with "AAV2 SpyTag + irrelevant mAb" or "AAV2 SpyTag + anti-ENTPD3" particles. Positive staining in pancreatic islets was detected in a pancreas sample from one mouse injected with "AAV2 SpyTag + anti-ENTPD3" particles. [Figure 14]Immunohistochemistry images of liver and tongue samples from C57BL / 6 mice 14 days after intravenous injection with phosphate-buffered saline (PBS), wild-type (wt) AAV9 particles carrying 2.0 x 10 viral genomes (vg) / animal, or SpyTag-tagged AAV2 particles carrying the CMV eGFP nucleic acid of interest and modified with either (1) SpyCatcher-anti-human ASGR1 antibody (AAV2 SpyTag + irrelevant mAb) or (2) SpyCatcher-anti-human ENTPD3 antibody binding to ENTPD3 (AAV2 SpyTag + anti-ENTPD3). Mice were sacrificed, and livers and tongues were collected and fixed in 10% neutral-buffered formalin. The organs were then embedded on slides, cryosectioned, and stained with anti-GFP antibody. Each image represents one mouse; three mice were injected and analyzed from the "AAV2 SpyTag + irrelevant mAb" and "AAV2 SpyTag + anti-ENTPD3" groups; all showed similar GFP expression patterns. These "AAV2" virus particles are mosaic virus particles composed at a 1:7 ratio between "SpyTag" capsid proteins, in which SpyTag was inserted immediately after residue G453, adjacent to a 10-amino acid linker (SEQ ID NO: 29), and capsids without SpyTag but containing a Myc tag amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 52), which reduces native receptor binding. The virus expresses eGFP as a marker of transduction. ENTPD3 has been reported to be expressed in the mouse tongue but not in the liver (data extracted from the public database GenePaint.org http: / / www.informatics.jax.org / assay / MGI:5423021 and the Riken FANTOM5 project, adult mouse dataset). eGFP expression was not observed in the liver of mice injected with AAV2 SpyTag + irrelevant mAb or AAV2 SpyTag + anti-ENTPD3 particles, but was detected in the tongue of all three mice injected with AAV2 SpyTag + anti-ENTPD3 particles. DETAILED DESCRIPTION OF THE INVENTION

[0047] WO2016 / 11291 describes the use of specific binding pairs (SpyCatcher:SpyTag) to produce virus-like particles (VLPs) derived from modified bacteriophage AP205, which display immunogenic antigens on the AP205 capsid at high density for vaccination purposes. In theory, such extensive modification of the viral capsid may be desirable to ensure that the natural tropism of the viral vector is substantially reduced or abolished for the purpose of retargeting the viral vector. However, displaying targeting ligands on the viral surface at such high density may hinder transduction efficiency. See Examples 4 and 5. It has been discovered that optimal transduction efficiency occurs when the degree of modification of the viral surface with members is reduced. Thus, genetically modified viral particles, compositions containing them, and methods for making and using them are provided herein.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0050] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds). Each heavy chain contains a heavy chain variable domain (V H ) and the heavy chain constant region (C H The heavy chain constant region comprises at least three domains: C H 1. C H 2. C HEach light chain comprises a light chain variable domain (C H ) and the light chain constant region (C L ). The heavy and light chain variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure contains two identical antigen-binding domains, each of which is a V H Domains and V L formed by the association of domains, each of which has its own C H and C L The domains together form the antibody Fv region. Single domain antibodies contain a single antigen-binding domain, e.g., a V H or V L The antigen-binding domain of an antigen, e.g., the portion of an antibody that recognizes and binds to the first member of a specific binding pair of an antigen, is also called the "paratope." It is a small region (of 5-10 amino acids) of the Fv region of an antibody, a portion of the fragment antibody binding (Fab region), and may contain portions of the heavy and / or light chains of an antibody. A paratope specifically binds to a first member of a specific binding pair when the paratope binds to the first member of the specific binding pair with high affinity. The term "high affinity" antibody refers to an antibody that specifically binds to a first member of a specific binding pair with a high affinity of about 10 -9 M or less (e.g., about 1 × 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or approximately 1 x 10 -12 K for the first member of the specific binding pair of D In one embodiment, K Dis measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0051] The phrase "complementarity-determining region" or "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of an organism's immunoglobulin genes, which amino acid sequences are normally (i.e., in wild-type animals) found between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T-cell receptor). CDRs can be encoded, for example, by germline sequences, or by rearranged or unrearranged sequences, e.g., by naive or mature B or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the animal germline), humanized, and / or modified with amino acid substitutions, additions, or deletions. In some circumstances (e.g., with respect to CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences), which are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B-cell nucleic acid sequence, e.g., as a result of splicing or joining of sequences (e.g., VDJ reassortment to form a heavy chain CDR3).

[0052] The term "inverted terminal repeat sequences" or "ITR" refers to the symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as origins of replication for viral DNA synthesis and are essential cis components for generating AAV vectors.

[0053] The term "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa and lambda light chains and VpreB, as well as surrogate light chains, unless otherwise specified. A light chain variable domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. A full-length light chain generally includes, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. The light chain variable domain is encoded by a light chain variable region gene sequence, which generally includes a V segment derived from a repertoire of V and J segments present in the germline. L Segment and J L The sequences, locations, and nomenclature of V and J light chain segments for various organisms can be found in the IMGT database, www.imgt.org. Light chains include, for example, light chains that do not selectively bind either the first or second member of a specific binding pair that is selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains or another light chain by binding and recognition of one or more first members of a specific binding pair that is selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Common or universal light chains include light chains derived from the human Vκ1-39Jκ gene or the human Vκ3-20Jκ gene, including somatically mutated (e.g., affinity matured) versions thereof. Exemplary human Vκ1-39Jκ genes include: LThe segments include the human Vκ1-39 gene segment, the human Vκ3-20 gene segment, the human Vλ1-40 gene segment, the human Vλ1-44 gene segment, the human Vλ2-8 gene segment, the human Vλ2-14 gene segment, and the human Vλ3-21 gene segment, including somatically mutated (e.g., affinity matured) versions thereof. Light chains can be made that include variable domains from one organism (e.g., human or rodent, e.g., rat or mouse, or avian, e.g., chicken) and constant regions from the same or a different organism (e.g., human or rodent, e.g., rat or mouse, or avian, e.g., chicken).

[0054] The term "about" or "approximately" includes being within a statistically significant range of a value. Such a range may be within 10-fold, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" depends on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0055] The term "capsid protein" includes proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are generally called VP1, VP2, and / or VP3, and are encoded by a single cap gene. For AAV, three AAV capsid proteins are generated in an overlapping manner from the cap open reading frame (ORF) through alternative mRNA splicing and / or alternative translation initiation codon usage, but all three proteins share a common stop codon. Warrington et al. (2004) J. Virol. 78:6595 (incorporated herein by reference in its entirety). AAV2 VP1 is generally translated from the ATG start codon (amino acid M1) on the 2.4 kb mRNA, while AAV2 VP2 and VP3 arise from a smaller 2.3 kb mRNA using a weaker ACG start codon (amino acid T138) to generate VP2 and read-through translation to the next available ATG codon (amino acid M203) to generate VP3, the most abundant capsid methane protein. Warrington, supra; Rutledge et al. (1998) J. Virol. 72:309-19 (incorporated herein by reference in its entirety). The amino acid sequences of adeno-associated virus capsid proteins are well known in the art and are generally conserved, particularly within dependoparvoviruses. See Rutledge et al., supra. For example, Rutledge et al. (1998), supra, provide in Figure 4B an amino acid sequence alignment for the VP1, VP2, and VP3 capsid proteins of AAV2, AAV3, AAV4, and AAV6, with the start sites of VP1, VP2, and VP3, respectively, indicated by arrows and the variable domains indicated by boxes.Thus, the amino acid positions provided herein may be provided relative to the VP1 capsid protein of AAV, and amino acid positions provided herein that are not further specified refer to the AAV2 sequence of the major encoded protein VP1 represented as SEQ ID NO: 1, although one skilled in the art would readily be able to determine the position of the same amino acid within the VP2 and / or VP3 capsid proteins of AAV, and the corresponding amino acid positions within different serotypes, respectively. Thus, the amino acid positions provided herein may be provided relative to the VP1 capsid protein of AAV, and the amino acid positions provided herein that are not further specified refer to the AAV2 sequence of the major encoded protein VP1 represented as SEQ ID NO: 9, although one skilled in the art would readily be able to determine the position of the same amino acid within the VP2 and / or VP3 capsid proteins of AAV, and the corresponding amino acid and positions within different AAV serotypes, respectively. Furthermore, one skilled in the art would be able to swap domains between capsid proteins of different AAV serotypes to form "chimeric capsid proteins."

[0056] Domain swapping between two AAV capsid protein constructs to generate "chimeric AAV capsid proteins" has been described. See, for example, Shen et al. (2007) Mol. Therapy 15(11):1955-1962 (incorporated herein by reference in its entirety). "Chimeric AAV capsid proteins" include AAV capsid proteins that contain amino acid sequences, e.g., domains from two or more different AAV serotypes, and can and / or do form AAV-like viral capsids / viral particles. Chimeric AAV capsid proteins are encoded by chimeric AAV capsid genes, e.g., nucleotides containing a plurality, e.g., at least two, nucleotide sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a distinct AAV serotype, and which together encode a functional chimeric AAV capsid protein. Reference to a chimeric capsid protein in relation to a particular AAV serotype indicates that the capsid protein contains one or more domains from a capsid protein of that serotype and one or more domains from a capsid protein of a different serotype. For example, an AAV2 chimeric capsid protein contains a capsid protein that contains one or more domains from an AAV2 VP1, VP2, and / or VP3 capsid protein and one or more domains from a VP1, VP2, and / or VP3 capsid protein of a different AAV.

[0057] A "mosaic capsid" contains at least two sets of VP1, VP2, and / or VP3 proteins, each set of which is encoded by a different cap gene.

[0058] In some embodiments, the mosaic capsids described herein comprise recombinant VP1, VP2, and / or VP3 proteins encoded by cap genes that have been genetically modified by the insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprise VP1, VP2, and / or VP3 proteins encoded by a reference cap gene, e.g., a wild-type reference cap gene that encodes wild-type VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins; a control reference cap gene that encodes VP1, VP2, and / or VP3 proteins identical to the recombinant VP1, VP2, and / or VP3 proteins but in the absence of the heterologous epitope; or a mutant wild-type reference cap gene that encodes substantially wild-type VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins but with mutations (e.g., introduced, substituted, deleted) that preferably reduce the tropism of the wild-type VP1, VP2, and / or VP3 proteins. In some embodiments, the reference cap gene encodes a chimeric VP1, VP2, and / or VP3 protein.

[0059] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences, including immunoglobulin heavy chain constant region sequences from any organism. A heavy chain variable domain, unless otherwise specified, contains three light chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs, and FRs, and combinations thereof. A typical heavy chain contains (from the N-terminus to the C-terminus) a CDR, CDRs, and FRs following the variable domain. H 1 domain, hinge, C H 2 domain, and C H The functional fragment of the heavy chain has three domains that specifically recognize the first member of the specific binding pair (e.g., a K in the micromolar, nanomolar, or picomolar range). DThe heavy chain variable domain is encoded by a variable region nucleotide sequence, and the gene sequence is generally a V region present in the germline. H Segment, D H Segment, and J H V derived from segment repertoire H Segment, D H Segment, and J H The sequences, locations, and nomenclature of V, D, and J heavy chain segments for various organisms can be found in the IMGT database, which is accessible via the internet on the worldwide web (www) at the URL "imgt.org".

[0060] The terms "heavy chain-only antibody," "heavy chain-only antigen binding protein," "single domain antigen binding protein," "single domain binding protein," and the like refer to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable region operably linked to a heavy chain constant region, where the heavy chain constant region is typically a functional C H Therefore, the terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," etc., refer to antibodies that: (i) lack a functional C H (ii) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region lacking one domain; or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which is a functional C H In various embodiments, the homodimeric single domain antigen binding protein includes both a homodimeric single domain antigen binding protein, which comprises a variable domain operably linked to a heavy chain constant region lacking one domain. In various embodiments, the homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which is a functional C HIn addition, each immunoglobulin-like chain of a single domain antigen-binding protein comprises an identical variable domain operably linked to an identical heavy chain constant region gene (e.g., IgG, IgA, IgE, IgD, or a combination thereof) encoding the C (and, optionally, hinge region). H heavy chain constant region (C) containing a deletion or inactivating mutation in H ) gene sequence, linked to a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segments (e.g., V L , J L ), or a combination thereof. Single domain antigen binding proteins containing variable domains derived from heavy chain gene segments are referred to as "V H Single domain antibodies or "V H Single domain antigen binding proteins comprising a variable domain derived from a light chain gene segment are sometimes referred to as "single domain antibody binding proteins," see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 2014 / 0289876; 2015 / 0197553; 2015 / 0197554; 2015 / 0197555; 2015 / 0196015; 2015 / 0197556; 2015 / 0197557, each of which is incorporated by reference in its entirety. L They may be referred to as "single domain antigen binding proteins," see, e.g., U.S. Patent Publication No. 2015 / 0289489, which is incorporated by reference in its entirety.

[0061] The term "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa (κ) and lambda (λ) light chains, as well as surrogate light chains, and VpreB, unless otherwise specified. A light chain variable domain typically comprises three light chain CDRs and four framework (FR) regions, unless otherwise specified. A full-length light chain generally comprises, from the amino terminus to the carboxyl terminus, a variable domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. The light chain variable domain is encoded by a light chain variable region nucleotide sequence, which is generally derived from a repertoire of light chain V and J gene segments present in the germline. L Gene segments and light chain J L The light chain V gene segment and the light chain J gene segment are included. The sequences, locations, and nomenclature of light chain V gene segments and light chain J gene segments for various organisms can be found in the IMGT database, which is accessible via the Internet on the worldwide web (www) at the URL "imgt.org." Light chains include, for example, light chains that do not selectively bind to either the first or second member of a specific binding pair selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair binding protein in which the light chain appears. Common or universal light chains include light chains derived from the human Vκ1-39Jκ5 gene or the human Vκ3-20Jκ1 gene, including somatically mutated (e.g., affinity matured) versions thereof.

[0062] As used herein, the phrase "operably linked" includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or otherwise cooperate with each other to participate in biological events, such juxtaposition achieving or enabling such interaction and / or cooperation. By way of example, a regulatory sequence (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it is positioned relative to the coding sequence such that its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, "operably linked" includes the covalent attachment of associated components or elements to each other. Those skilled in the art will readily appreciate that in some embodiments, covalent attachment is not required to achieve effective operable linkage. For example, in some embodiments, nucleic acid regulatory sequences that are operably linked to the coding sequences they control are contiguous with the nucleotide of interest. Alternatively or additionally, in some embodiments, one or more such regulatory sequences act in trans or at a distance to control the coding sequence of interest. In some embodiments, the term "expression control sequence," as used herein, refers to polynucleotide sequences necessary and / or sufficient to affect the expression and processing of coding sequences to which they are linked. In some embodiments, expression control sequences may be or include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak sequences); sequences that enhance protein stability; and / or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are preferentially or only active in a particular host cell or organism, or type thereof. By way of example, in prokaryotes, control sequences typically include a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include a promoter, an enhancer, and / or a transcription termination sequence.Those skilled in the art will understand that in many embodiments, the term "control sequences" refers to components essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0063] The term "recombinant capsid protein" includes a capsid protein having at least one mutation compared to the corresponding capsid protein of a wild-type virus, which may be a reference and / or control virus for comparative studies. Recombinant capsid proteins include capsid proteins containing a heterologous amino acid sequence that may be inserted into and / or displayed by a capsid protein. "Heterologous" in this context means heterologous compared to the virus from which the capsid protein is derived. The inserted amino acid may simply be inserted between two given amino acids of the capsid protein. An amino acid insertion may also be performed in conjunction with a deletion of a given amino acid of the capsid protein at the insertion site (e.g., one or more capsid protein amino acids are replaced by five or more heterologous amino acids).

[0064] "Retargeting" or "reorientation" can include scenarios in which a wild-type vector targets some cells within a tissue and / or some cells within some organs within an organism, where general targeting of a tissue or organ is reduced or abolished by the insertion of a heterologous epitope, and retargeting of more specific cells in a tissue or specific organ within an organism is achieved by a targeting ligand that binds to a marker expressed by the specific cells. Such retargeting or redirection can also include scenarios in which a wild-type vector targets a tissue, where tissue targeting is reduced or abolished by the insertion of a heterologous epitope, and retargeting to an entirely different tissue is achieved by a targeting ligand.

[0065] A "specific binding pair," "protein:protein binding pair," and the like, includes two proteins (e.g., a first member (e.g., a first polypeptide)) and a second cognate member (e.g., a second polypeptide) that interact to form a covalent isopeptide bond, bound under conditions that allow or promote isopeptide bond formation, the term "cognate" referring to components that function together, i.e., react together, to form an isopeptide bond. Thus, two proteins that react together efficiently to form a covalent isopeptide bond under conditions that allow or promote isopeptide bond formation may also be referred to as a "complementary" pair of peptide linkers. Specific binding pairs that can interact to form covalent isopeptide bonds are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506, and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, Isopeptag:Pyrin C, SnoopTag:SnoopCatcher, etc. Generally, a peptide tag refers to a member of a protein:protein binding pair, which is generally less than 30 amino acids in length, that forms a covalent isopeptide bond with a second cognate protein, which is generally larger, but may also be shorter than 30 amino acids in length, such as the SpyTag:KTag system.

[0066] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, where at least one of the amino groups is not derived from the protein backbone or, alternatively, is not found to be part of the protein backbone. Isopeptide bonds can form within a single protein, or between two peptides or between a peptide and a protein. Thus, isopeptide bonds can form intramolecularly within a single protein, or intramolecularly, i.e., between two peptide / protein molecules, for example, between two peptide linkers. Typically, an isopeptide bond can occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid residue. Each residue in a pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond can be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond can occur between the side chain amine of lysine and the carboxamide group of asparagine or the carboxyl group of aspartic acid.

[0067] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zakeri et al. (2012) PNAS 109:E690-E697 (each of which is incorporated herein by reference in its entirety) and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein FbaB. By splitting the domain, Zakeri et al. obtained a peptide called "SpyTag" with the sequence AHIVMVDAYKPTK (SEQ ID NO: 1), which forms an amide bond to its cognate protein, "SpyCatcher," a 112-amino acid polypeptide with the amino acid sequence set forth in SEQ ID NO: 3 (Zakeri (2012), supra). Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase (Fierer (2014) PNAS 111:E1176-1181). SpyLigase was engineered by deleting the β-strand from SpyCatcher that contains a reactive lysine, resulting in KTag, a 10-residue peptide tag with the amino acid sequence ATHIKFSKRD (SEQ ID NO: 2). SpyTag002: The SpyCatcher002 system was developed by Keeble et al. al (2017) Angew Chem Int Ed Engl 56:16521-25, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, represented as SEQ ID NO: 54, and binds to SpyCatcher002 (SEQ ID NO: 55).

[0068] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. The D4 Ig-like domain of RrgA, an adhesin from S. pneumoniae, was split to form SnoopTag (residues 734-745, SEQ ID NO: 5) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher results in a specific native isopeptide bond between the complementary proteins. See Veggiani (2016) supra.

[0069] The isopeptag:pilin-C specific binding pair was derived from the major pilin protein Spy0128 from Streptococcus pyogenes (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). The isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, represented as SEQ ID NO: 7, and binds to Pilin-C (residues 18-299 of SpyO128). Incubation of SnoopTag and SnoopCatcher results in the creation of a specific native isopeptide bond between the complementary proteins. See Zakeir and Howarth (2010) supra.

[0070] The term "peptide tag" includes a polypeptide that is (1) heterologous to the protein tagged with the peptide tag, (2) a member of a specific protein:protein binding pair capable of forming an isopeptide bond, and (3) is 50 amino acids or less in length.

[0071] The term "target cell" includes any cell in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit receptors on their surface that allow the cells to be targeted by targeting ligands, as described below.

[0072] Terms such as "transduction" or "infection" refer to the introduction of nucleic acid into the nucleus of a target cell by a viral vector. Terms such as "transduction efficiency" related to transduction and the like refer to the fraction (e.g., percentage) of cells that express a target nucleotide after incubation with a set number of viral vectors containing the target nucleotide. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting transduced with a fluorescent reporter gene, PCR for the expression of the target nucleotide, etc.

[0073] As used herein, the term "wild-type" includes entities having a structure and / or activity found in nature in a "normal" (as opposed to mutant, diseased, altered, etc.) state or context. Those skilled in the art will understand that a wild-type viral vector, e.g., a wild-type capsid protein, can be used as a reference viral vector in comparative studies. Generally, the reference viral capsid protein / capsid / vector is identical to the test viral capsid protein / capsid / vector, but for any alterations whose effects are being tested. For example, to determine the effect on transduction efficiency of inserting a first member of a specific binding pair into a test viral vector, the transduction efficiency of the test viral vector (in the presence or absence of an appropriate targeting ligand) can be compared to the transduction efficiency of a reference viral vector that is identical to the test viral vector in every instance (e.g., additional mutations, nucleotides of interest, number of viral vectors and target cells, etc.) except for the presence of the first member of the specific binding pair (in the absence or presence of an appropriate targeting ligand, as needed).

[0074] The retargeting strategy described herein offers the advantages of both the scaffold and direct recombination approaches described above, while resolving many of the inherent shortcomings of both. This strategy utilizes a specific binding pair in which a first member and a second cognate member specifically bind to each other, forming a covalent bond upon binding that permanently links the viral particle to any targeting ligand fused to the cognate member. Using such genetically modified viral particles, targeting is maintained as long as the viral capsid remains intact. For example, one advantage of the system provided herein compared to other scaffold approaches is the ability to attach "adapters" (e.g., targeting ligands) to recombinant viral particles similar to direct recombination approaches. However, in contrast to direct recombination approaches, the system described herein maintains the flexibility of the scaffold-adapter approach in that recombinant viral particles can maintain the variability found in adapters, for example, cognate members can be fused to targeting ligands, and then different fusion proteins can be attached to viral particles according to target cells.

[0075] Recombinant viral capsid proteins and viral vectors and nucleic acids Provided herein are recombinant viral particles (e.g., viral capsid proteins, and recombinant viral capsids and / or recombinant viral vectors comprising the recombinant viral capsid proteins) that have been genetically engineered to display a heterologous amino acid sequence comprising a first member of a specific binding pair, wherein the amino acid sequence is less than 50 amino acids in length, and the recombinant viral capsid / particle protein reduces or abolishes native tropism. In some embodiments, the viral particle further comprises a second cognate member of the specific binding pair, wherein the first and second members are covalently linked and the second member is fused to a targeting ligand.

[0076] In some embodiments, the heterologous amino acid sequence comprises a first member of a specific binding pair and one or more linkers. In some embodiments, the heterologous amino acid sequence comprises the first member of a specific binding pair adjacent to a linker, e.g., the heterologous amino acid sequence comprises the N-terminus to the C-terminus of the first linker, the first linker of the specific binding pair, and a second linker. In some embodiments, the first and second linkers are each independently at least one amino acid in length. In some embodiments, the first and second linkers are identical.

[0077] Generally, heterologous amino acid sequences described herein, e.g., comprising the first member of a specific binding pair by itself or in combination with one or more linkers, are from about 5 amino acids in length to about 50 amino acids in length. In some embodiments, the heterologous amino acid sequence is at least 5 amino acids in length. In some embodiments, the heterologous amino acid sequence is 6 amino acids in length. In some embodiments, the heterologous amino acid sequence is 7 amino acids in length. In some embodiments, the heterologous amino acid sequence is 8 amino acids in length. In some embodiments, the heterologous amino acid sequence is 9 amino acids in length. In some embodiments, the heterologous amino acid sequence is 10 amino acids in length. In some embodiments, the heterologous amino acid sequence is 11 amino acids in length. In some embodiments, the heterologous amino acid sequence is 12 amino acids in length. In some embodiments, the heterologous amino acid sequence is 13 amino acids in length. In some embodiments, the heterologous amino acid sequence is 14 amino acids in length. In some embodiments, the heterologous amino acid sequence is 15 amino acids in length. In some embodiments, the heterologous amino acid sequence is 16 amino acids in length. In some embodiments, the heterologous amino acid sequence is 17 amino acids in length. In some embodiments, the heterologous amino acid sequence is 18 amino acids in length. In some embodiments, the heterologous amino acid sequence is 19 amino acids in length. In some embodiments, the heterologous amino acid sequence is 20 amino acids in length. In some embodiments, the heterologous amino acid sequence is 21 amino acids in length. In some embodiments, the heterologous amino acid sequence is 22 amino acids in length. In some embodiments, the heterologous amino acid sequence is 23 amino acids in length. In some embodiments, the heterologous amino acid sequence is 24 amino acids in length. In some embodiments, the heterologous amino acid sequence is 25 amino acids in length. In some embodiments, the heterologous amino acid sequence is 26 amino acids in length. In some embodiments, the heterologous amino acid sequence is 27 amino acids in length. In some embodiments, the heterologous amino acid sequence is 28 amino acids in length. In some embodiments, the heterologous amino acid sequence is 29 amino acids in length.In some embodiments, the heterologous amino acid sequence is 30 amino acids in length. In some embodiments, the heterologous amino acid sequence is 31 amino acids in length. In some embodiments, the heterologous amino acid sequence is 32 amino acids in length. In some embodiments, the heterologous amino acid sequence is 33 amino acids in length. In some embodiments, the heterologous amino acid sequence is 34 amino acids in length. In some embodiments, the heterologous amino acid sequence is 35 amino acids in length. In some embodiments, the heterologous amino acid sequence is 36 amino acids in length. In some embodiments, the heterologous amino acid sequence is 37 amino acids in length. In some embodiments, the heterologous amino acid sequence is 38 amino acids in length. In some embodiments, the heterologous amino acid sequence is 39 amino acids in length. In some embodiments, the heterologous amino acid sequence is 40 amino acids in length. In some embodiments, the heterologous amino acid sequence is 41 amino acids in length. In some embodiments, the heterologous amino acid sequence is 42 amino acids in length. In some embodiments, the heterologous amino acid sequence is 43 amino acids in length. In some embodiments, the heterologous amino acid sequence is 44 amino acids in length. In some embodiments, the heterologous amino acid sequence is 45 amino acids in length. In some embodiments, the heterologous amino acid sequence is 46 amino acids in length. In some embodiments, the heterologous amino acid sequence is 47 amino acids in length. In some embodiments, the heterologous amino acid sequence is 48 amino acids in length. In some embodiments, the heterologous amino acid sequence is 49 amino acids in length. In some embodiments, the heterologous amino acid sequence is 50 amino acids in length.

[0078] In some embodiments, the specific binding pair is a SpyTag:SpyCatcher binding pair, where the first member is SpyTag and the second cognate member is SpyCatcher. In some embodiments, the specific binding pair is a SpyTag:KTag, where the first member is SpyTag and the second cognate member is KTag. In some embodiments, the specific binding pair is a SpyTag:KTag, where the first member is KTag and the second cognate member is SpyTag. In some embodiments, the specific binding pair is an Isopeptag:Pilin-C, where the first member is an Isopeptag and the second cognate member is Pilin-C or a portion thereof. In some embodiments, the specific binding pair is a SnoopTag:SnoopCatcher, where the first member is SnoopTag and the second cognate member is SnoopCatcher.

[0079] In some embodiments, the recombinant viral capsid protein described herein is an Ad capsid protein, e.g., a capsid protein of an Ad serotype selected from the group consisting of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, and Ad7. In some embodiments, the recombinant viral capsid protein is derived from the Ad2 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from the Ad5 capsid gene. In some embodiments, the recombinant Ad viral capsid protein described herein comprises a first member of a specific binding pair in the fiber protein domain, e.g., at the carboxy terminus of the fiber protein, the fiber knob, and / or the HI loop of the fiber knob.

[0080] In some embodiments, the recombinant viral capsid protein described herein is derived from an adeno-associated virus (AAV) capsid protein gene, such as a capsid gene of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene or an AAV9 capsid gene. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV2 VP1 capsid protein, the wild-type amino acid sequence of which is represented by SEQ ID NO:9. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9 VP1 capsid protein, the wild-type amino acid sequence of which is represented by SEQ ID NO:31.

[0081] Generally, the recombinant viral capsid proteins described herein comprise a first member of a specific binding pair inserted into and / or displayed by the capsid protein such that the first member of the specific binding pair reduces and / or abolishes the natural tropism of the capsid protein or a capsid containing it. In some embodiments, the first member of the specific binding pair is inserted into a region of the capsid protein responsible for the natural tropism of a wild-type reference capsid protein, e.g., a region of the capsid protein responsible for a cellular receptor. In some embodiments, the first member of the specific binding pair is inserted into and / or displayed by the knob domain of an Ad fiber protein. In some embodiments, the first member of the specific binding pair is inserted into and / or displayed by the HI loop of an Ad fiber protein. In some embodiments, the first member of the specific binding pair is inserted after an amino acid position selected from the group consisting of G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the first member of the specific binding pair is inserted and / or displayed between amino acids N587 and R588 of the AAV2 VP1 capsid. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises the amino acid sequence set forth as SEQ ID NO: 13, 15, 17, 19, 21, 23, 25, 27, 29, 35, 37, or 39. Additional suitable insertion sites identified using AAV2 are well known in the art (Wu et al. (2000) J. Virol. 74:8635-8647) and include I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716.The recombinant viral capsid protein described herein can be an AAV2 capsid protein comprising a first member of a specific binding pair inserted at a position selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Additional suitable insertion sites identified by using additional AAV serotypes are well known and include I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), and I-585 (AAV5). In some embodiments, the recombinant viral capsid protein described herein can be an AAV2 capsid protein comprising a first member of a specific binding pair inserted at a position selected from the group consisting of I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

[0082] As used herein, the designation I-### refers to an insertion site, designating the amino acid number relative to the VP1 protein of an AAV capsid protein as ###, although such insertions can be located directly N- or C-terminal, preferably one amino acid C-terminal in a sequence five amino acids N- or C-terminal to a given amino acid, preferably three, more preferably two, and especially one amino acid(s) N- or C-terminal to a given amino acid. Additionally, while the positions referred to herein are with respect to the VP1 protein encoded by an AAV capsid gene, corresponding positions (and mutations thereof) can be easily identified for the VP2 and VP3 capsid proteins encoded by capsid genes by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by reference AAV capsid genes.

[0083] Thus, insertion of the coding nucleic acid at one of these sites in the cap gene at the corresponding position leads to insertion in VP1, VP2, and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with shifted start codons. Thus, for example, for AAV2, according to this nomenclature, only insertions of amino acids 1-138 are inserted into VP1, insertions of 138-203 are inserted into VP1 and VP2, and insertions of 203 to the C-terminus are inserted into VP1, VP2, and VP3, and of course, this also applies to insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0084] Additionally, due to the high degree of conservation, at least in large stretches, and the large number of closely related family members, corresponding insertion sites in AAVs other than those listed can be identified by performing amino acid alignments or by comparison of capsid structures. See, e.g., Rutledge et al. (1998) J. Virol. 72:309-19, and U.S. Patent No. 9,624,274 (each of which is incorporated herein by reference in its entirety) for exemplary alignments of different AAV capsid proteins.

[0085] In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces or abolishes the natural tropism of the viral vector, e.g., transduction of cells naturally permissive for infection by the wild-type reference viral vector and / or target cells is undetectable in the absence of covalent attachment to the second cognate member of the binding pair fused to an appropriate targeting ligand. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector, e.g., compared to transduction of cells naturally permissive for infection by the wild-type reference viral vector. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 10%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 20%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 30%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 40%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 50%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 60%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 70%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 80%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 90%.In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 95%. In some embodiments, the insertion (presentation) of the first member of the specific binding pair reduces the natural tropism of the viral vector by at least 90%. In embodiments in which the insertion (presentation) of the first member of the specific binding pair does not completely abolish the natural tropism of the recombinant viral capsid, the natural tropism of such recombinant viral capsid can be further reduced by a second, different mutation. For example, in one embodiment, the recombinant viral capsid protein described herein can be derived from the AAV9 serotype, can include a first member of a specific binding pair, and can further include a mutation, e.g., a W503A mutation.

[0086] This detachment of the virus from its natural host is important, especially when systemic administration of the viral vector is intended versus local or locoregional administration, because uptake of the viral vector by natural host cells limits the effective dose of the viral vector. For AAV2 and AAV6, HSPG has been reported to be the main receptor for viral uptake in many cells, especially hepatocytes. For AAV2, HSPG binding activity relies on a group of five basic amino acids: R484, R487, R585, R588, and K532 (Kern et al., (2003) J Virol. 77(20):11072-81). Therefore, in the case of HSPG as the main receptor for viral vector binding to target cells, preferred point mutations are those that reduce the transduction activity of the viral vector for a given target cell mediated by the natural receptor by at least 50%, preferably at least 80%, and particularly at least 95%.

[0087] As a result, a preferred additional mutation for HSPG-binding viral vectors is one that removes or replaces the R or K involved in HSPG binding of the respective virus with a basic amino acid such as R, K, or H, preferably a non-basic amino acid such as A, D, G, Q, S, and T, preferably A, or an amino acid present at the corresponding position in a different but highly conserved AAV serotype that lacks such an amino acid at this position. As a result, preferred amino acid substitutions are R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A, or R588T, particularly R585A and / or R588A for AAV2, and K531A or K531E for AAV6. In a particularly preferred embodiment of the present invention, the AAV2 capsid protein mutants contain two additional point mutations, R585A and R588A, sufficient to significantly confer HSPG-binding activity, allowing efficient egress from HSPG-expressing cells and increasing the specificity of each mutant virus for its new target cells.

[0088] Targeting Ligands The viral particles described herein further comprise a second member of a specific binding pair that specifically forms a covalent bond with the first member of the specific binding pair inserted into / displayed by the recombinant viral capsid protein, where the second member is fused to a targeting ligand. In some embodiments, the targeting ligand binds to a receptor expressed on the surface of a cell surface protein on a cell, e.g., a (human) eukaryotic cell (e.g., a target cell). In some embodiments, the targeting ligand binds to a receptor expressed primarily (e.g., only) by (human) kidney cells. In some embodiments, the targeting ligand binds to a receptor expressed primarily (e.g., only) by (human) brain cells. In some embodiments, the targeting ligand binds to a receptor expressed primarily (e.g., only) by (human) lymphocytes. In some embodiments, the targeting ligand binds to a receptor expressed primarily (e.g., only) by (human) T cells. In some embodiments, the targeting ligand binds to a receptor expressed primarily (e.g., only) by (human) B cells. In some embodiments, the targeting ligand binds to a receptor expressed primarily (e.g., only) by (human) dendritic cells. In some embodiments, the targeting ligand binds to a receptor that is primarily (e.g., only) expressed by (human) macrophages. In some embodiments, the targeting ligand binds to a receptor that is primarily (e.g., only) expressed by (human) NK cells. In some embodiments, the targeting ligand binds to a receptor that is primarily (e.g., only) expressed by (human) kidney cells. In some embodiments, the targeting ligand binds to a receptor that is primarily (e.g., only) expressed by (e.g., human) cancer cells. In some embodiments, the targeting ligand binds to a receptor that is primarily (e.g., only) expressed by (human) cells infected with a heterologous pathogen.

[0089] There are numerous cell surface proteins, e.g., cell surface receptors, that can be suitable for targeting by targeting ligands, for which targeting ligands, e.g., antibodies or portions thereof, are already available. Such structures include class I and class II major histocompatibility antigens, receptors for various cytokines (e.g., receptors for IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), cell type-specific sex hormones, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), colony-stimulating growth factors, endothelial growth factors, epidermal growth factors, fibroblast growth factors, glial-derived neurotrophic factors, glial cell growth factors, gro-beta / mip receptors, and the like. 2, hepatocyte growth factor, insulin-like growth factor, interferons (α-IFN, β-IFN, γIFN, consensus IFN), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte growth factor, leukemia inhibitory factor, macrophage / monocyte chemotactic and activating factor, nerve growth factor, neutrophil-activating protein 2, platelet-derived growth factor, stem cell factor, transforming growth factor, tumor necrosis factor, vascular endothelial growth factor, lipoprotein (IL-1) receptors, other or other type 1 transmembrane receptors such as PRLR, G-protein coupled receptors such as GCGR These include, but are not limited to, receptors, ion channels such as Nav1.7, ASIC1, or ASIC2, cell adhesion molecules, transport molecules for metabolic substances such as amino acids, light receptors for B or T lymphocytes (e.g., B cell receptors and associated proteins (e.g., CD19, CD20, etc.), and T cell receptors and associated proteins (e.g., CD3, CD4, CD8, etc.), tetraspanin proteins (e.g., CD63). The recombinant viral capsids described herein enable specific infection of cell types by using targeting ligands that bind to differentiated cell surface antigens as targets for the viral-vector complex.

[0090] In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) kidney cells, i.e., at (e.g., only) liver-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) brain cells, i.e., at (e.g., only) brain cell-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) hematopoietic cells, i.e., at (e.g., only) hematopoietic cell-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) T cells, i.e., at (e.g., only) T cell-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) B cells, i.e., at (e.g., only) B cell-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) dendritic cells, i.e., at (e.g., only) dendritic cell-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily by (human) macrophages, i.e., at (e.g., only) macrophage-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily (e.g., only) by (human) NK cells, i.e., a protein expressed primarily (e.g., only) by (human) kidney cells, i.e., a protein expressed primarily (e.g., only) by kidney-specific markers. In some embodiments, the targeting ligand binds to a receptor expressed primarily (human) pancreatic cells, i.e., a pancreas-specific marker. In some embodiments, the targeting ligand binds to a receptor expressed primarily (human) intestinal cells, i.e., a receptor expressed primarily (e.g., only) by intestinal-specific markers. In some embodiments, the targeting ligand binds to a protein expressed primarily (e.g., only) by (human) cancer cells, i.e., a protein expressed primarily (e.g., only) by tumor-associated antigens. In some embodiments, the targeting ligand binds to a protein expressed primarily (e.g., only) by (human) cells infected with a heterologous pathogen.Proteins that are (1) specifically expressed by or whose expression is enriched in cells / tissues / organs and (2) recognized by antigen binding proteins useful as targeting ligands described herein are well known. www.proteinatlas.org But you can see, Uhlen et al. (2010) Nat. Biotech. 28:1248-50 (incorporated herein by reference in its entirety) See also Table 2 below, which provides exemplary and non-limiting organ-specific markers for which antigen binding proteins that may be useful as targeting ligands are available, and the cells / tissues / organs that express such markers. [Table 2-1] [Table 2-2]

[0091] In some embodiments, the targeting ligand binds to a receptor expressed by (human) liver cells, e.g., an asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the targeting ligand binds to a receptor expressed by (human) brain cells. In some embodiments, the targeting ligand binds to a receptor expressed by (human) T cells, e.g., CD3, e.g., CD3ε. In some embodiments, the targeting ligand binds to a receptor expressed by (human) kidney cells, e.g., a receptor expressed by (human) muscle cells, such as an integrin. In some embodiments, the targeting ligand binds to a receptor expressed by (human) cancer cells, e.g., tumor-associated antigens, e.g., E6 and E7. In some embodiments, the targeting ligand binds to the human glucagon receptor (hGCGR).

[0092] In some embodiments, the targeting ligand binds to a tumor-associated antigen expressed by a tumor cell. Non-limiting examples of specific tumor-associated antigens include, for example, AFP, ALK, BAGE protein, β-catenin, brc-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CCR5, CD19, CD20, CD30, CD40, CDK4, CEA, CTLA4, cyclin-B1, CYP1B1, EGFR, EGFRvIII, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, Fra-1, FOLR1, GAGE ​​protein (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gp100, Her2, HLA / B-raf, HLA / k-ras, HLA / MAGE-A3, hTERT, LMP2, These include MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesothelin, ML-IAP, Muc1, Muc2, Muc3, Muc4, Muc5, Muc16 (CA-125), MUM1, NA17, NY-BR1, NY-BR62, NY-BR85, NY-ESO1, OX40, p15, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, SART-1, SART-3, Steap-1, Steap-2, survivin, TAG-72, TGF-β, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, and uroplakin-3.

[0093] In some embodiments, the targeting ligand binds to a CD marker associated with the immune response, e.g., CD3, CD4, CD8, CD19, CD20, etc.

[0094] One embodiment of the present invention is a multimeric structure comprising the recombinant viral capsid proteins of the present invention. The multimeric structure comprises at least five, preferably at least 10, more preferably at least 30, and most preferably at least 60 recombinant viral capsid proteins, each comprising a first member of a specific binding pair as described herein. These can form stationary viral capsids (empty viral particles) or viral vectors (capsids that encapsulate a nucleotide of interest). The formation of a viral vector capable of packaging a viral genome is a highly preferred feature for using the recombinant viral capsids described herein as viral vectors.

[0095] One embodiment of the present invention is a nucleic acid encoding the capsid protein described above. The nucleic acid is preferably a vector comprising the claimed nucleic acid. The nucleic acid, particularly the vector, is required for the recombinant expression of the capsid protein of the present invention.

[0096] A further embodiment of the present invention is the use of at least one recombinant viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., a viral vector) for the production of and use as a gene transfer vector.

[0097] Use and Preparation Another embodiment of the recombinant viral capsid proteins described herein is their use to deliver a target nucleotide, such as a reporter gene or a therapeutic gene, to a target cell. Generally, the target nucleotide can be a transfer plasmid, which generally includes 5' and 3' inverted terminal repeat (ITR) sequences flanking the reporter gene(s) or therapeutic gene(s) (which, when contained within an AAV vector, can be under the control of a viral or non-viral promoter). In one embodiment, the target nucleotide is a transfer plasmid including 5' to 3': 5' ITR, promoter, gene (e.g., reporter and / or therapeutic gene), and 3' ITR.

[0098] Non-limiting examples of useful promoters include, for example, the cytomegalovirus (CMV) promoter, the spleen focus-forming virus (SFFV) promoter, the elongation factor 1 alpha (EF1a) promoter (1.2 kb EF1a promoter, or 0.2 kb EF1a promoter), the chimeric EF1a / IF4 promoter, and the phosphoglycerate kinase and phosphoglycerate kinase (PGK) promoter. Internal enhancers may also be present in the viral construct to increase expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer may be used in combination with the chicken β-actin promoter.

[0099] Various reporter genes (or detectable moieties) can be encapsulated in the multimeric structure comprising the recombinant viral capsid protein described herein. Exemplary reporter genes include, for example, β-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or combinations thereof. Although the methods described herein demonstrate the construction of targeting vectors with the use of a reporter gene encoding green fluorescent protein, one of skill in the art upon reading this disclosure will understand that the non-human animals described herein can be produced in the absence of a reporter gene or with any reporter gene known in the art.

[0100] Various therapeutic genes can also be packaged within the multimeric structures comprising the recombinant viral capsid proteins described herein, e.g., as part of a transfer vector. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.

[0101] A further embodiment of the present invention is a process for the preparation of a recombinant capsid protein, the method comprising: a) expressing a nucleic acid encoding a recombinant capping system protein under suitable conditions; b) isolating the expressed capsid protein of step a).

[0102] In some embodiments, the viral particles described herein comprise mosaic capsids, e.g., comprising genetically modified capsid proteins described herein (e.g., in the presence or absence of covalent attachment to a targeting ligand), in a specific ratio with a reference capsid protein. Methods for producing such mosaic viral particles include: a) expressing under suitable conditions a nucleic acid encoding a recombinant capsid protein and a nucleotide encoding a reference capsid protein in a ratio of 1:1 to 10:1 (wt / wt); b) isolating the expressed capsid protein of step a).

[0103] In general, a mosaic capsid formed according to the present methods will have a modified capsid protein:reference capsid protein ratio similar to the ratio (wt:wt) of the mosaic capsid-encoding nucleic acid used to generate the mosaic capsid. Thus, in some embodiments, the compositions described herein include a recombinant viral capsid protein and a reference capsid protein (or a combination of reference capsid proteins) in a ratio ranging from 1:1 to 1:15, or the methods described herein combine a recombinant viral capsid protein and a reference capsid protein (or a combination of reference capsid proteins) in a ratio ranging from 1:1 to 1:15. In some embodiments, the ratio is 1:2. In some embodiments, the ratio is 1:3. In some embodiments, the ratio is 1:4. In some embodiments, the ratio is 1:5. In some embodiments, the ratio is 1:6. In some embodiments, the ratio is 1:7. In some embodiments, the ratio is 1:8. In some embodiments, the ratio is 1:9. In some embodiments, the ratio is 1:10. In some embodiments, the ratio is 1:11. In some embodiments, the ratio is 1:12. In some embodiments, the ratio is 1:13. In some embodiments, the ratio is 1:14. In some embodiments, the ratio is 1:15.

[0104] A further embodiment of the present invention is a method for altering the tropism of a virus, comprising: (a) inserting a nucleic acid encoding a heterologous amino acid sequence into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the heterologous amino acid sequence; and / or (b) culturing packaging cells under conditions sufficient to produce a viral vector, wherein the packaging cells contain the nucleic acid. A further embodiment of the present invention is a method for presenting a targeting ligand on the surface of a capsid protein, comprising: (a) expressing a nucleic acid encoding a recombinant viral capsid protein described herein (and optionally including nucleotides encoding a reference capsid protein) under suitable conditions, wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair; (b) isolating the expressed capsid protein comprising the first member of the specific binding pair of step (a) or a capsid comprising the same; and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under conditions suitable to allow formation of an isopeptide bond between the first and second members, wherein the second cognate member of the specific binding pair is fused to the targeting ligand.

[0105] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid containing the nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementary adeno-associated viral vector from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral vector from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the viral vector with a nuclease, e.g., DNase I and MgCl2, containing the supernatant, (c) concentrating the viral vector, (d) purifying the viral vector, or (e) any combination of (a)-(d).

[0106] Packaging cells useful for producing the viral vectors described herein include, for example, animal cells that are permissive for the virus, or cells that have been modified to be permissive for the virus, or packaging cell constructs, for example, through the use of a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral vectors described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), SV40 Large T-antigen-containing HEK-293 cells (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), epithelial glioblastoma-astrocytoma cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH Examples of such cells include 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, and CAP-T cells.

[0107] L929 cells, the FLY virus packaging cell line reviewed in Cosset et al (1995) J Virol 69, 7430-7436, NS0 (mouse myeloma) cells, human amniotic cells (e.g., CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia pastoris), plant cells (including but not limited to tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g., High5)), or bacterial cells (including but not limited to E. coli).

[0108] For additional packaging cells and systems, packaging techniques and vectors for packaging nucleic acid genomes into pseudotyped viral vectors, see, e.g., Polo, et al., Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express the viral components or transiently transfecting cells with plasmids.

[0109] Further embodiments include methods for redirecting viruses and / or delivering reporter or therapeutic genes to target cells, including methods for transducing cells in vitro or in vivo, comprising contacting the target cell with a viral vector comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro. In other embodiments, the target cell is in vivo in a subject, e.g., a human.

[0110] target cell The recombinant viral vectors disclosed herein can be used to target a wide variety of cells for delivery of a nucleotide of interest. Target cells will generally be selected based on the nucleotide of interest and the desired effect.

[0111] In some embodiments, the subject nucleotides can be delivered to target cells to produce proteins that ameliorate organismal defects, such as enzyme deficiencies and immunosuppression, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that normally produce proteins in an animal are targeted. In other embodiments, cells in the region where the protein is most beneficial are targeted.

[0112] In other embodiments, target nucleotides, such as genes encoding siRNA, can inhibit the expression of specific genes in target cells.Target nucleotides can, for example, inhibit the expression of genes involved in pathogen life cycle.Therefore, cells that are susceptible to infection by pathogens or infected by pathogens can be targeted.In other embodiments, target nucleotides can inhibit the expression of genes involved in the production of toxins in target cells.

[0113] In other embodiments, the nucleotide of interest may encode a toxic protein that kills the cell in which it is expressed, in which case tumor cells or other unwanted cells may be targeted.

[0114] In yet another embodiment, the subject nucleotides encode a therapeutic protein.

[0115] Once a specific population of target cells in which expression of a target nucleotide is desired is identified, a target receptor that is specifically expressed on the target cell population is selected. The target receptor can be expressed only on a population of cells, or can be expressed to a greater extent on this population of cells than on other populations of cells. The more specific the expression, the more specifically delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and therefore of the gene delivery) can vary. For example, to introduce a toxic gene, high specificity is most preferred to avoid killing non-target cells. For expression of a protein for harvesting or expression of a secreted product where a global effect is desired, less marker specificity may be required.

[0116] As discussed above, the target receptor can be any receptor for which a targeting ligand can be identified or created. Preferably, the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target receptor can be a carbohydrate or other molecule that can be recognized by a binding partner. If the binding partner (e.g., a ligand) for the target receptor is already known, it can be used as an affinity molecule. However, if the binding molecule is unknown, an antibody against the target receptor can be generated using standard procedures. The antibody can then be used as a targeting ligand.

[0117] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the particular use (e.g., therapy, expression of recovered proteins, and conferring disease resistance) and (2) expression of markers with the desired amount of specificity.

[0118] Target cell is not limited in any way, and includes both germline cells and cell lines and somatic cells and cell lines.Target cell can be stem cell from any source.When target cell is germline cell, target cell is preferably selected from the group consisting of single cell embryo and embryonic stem cell (ES).

[0119] Pharmaceutical Compositions, Dosage Forms, and Administration A further embodiment provides a pharmaceutical product comprising at least one recombinant viral capsid protein and a suitable targeting ligand according to the invention, and / or a nucleic acid according to the invention. Preferably, such a pharmaceutical product is useful in gene transfer particles.

[0120] Also disclosed herein are pharmaceutical compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and / or excipient. Additionally, disclosed herein are pharmaceutical dosage forms comprising the viral particles described herein.

[0121] As discussed herein, the viral particles described herein can be used for a variety of therapeutic applications (in vivo and in vitro) and as research tools.

[0122] Pharmaceutical compositions based on the viral particles disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The viral particles can be formulated for administration, for example, by injection, inhalation, or isolation (either through the mouth or nose), or by oral, buccal, parenteral, or rectal administration, or by direct administration to a tumor.

[0123] Pharmaceutical compositions can be formulated for various modes of administration, including systemic, topical, or localized administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, pharmaceutical compositions can be formulated in a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, pharmaceutical compositions can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also suitable.

[0124] For oral administration, pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., cationized oils, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as appropriate.

[0125] Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Preparations for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers, with optional preservatives added. Pharmaceutical compositions can be formulated as suspensions, solutions, or emulsions in oily or aqueous media, and can contain other agents, including suspending agents, stabilizing agents, and / or dispersing agents.

[0126] Additionally, pharmaceutical compositions can be formulated as depot preparations. These long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of localized, non-invasive delivery of drugs over long periods of time. This technology can include microspheres with precapillary sizes that can be injected into any selected part of an organ via a coronary catheter without causing inflammation or ischemia. The administered therapeutic agent is gradually released from the microspheres and absorbed by surrounding cells present in the selected tissue.

[0127] Systemic administration can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives.In addition, detergents can be used to promote penetration.Transmucosal administration can be achieved by using intranasal sprays or suppositories.For topical administration, the viral particles described herein can be formulated into ointments, salves, gels or creams that are generally known in the art.To accelerate healing, cleaning solutions can also be used locally to treat wounds or inflammation.

[0128] Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical form must be sterile and fluid. It must also be stable under the conditions of manufacture and specific storage parameters (e.g., refrigeration and freezing), and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0129] When the formulations disclosed herein are used as therapeutic agents for promoting immune responses in subjects, the therapeutic agents can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0130] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use of agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

[0131] Sterile injectable solutions can be prepared by incorporating the active compound or construct in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0132] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solution types described above, although sustained-release capsules or microparticles and microspheres may also be used.

[0133] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration.In this context, those skilled in the art will know which sterile aqueous medium can be used in light of this disclosure.For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid, or injected into the proposed infusion site.

[0134] The person responsible for administration will in any event determine the appropriate dose for the individual subject. For example, a subject can be administered the viral particles described herein daily or weekly for a period of time, or monthly, twice a year, or yearly, depending on need or exposure to pathogenic microorganisms or the subject's condition (e.g., cancer).

[0135] In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, subcutaneous, or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration, liposomal formulations, sustained-release capsules, biodegradable forms, and any other form currently in use.

[0136] Nasal or inhalable solutions or sprays, aerosols, or inhalants may also be used. Nasal solutions can be aqueous solutions designed for administration to the nasal passages in drops or sprays. Nasal solutions can be prepared to mimic nasal secretions in many respects. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, if necessary, antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers can be included in the formulation. Various commercially available nasal preparations are known and can contain, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

[0137] Oral formulations may contain excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. In certain defined embodiments, oral pharmaceutical compositions may contain inert diluents or assimilable edible carriers, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be directly incorporated with dietary food. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0138] Tablets, troches, pills, capsules, etc. may also contain the following: binders such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint oil, cornstarch oil, or cherry flavoring. When the dosage unit form is a capsule, in addition to materials of the above type, a liquid carrier may be contained. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain sucrose as a sweetener, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavor.

[0139] Further embodiments disclosed herein may relate to kits for use with the methods and compositions.The kit may also include suitable containers, such as vials, tubes, minitubes or microtubes, test tubes, flasks, bottles, syringes, or other containers.If additional components or agents are provided, the kit may include one or more additional containers into which the agents or components can be placed.The kits herein will also typically include a means for containing the virus particles and any other reagent containers in sealed containers for commercial sale.Such containers may include injection or blow-molded plastic containers into which the desired vials are held.Optionally, the compositions described may require one or more additional active agents, such as anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antitumor agents.

[0140] The compositions disclosed herein can be administered by any means known in the art, including administration to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, injection, infusion, continuous infusion, localized perfusion, via catheter, lavage, in a cream, or in a lipid composition.

[0141] Any method known to those skilled in the art can be used for large-scale production of the viral particles, packaging cells, and particle constructs described herein. For example, master and working seed stocks can be prepared under GMP conditions in a suitable primary CEF or by other methods. Packaging cells can be plated in large-surface-area flasks and grown to near confluence, resulting in purified viral particles. The cells can be harvested and the viral particles released into the culture medium, where they are isolated and purified, or intracellular viral particles can be released by mechanical disruption (cell debris can be removed by large-pore depth filtration and endonuclease-digested host cell DNA). The viral particles can then be purified and concentrated by tangential flow filtration, followed by diafiltration. The resulting concentrated bulk can be formulated by diluting with a buffer containing a stabilizer, filled into vials, and lyophilized. Compositions and formulations can be stored for later use. For use, the lyophilized viral particles can be reconstituted by adding a diluent.

[0142] The particular additional agents used in the combination therapy can be formulated and administered by any means known in the art.

[0143] The compositions disclosed herein may also include adjuvants such as aluminum salts and other mineral adjuvants, tensin-activating agents, bacterial derivatives, vehicles, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants can stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy. [Example]

[0144] These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0145] material and method Cell lines and antibodies All 293 cell lines were maintained in DMEM supplemented with 10% FBS, 1% Pen / Strep, and 1% L-glutamine. The 293 hErbB2 and 293hASGR1 / 2 cell lines were generated by lentiviral transduction of the parental 293 cell line with vectors expressing the corresponding cDNAs. All cell lines were obtained from the Regeneron TC Core Facility. The B1 antibody recognizes a linear epitope shared by AAV VP1, VP2, and VP3.

[0146] AAV capsid protein constructs GeneBlocks encoding the desired SpyTag insert, flanking linker amino acids, and additional mutations were purchased from IDT and cloned into BsiWI- and XcmI-digested pAAV2-CAP wt or pAAV9-CAP wt using Gibson Assembly according to the manufacturer's protocol (NEB).

[0147] SpyCatcher antibody fusion GeneBlocks encoding SpyCatcher were purchased from IDT and the coding sequences were cloned in frame using Gibson Assembly into expression plasmids for scFv or antibody heavy chains at the C-terminus of each construct, separated by the flexible amino acid linker GSGESG (SEQ ID NO: 48).

[0148] Preparation of AAV viral vectors Virus was produced by transfecting 293T packaging cells using PEI Pro with the following plasmids: pAd helper, an AAV2 ITR-containing genome plasmid encoding a reporter protein, and the pAAV-CAP plasmid encoding the AAV Rep and Cap genes, with or without additional plasmids encoding either scFv or antibody heavy and light chains. The scFv and antibody heavy chain constructs were all fused to SpyCatcher at the C-terminus as described above. Transfections were performed in OptiMEM, and after 8 hours the medium was changed to DMEM supplemented with 10% FBS, 1% Pen / Strep, and 1% L-Glut.

[0149] Transfected packaging cells were incubated at 37°C for 3 days, and then virus was recovered from cell lysates using a standard freeze-thaw protocol. Briefly, packaging cells were lifted by disruption and pelleting. The supernatant was removed, and the cells were resuspended in a solution of 50 mM Tris-HCl, 150 mM NaCl, and 2 mM MgCl2 [pH 8.0]. Intracellular virus particles were released by cell lysis via three successive freeze-thaw cycles, which consisted of shuttling the cell suspension between a dry ice / ethanol bath and a 37°C water bath with vigorous agitation. Viscosity was reduced by treating the lysate with EMD Millipore Benzonase (50 U / ml cell lysate) for 60 minutes at 37°C with occasional mixing. Cell debris was then pelleted by centrifugation, and the resulting supernatant was filtered through a 0.22 μm PVDF Millex-GV filter directly into the upper chamber of an Amicon Ultra-15 centrifugal filter unit with an Ultracel-100 membrane (100 kDa MWCO) filter cartridge. The filter unit was centrifuged at 5-10 min intervals until the desired volume was reached in the upper chamber. The concentrated crude virus was then pipetted into a low-protein binding tube and stored at 4°C. Titers (viral genomes per milliliter, vg / mL) were determined by qPCR using a standard curve of known virus concentrations.

[0150] Cell infection / transduction and flow cytometry analysis To infect cells, viral particles were added directly to the medium of the cells in culture, and the mixture was incubated overnight at 37°C. The medium in each well was replaced after 24 hours, and the cells were incubated for 5 days. On day 5 post-infection, cells were trypsinized and resuspended in PBS with 2% FBS, and the percentage of GFP+ cells was collected on a BD FACSCanto flow cytometer and analyzed using FlowJo software.

[0151] Western blot analysis Reactions between SpyTag-tagged proteins VP1, VP2, and VP3 and SpyCatcher-tagged antibodies or scFvs were monitored by Western blot analysis. An equal volume of Novex® Tris-Glycine SDS sample buffer containing a reducing agent was added to the crude virus preparation, and the sample was heated to 85°C for 5 minutes, then cooled to room temperature and loaded onto a precast 4-12% Tris-Glycine gel (Invitrogen). Proteins were separated by reducing SDS-PAGE and blotted onto PVDF via wet loading. Membranes were blocked with Li-Cor Odyssey TBS blocking buffer and probed with mouse monoclonal B1 antibody (ARP American Research Products, Inc.) diluted 1:100 in TBST overnight at 4°C. Blots were washed with TBST, probed with infrared-conjugated anti-mouse secondary antibody, and imaged on the Li-Cor Odyssey.

[0152] Example 1 Conjugation of scFv to a peptide inserted into the AAV2 capsid at residue N587 directs antigen-specific targeting Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table A-1] [Table A-2]

[0153] Cells incubated with the viral particles described above were assessed by flow cytometry analysis for infection.

[0154] AAV2 carrying heparin-binding mutations (HBM) R585A and R588A and a SpyTag peptide at capsid position N587 was generated in the presence or absence of C6.5-SpyCatcher, a HER2-binding scFv, and fused to SpyCatcher at its C-terminus. AAV2 conjugated to the HER2-targeting scFv specifically infects HER2+ cells with very little background infection of HER2- cells. (Figure 1)

[0155] Example 2. Antibody Binding to a Peptide Inserted into the AAV2 Capsid at Residue N587 Directs Antigen-Specific Targeting Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table B]

[0156] Wild-type AAV2 and AAV2 carrying heparin-binding mutations (HBM) R585A and R588A were generated in the presence or absence of heavy and light chains of an antibody encoding SpyCatcher-Herceptin, an antibody that binds to HER2, and a SpyTag peptide at capsid position N587 was fused to SpyCatcher at the C-terminus of the heavy chain. Cells infected with the above-described viral particles were evaluated by flow cytometry analysis to observe transduction. AAV2 conjugated to a HER2-targeting antibody specifically infected HER2+ cells, with very little background infection of HER2- cells. (Figure 2)

[0157] Example 3. Conjugation of antibodies to a peptide inserted into the AAV2 capsid at residue G453 directs antigen-specific targeting Residues N587 and G453 are located on exposed regions of the AAV2 capsid that form protein spikes that extend away from the virion surface. Because the residues are located on two different spikes, we investigated whether a SpyTag inserted after residue G453 would function similarly to a SpyTag inserted after residue N587. Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table C]

[0158] Wild-type AAV2 and AAV2 carrying heparin-binding mutations (HBM) R585A and R588A were generated in the presence or absence of heavy and light chains of an antibody encoding SpyCatcher-Herceptin, an antibody that binds to HER2, and the SpyTag peptide at capsid position G453 was fused to SpyCatcher at the C-terminus of the heavy chain. Mosaic viruses were generated by mixing SpyTag-expressing capsids with HBM capsids. Cells infected with the above-described viral particles were evaluated by flow cytometry analysis to observe transduction. AAV2 conjugated to a HER2-targeting antibody specifically infected HER2+ cells, with very little background infection of HER2- cells. (Figure 3)

[0159] Example 4 Increasing modification of AAV virions with scFv reduces their infectivity In an attempt to optimize the efficiency of the SpyTag-SpyCatcher reaction, we improved the delivery of SpyTag to the viral surface by flanking the peptide tag with flexible linker amino acids on each side. We generated a panel of N587 SpyTag insertion mutants flanking increasing linker lengths, and used these AAV2 Rep-Cap constructs to prepare viruses fused to SpyCatcher at their C-terminus in the presence or absence of C6.5-SpyCatcher, a HER2-binding scFv. The reaction between SpyTag-tagged AAV2 proteins VP1, VP2, and VP3 and SpyCatcher-tagged C6.5 was monitored by Western blotting, and SpyTag-tagged capsid proteins reacted with SpyCatcher-tagged scFvs showed an increase in size by SDS-PAGE. Cells infected with the above-described viral particles were assessed by flow cytometry analysis to measure transduction.

[0160] Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table D]

[0161] The pAAV2-CAP N587 linker SpyTag construct contains: pAAV2-CAP N587 Linker 1 SpyTag HBM pAAV2-CAP N587 Linker 2 SpyTag HBM pAAV2-CAP N587 Linker 4 SpyTag HBM pAAV2-CAP N587 Linker 6 SpyTag HBM pAAV2-CAP N587 Linker 8 SpyTag HBM pAAV2-CAP N587 Linker 10 SpyTag HBM

[0162] When SpyTag was not flanked by linker amino acids, the VP-SpyTag-SpyCatcher-scFv complex was undetectable by Western blotting, and the virus achieved low levels of specific transduction of HER2+ cells (Figure 4). As the linker length increased (1 to 6 amino acids), the VP-SpyTag-SpyCatcher-scFv complex became detectable by Western blotting, and the viral transduction efficiency increased (Figure 4). However, when SpyTag was flanked by the two longest linkers (8 to 10 amino acids), although nearly all VP proteins reacted with SpyCatcher-Vh by Western blotting, these fully decorated viruses no longer efficiently transduced cells (Figure 4). Thus, excessive modification of AAV particles with scFv was detrimental to their ability to transduce target cells, and only a small number of conjugated scFvs was required to retarget the virus to target cells.

[0163] Example 5. Increasing antibody modification of AAV virions reduces their infectivity Using a panel of N587 SpyTag insertion mutations flanking the linker length, these AAV2 Rep-Cap constructs were used to prepare viruses in the presence or absence of antibody heavy and light chains encoding SpyCatcher-Herceptin, an antibody that binds to HER2, fused to SpyCatcher at the C-terminus of the heavy chain. Reactions between SpyTag-tagged AAV proteins VP1, VP2, and VP3 and SpyCatcher-tagged Herceptin heavy chain (Vh) were monitored by Western blotting; SpyTag-tagged capsid proteins that reacted with SpyCatcher-tagged antibodies exhibited a size shift on SDS-PAGE.

[0164] Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table E]

[0165] The pAAV2-CAP N587 linker SpyTag construct contains: pAAV2-CAP N587 SpyTag HBM pAAV2-CAP N587 Linker 1 SpyTag HBM pAAV2-CAP N587 Linker 2 SpyTag HBM pAAV2-CAP N587 Linker 4 SpyTag HBM pAAV2-CAP N587 Linker 6 SpyTag HBM pAAV2-CAP N587 Linker 8 SpyTag HBM pAAV2-CAP N587 Linker 10 SpyTag HBM

[0166] When SpyTag was flanked by no or very short linker amino acids, VP-SpyTag-SpyCatcher-Vh complexes were not detectable by Western blotting, yet the virus specifically infected HER2+ cells with efficiency approaching wild-type levels (Figure 5). Conversely, when SpyTag was flanked by longer linkers (six or more amino acids), nearly all VP proteins reacted with SpyCatcher-Vh by Western blotting, but these fully decorated viruses no longer infected (Figure 5). Increased antibody modification of AAV particles is detrimental to their ability to transduce target cells, and only a small number of conjugated antibodies is required to retarget the virus to target cells.

[0167] Example 6 Mosaicism regulates the transduction efficiency of viral particles Although the long, flexible linker allows for efficient reaction of SpyTag-tagged AAV capsids with SpyCatcher-fused scFvs, over-modification of AAV particles with scFvs is detrimental to their ability to transduce target cells. Therefore, the number of SpyTags on each virion was reduced while maintaining SpyTag delivery and efficient reactivity by producing mosaic AAV particles, which are mixtures of different ratios of highly reactive SpyTag-tagged constructs with non-SpyTag-tagged capsid constructs, all of which carry the R585A R588A heparin-binding mutation (HBM). Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table F]

[0168] The ratio between pAAV2-CAP N587 Linker10 SpyTag HBM and pAAV2-CAP R585A R588A plasmids was either 1:0 (4µg:0µg), 3:1 (3µg:1µg), 1:1 (2µg:2µg), or 1:3 (1µg:3µg) representing pure pAAV2-CAP N587 Linker10 SpyTag HBM virions in the transfection mix. The ratio between pAAV2-CAP G453 Linker10 SpyTag HBM and pAAV2-CAP R585A R588A plasmids was either 1:0 (4µg:0µg), 1:3 (1µg:3µg), or 1:7 (0.5µg:3.5µg) representing pure pAAV2-CAP G453 Linker10 SpyTag HBM virions in the transfection mix. The reaction between SpyTag-tagged AAV proteins VP1, VP2, and VP3 and SpyCatcher-tagged anti-HER scFv or SpyCatcher-tagged Herceptin heavy chain (Vh) was monitored by Western blotting. SpyTag-tagged capsid proteins reacting with SpyCatcher-tagged scFv or antibodies will show a size shift on SDS-PAGE. The reaction of the N587 SpyTag linker panel with SpyCatcher anti-HER2 scFv is shown in Figure 6. The reaction of the N587 SpyTag linker panel with SpyCatcher anti-HER2 antibody is shown in Figure 7. Cells infected with the mosaic virus particles described above were evaluated by flow cytometry analysis to measure transduction (Figures 6-7). As the amount of SpyTag capsids flanked by the highly reactive linker 10 decreased and the number of non-SpyTag capsids increased, a decrease in the amount of VP-SpyTag-SpyCatcher-scFv complex was observed by Western blotting, which was coupled with an increase in viral transduction efficiency (Figures 6-7). An inverse relationship between the number of antibodies decorating virions and transduction efficiency by retargeted viruses was demonstrated.

[0169] The reactivity of G453 SpyTag HBM and G453 Linker 10 SpyTag HBM with SpyCatcher-anti-HER2 antibody is shown in Figure 8. Both SpyTag insertions at G453, either SpyTag alone or adjacent to Linker 10, reacted very efficiently with SpyCatcher-tagged Herceptin as measured by Western blotting, suggesting that the G453 insertion site is more naturally deliverable than N587, which does not readily react unless adjacent to a linker amino acid. As observed with the N587 linker panel, when virus was heavily modified by SpyCatcher-Herceptin antibody, it no longer infected. Thus, high levels of antibody modification of AAV particles are detrimental to their ability to transduce target cells, and SpyTag inserted after G453 is more naturally deliverable than SpyTag inserted at N587.

[0170] Example 7 Specific targeting can be achieved in vitro using the SpyTag-SpyCatcher system with additional antibody-targeting pairs The ability of the SpyTag-SpyCatcher approach to retarget AAV to targets other than HER2 was investigated. SpyCatcher-tagged antibodies targeting additional cell surface proteins were cloned and the ability of these antibodies to retarget SpyTag-tagged AAV to cell types expressing these additional targets was investigated. In experiments targeting ASGR1 and CD63, each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table G]

[0171] In experiments targeting PTPRN, each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table H]

[0172] In experiments targeting ENTPD3 and CD20, each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table I]

[0173] We tested SpyCatcher-Vh and Vk plasmids encoding antibody heavy and light chains recognizing the human proteins ASGR1, CD63, PTPRN, ENTPD3, and CD20. To produce mosaic AAV particles with a small number of exposed SpyTags, SpyTag and non-SpyTag plasmids were present in the transfection mix at a 1:7 ratio, previously determined to be the ideal ratio of SpyTag to non-SpyTag capsids for AAV retargeting using antibodies. Cells expressing ASGR1, CD63, or PTPRN were infected with the mosaic AAV2 particles described above, and transduction was measured by flow cytometry. AAV2 conjugated to ASGR1-, CD63-, and PTPRN-specific antibodies were able to specifically infect cognate target cells expressing ASGR1, CD63, and PTPRN, respectively, and displayed very low background infection in the absence of antibodies. Cells expressing ENTPD3 or CD20 were infected with the AAV2 particles described above, and transduction was measured by luciferase assay using standard protocols. AAV2 conjugated to ENTPD3- and CD20-specific antibodies could specifically infect cognate target cells expressing ENTPD3 and CD20, respectively, and displayed very low background infection in the absence of antibodies. (Figure 9)

[0174] Example 8 The SpyTag-SpyCatcher system can be adapted for retargeting of AAV9 We tested the compatibility of the SpyTag-SpyCatcher system with other AAV serotypes. AAV9 is a widely used serotype that produces high-titer virus and is highly efficient at transducing mouse tissues. Residues important for receptor binding differ between AAV2 and AAV9, as AAV2 binds heparin sulfate proteoglycans and AAV9 binds galactose. Residues known to be important in receptor binding were determined from available literature (Bell, CL, Gurda, BL, Van Vliet, K., Agbandje-McKenna, M., & Wilson, JM (2012). Identification of the galactose binding domain of the adeno-associated virus serotype 9 capsid. Journal of Virology, 86(13), 7326-7333. http: / / doi.org / 10.1128 / JVI.00448-12) and include N470, D271, N272, Y446, and W503. The W503A mutation was selected as the receptor-binding mutation for use in generating mutant constructs because this single amino acid mutation significantly reduced receptor binding. Regions of the AAV9 capsid orthologous to the two protrusions (variable loops) within which AAV2 N587 and G453 reside were also identified, with the corresponding residues in AAV9 being A589 and G453. SpyTag was inserted into these two sites in AAV9, with and without flanking linker amino acids, in combination with the receptor-binding mutation W503A.

[0175] Each virus was produced as described above by transfecting one 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table J]

[0176] AAV9 wt, AAV9-CAP A589SpyT_W503A, AAV9-CAP A589 linker 10SpyT_W503A, and AAV9-CAP G453 For linker 10SpyT_W503A, 4 μg of each plasmid was used for each transfection.

[0177] For mosaic virus, 3.5 μg of pAAV9-CAP W503A and 0.5 μg of either pAAV9-CAP A589 linker10SpyT_W503A or pAAV9-CAP G453 linker10SpyT_W503A were used for each transfection to achieve a 1:7 ratio of SpyTag to non-SpyTag Rep-Cap plasmid.

[0178] Cell transduction, flow cytometry analysis, and Western blot analysis were performed as described above.

[0179] AAV9 RC A589 and G453 SpyTag insertion supported reaction with SpyCatcher-Herceptin and mediated specific transduction of HER2+ cells (Figure 10). Similar to AAV2, SpyTag inserted into AAV9 RC A589 without flanking linkers exhibited very poor delivery and poor reaction with SpyCatcher (Figure 10). Conversely, adding amino acid linkers on either side of the SpyTag allowed for more robust reactivity with SpyCatcher, and mosaic particles bearing several highly reactive SpyTags were highly efficient at transducing target cells (Figure 10).

[0180] Example 9 In vivo retargeting of SpyTag-tagged AAV particle-SpyCatcher-Vh complexes To determine whether VP-SpyTag-SpyCatcher-Vh can be retargeted to hepatocytes expressing hASGR1 in vivo, mice genetically modified to express hASGR1 on a C57BL / 6 background and control wild-type mice were intraperitoneally injected with wild-type AAV alone or VP-SpyTag-SpyCatcher-Vh viral particles (as pure or mosaic particles, with or without an amino acid linker) carrying a reporter gene, such as green fluorescent protein or firefly luciferase. Controls included mice injected with phosphate-buffered saline (PBS). To determine whether VP-SpyTag-SpyCatcher-Vh could be released from the liver and retargeted to other organs, wild-type AAV alone or VP-SpyTag-SpyCatcher-Vh viral particles (as mosaic particles) carrying a reporter gene, such as green fluorescent protein, were intravenously injected. To demonstrate detachment from the liver and retargeting to another organ, we used a gene that is not known to be expressed in the liver but in pancreatic islet cells (Syed et al. 2013, Am J Physiol Endocrinol Metab 305:E1319-E1326, 2013) and tongue from publicly available databases (GenePaint.org http: / / www.informatics.jax.org / assay / MGI:5423021 The protein ENTPD3 was chosen because it is expressed in other organs, such as the rat and the Riken FANTOM5 project (data extracted from the adult mouse dataset).

[0181] SpyCatcher-tagged antibodies targeting human CD3, human CD63, human ASGR1 (none of which are expressed in wild-type mice), or human ENTPD3 (which also recognizes the mouse protein) were cloned and tested for their ability to retarget SpyTagged AAV2 carrying either eGFP or firefly luciferase in vivo. Each virus was produced as described above by transfecting 15 cm plates of 293T packaging cells with the following plasmids and quantities: [Table K-1] [Table K-2]

[0182] SpyCatcher-Vh and Vk plasmids encoding antibody heavy and light chains recognizing the human proteins ASGR1, CD3, or CD63 were tested in mice genetically modified to express hASGR1 on a C57BL / 6 background. SpyCatcher-Vh and Vk plasmids encoding antibodies recognizing the human protein ASGR1 (as a non-targeting control) or the mouse and human protein ENTPD3 were tested in wild-type mice. Ten days after infection, the mice were sacrificed, and reporter gene expression was examined. The livers, spleens, and kidneys were fixed and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). To test animal bioluminescence, 14 days after infection, live animals were anesthetized using isoflurane and imaged 10 minutes later using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). Figures 11 and 12 show that infection with the AAV2-SpyTag-SpyCatcher-Vh complex was detected only in the livers of hASGR1-expressing mice injected with hASGR1-retargeted AAV, but not in the livers of wild-type mice that do not express hASGR1. No positive EGF was detected in other organs (data not shown).

[0183] Figure 13 shows immunohistochemical staining for eGFP expression in the liver and pancreas of wild-type mice after injection of AAV conjugated to antibodies targeting ENTPD3 or hASGR1 as a non-targeting control, since hASGR1 is not expressed in wild-type mice. Four weeks after infection, organs were harvested from infected animals, fixed in 10% neutrophil-buffered formalin for 48 hours, and then stained for eGFP via immunohistochemistry. Figure 13 shows that the AAV2-SpyTag-SpyCatcher-Vh complex was shed from the liver of wild-type mice, and all mice injected with AAV conjugated to antibodies targeting ENTPD3 and hASGR1 showed a similar loss of eGFP expression in the liver. Mice injected with AAV conjugated to antibodies targeting ENTPD3 had eGFP-expressing cells in the pancreatic islets, where ENTPD3 is thought to be expressed.

[0184] Figure 14 shows immunohistochemical staining for eGFP expression in the liver and tongue of wild-type mice after injection of AAV conjugated to antibodies targeting ENTPD3 or hASGR1 as a non-targeting control, since hASGR1 is not expressed in wild-type mice. 14 days after infection, organs were harvested from infected animals, fixed in 10% neutrophil-buffered formalin for 48 hours, and then stained for eGFP via immunohistochemistry. Figure 14 shows that the AAV2-SpyTag-SpyCatcher-Vh complex was shed from the liver of wild-type mice, and all mice injected with AAV conjugated to antibodies targeting ENTPD3 and hASGR1 showed a similar loss of eGFP expression in the liver. All three mice injected with AAV conjugated to a non-targeting, irrelevant antibody (anti-ASGR1) showed no staining in the tongue, and all three mice conjugated to anti-ENTPD3 showed eGFP-expressing cells in the tongue, where ENTPD3 is likely expressed.

[0185] Example 10: Delivery of suicide genes to cells expressing a targeting ligand We also describe the ability of the VP-SpyTag-SpyCatcher-Vh complex to specifically deliver therapeutic cargo, such as one or more suicide genes, biological therapies (e.g., antibodies), CRISPR / Cas gene editing systems, or shRNAs, to targeted cell types.

[0186] To test the ability of the VP-SpyTag-SpyCatcher-Vh complex to deliver suicide genes to specific cells, we used the HER2+ breast cancer xenograft nude mouse model described by Wang et al. ((2010) Cancer Gene Therapy 17:559-570).

[0187] VP-SpyTag-SpyCatcher-Vh complexes carrying a suicide gene (SG) are produced as described in Materials and Methods.

[0188] The cell lines BT474 breast cancer, SK-BR-3 breast cancer, and Calu-3 lung cancer are HER2-positive human tumor cell lines (Bunn PA et al., (2001) Clin Cancer Res. 7:3239-3250; Pegram M, et al. (1999) Oncogene 18:2241-2251; Spiridon CI, et al., (2002) Clin Cancer Res. 8:1720-1730). A-673 rhabdomyosarcoma and HeLa cervical carcinoma are HER2-negative human tumor cell lines, and BEAS-2b is an immortalized bronchial epithelial HER2-negative cell line (Jia LT et al. (2003) Cancer Res. 63:3257-3262; Kern JA, et al., (1993) Am J Respir Cell Mol Biol 9:448-454; Martinez-Ramirez A, et al., (2003) Cancer Genet Cytogenet. 2003;141:138-142). All of these cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in media recommended by ATCC.

[0189] Mice: Obtain female nude mice aged 6-8 weeks and house them under specific pathogen-free conditions. On day 0, mice were inoculated with (1) 10 7 BT474, SK-BR-3, Calu-3, A-673, or HeLa were injected subcutaneously into the right flank and treated intravenously with (2) intravenous VP-SpyTag-SpyCatcher-Vh complexes carrying a reporter (e.g., EGFP) or suicide gene. Untreated animals (animals injected with tumor cells alone), animals injected with wild-type AAV particles carrying a reporter or suicide gene, and animals injected with SpyTag viral particles alone served as controls. All animals were treated with the appropriate prodrug one day after injection and treatment. The size of each tumor was measured twice weekly using calipers, and tumor volume was calculated as length × width. 2 The calculation is based on the following: disease status, tumor ulceration, tumor diameter 15 mm, or tumor volume 1000 mm. 3 When the expression level reaches 100%, the mice are sacrificed and the date of sacrifice is recorded as the date of death. The livers, spleens, kidneys, and tumors of animals injected with viral particles carrying the reporter gene are fixed to visualize reporter gene expression.

[0190] While targeted delivery of suicide-inducing genes has been described (Zarogoulidis P., et al. (2013) J. Genet. Syndr. Gene Ther. 4:16849), this example describes the delivery of suicide genes to cells expressing a targeted HER2 ligand using the viral particles described herein. In additional experiments, suicide genes are delivered to other cell types expressing one or more other targeting ligands using the viral particles described herein. Illustrative, non-limiting examples of reporters suitable for targeting include reporters that mediate endocytosis of viral particles, such as calcinin embryonic antigen (CEA) (Qiu Y, et al. (2012) Cancer Lett. 316:31-38) and vascular endothelial growth factor (VEGFR) reporters (Leng A, et al. (2013) J. Genet. Syndr. Gene Ther. 4:16849). (2013) Tumor Biol. 32:1103-1111; Liu T, et al. (2011) Exp Mol Pathol. 91:745-752). Additional receptors that can be targeted include epidermal growth factor receptor (EGFR) (Heimberger AB, et al. (2009) Expert Opin Biol Ther.9:1087-1098), surface antigen classification 44 (CD44) (Heider KH, et al. (2004) Cancer Immunol Immunother. 53: 567-579), cluster of differentiation 133 (CD133 aka AC133) (Zhang SS, et al. (2012) BMC Med.; 10: 85), folate receptor (FR) (Duarte S, et al., (2011) J Control Release 149 (3): 264-72), transferrin receptor (TfR) or cluster of differentiation 71 (CD71) (Habashy HO, et al., Breast Cancer Res Treat. 119 (2): 283-93), mucin (Torres MP, et al., (2012) Curr Pharm Des. 2012; 18 (17): 2472-81), stage-specific fetal antigen 4 (SSEA-4) (Malecki M., et al., (2012) J Stem Cell Res Ther. 2(5)), and tumor resistance antigen 1-60 (TRA-1-60) (Malecki M., et al., (2013) J Stem Cell Res Ther. 3:134).

[0191] While the present invention has been particularly shown and described with reference to several embodiments, those skilled in the art will understand that changes in form and detail may be made in the various embodiments disclosed herein without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to serve as limitations on the scope of the claims.

[0192] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some preferred methods and materials are now described. All publications cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention provides, for example, the following items. (Item 1) A recombinant viral capsid protein, comprising a first member of a protein:protein binding pair operably linked to said capsid protein, optionally said first member being a peptide tag, and optionally said first member and / or a mutation reducing or abolishing the natural tropism of said capsid protein. (Item 2) 2. The recombinant viral capsid protein of claim 1, further comprising a second cognate member of the protein:protein binding pair, wherein the first and second members are covalently linked. (Item 3) 3. The recombinant viral capsid protein according to item 2, wherein the covalent bond is an isopeptide bond. (Item 4) 4. The recombinant viral capsid protein of item 2 or 3, wherein the second member is operably linked to a targeting ligand, and optionally, the targeting ligand is a binding moiety. (Item 5) 5. The recombinant viral capsid protein according to any one of items 1 to 4, wherein the first member is adjacent to a first and / or second linker that connects the first member to the capsid protein, and the first and / or second linkers are each independently at least one amino acid in length. (Item 6) 6. The recombinant viral capsid protein of item 5, wherein the first and second linkers are not identical. (Item 7) 6. The recombinant viral capsid protein of item 5, wherein the first and second linkers are identical and 10 amino acids in length. (Item 8) 8. The recombinant viral capsid protein according to any one of items 1 to 7, further comprising a mutation at an amino acid position involved in binding of the viral capsid protein to its native receptor, wherein the mutation comprises an insertion of a heterologous peptide into the capsid protein, a substitution of one or more amino acids of the capsid protein with a heterologous peptide, a deletion of one or more amino acids of the capsid protein, or a combination thereof. (Item 9) The transduction efficiency of the recombinant viral capsid protein is (i) reduced by 10%; (ii) reduced by 20%; (iii) reduced by 30%; (iv) reduced by 40%; (v) reduced by 50%; (vi) reduced by 60%; (vii) reduced by 70%; (viii) reduced by 80%; (ix) reduced by 90%; or (x) The recombinant viral capsid protein according to any one of items 1 to 8, which is disabled. (Item 10) 10. The recombinant viral capsid protein of any one of items 1 to 9, wherein the viral capsid protein is derived from a capsid gene of an adeno-associated virus (AAV), the capsid gene encoding an AAV VP1, VP2, and / or VP3 capsid protein, and optionally further comprises mutations at amino acid positions involved in capsid binding. (Item 11) 11. The recombinant viral capsid protein of item 10, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 12) 11. The recombinant viral capsid protein of item 10, wherein the adeno-associated virus is AAV2. (Item 13) 11. The recombinant viral capsid protein of item 10, wherein the adeno-associated virus is AAV9. (Item 14) the protein:protein binding pair being (i) SpyTag:SpyCatcher, (ii) SpyTag:KTag, (iii) Isopeptag: Pilin-C; (iv) SnoopTag:SnoopCatcher, or (v) The recombinant viral capsid protein according to any one of items 1 to 13, which is SpyTag002:SpyCatcher002. (Item 15) 15. The recombinant viral capsid protein of any one of items 1 to 14, wherein the first member and any linker together are about 50 amino acids or less in length. (Item 16) 16. The recombinant viral capsid protein of any one of items 1 to 15, wherein the first member is SpyTag. (Item 17) 17. The recombinant viral capsid protein according to any one of items 2 to 16, wherein the second cognate member is SpyCatcher. (Item 18) 17. The recombinant viral capsid protein of any one of items 2 to 16, wherein the second cognate member is KTag. (Item 19) 16. The recombinant viral capsid protein of any one of items 2 to 15, wherein the first member is KTag and the second cognate member is SpyTag. (Item 20) the first member is a SnoopTag and the second cognate member is a SnoopCatcher; 16. The recombinant viral capsid protein according to any one of items 2 to 15. (Item 21) 16. The recombinant viral capsid protein according to any one of items 2 to 15, wherein the first member is an isopeptag and the second cognate member is pilin-C. (Item 22) 16. The recombinant viral capsid protein according to any one of items 2 to 15, wherein the first member is SpyTag002 and the second cognate member is SpyCatcher002. (Item 23) 23. The recombinant viral capsid protein according to any one of items 1 to 22, wherein the recombinant viral capsid protein or a viral capsid comprising the recombinant viral capsid protein has reduced or no ability to infect target cells in the absence of a suitable targeting ligand, compared to the viral capsid protein or capsid comprising the suitable targeting ligand. (Item 24) 24. The recombinant viral capsid protein according to any one of items 4 to 23, wherein the binding moiety is an antibody or a portion thereof. (Item 25) 25. The recombinant viral capsid protein according to item 24, wherein the antibody or a portion thereof is fused to SpyCatcher. (Item 26) 26. The recombinant viral capsid protein of item 25, wherein the antibody or portion thereof is fused to a linker at its C-terminus, and the linker is fused to SpyCatcher at its C-terminus. (Item 27) 27. The recombinant viral capsid protein of item 26, wherein the linker comprises the sequence represented by SEQ ID NO: 48 (GSGESG). (Item 28) 28. The recombinant viral capsid protein according to any one of items 4 to 27, wherein the targeting ligand comprises the amino acid sequence represented as SEQ ID NO: 46. (Item 29) The targeting ligand specifically binds to a cell surface molecule, and optionally the cell surface marker is (i) asialoglycoprotein 1 (ASGR1), (ii) ENTPD3, (iii) PTPRN, (iv) CD20, (v) CD63, or (vi) The recombinant viral capsid protein according to any one of items 4 to 28, which is Her2. (Item 30) 30. The recombinant viral capsid protein of item 29, wherein the cell surface molecule is asialoglycoprotein 1 (ASGR1). (Item 31) 30. The recombinant viral capsid protein of item 29, wherein the cell surface molecule is CD63. (Item 32) 30. The recombinant viral capsid protein of item 29, wherein the cell surface molecule is ENTDP3. (Item 33) A recombinant viral capsid comprising the recombinant viral capsid protein according to any one of items 1 to 32. (Item 34) 34. The recombinant viral capsid of item 33, further comprising a reference viral capsid protein lacking any member of the specific binding pair. (Item 35) 35. The recombinant viral capsid of item 34, wherein the recombinant viral capsid protein and the reference viral capsid protein each contain a mutation in at least one residue involved in binding of the viral particle to its natural ligand. (Item 36) 36. The recombinant viral capsid according to item 34 or 35, comprising the recombinant viral capsid protein and the reference viral capsid protein in a ratio of 1:1 to 1:15. (Item 37) 37. A recombinant viral vector comprising a nucleotide of interest encapsulated by the recombinant viral capsid according to any one of items 33 to 36. (Item 38) 38. The recombinant viral particle of item 37, wherein the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. (Item 39) 38. The recombinant viral particle according to item 37, wherein the nucleotide of interest is under the control of a human promoter. (Item 40) 38. The recombinant viral particle of item 37, wherein the nucleotide of interest is under the control of a non-human promoter. (Item 41) 41. The recombinant viral particle according to any one of items 37 to 40, wherein the target nucleotide is a reporter gene. (Item 42) 42. The recombinant viral particle of item 41, wherein the reporter gene encodes a green fluorescent protein. (Item 43) 42. The recombinant viral particle of item 41, wherein the reporter gene encodes green fluorescent protein, β-galactosidase (encoded by the lacZ gene), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof. (Item 44) 41. The recombinant viral particle of any one of items 37 to 40, wherein the nucleotides of interest are selected from the group consisting of nucleotides encoding a therapeutic protein, a suicide gene, an antibody or fragment thereof, nucleotides encoding a CRISPR / Cas system or part(s) thereof, nucleotides encoding an antisense RNA, and nucleotides encoding an shRNA. (Item 45) A composition comprising: (a) the viral capsid according to any one of items 33 to 36, or the viral vector according to any one of items 37 to 44, and (b) a pharmaceutically acceptable carrier. (Item 46) 46. ​​A method for delivering a nucleotide of interest to a target cell, comprising contacting the target cell with the viral particle of any one of items 37 to 44 or the composition of item 45, wherein the viral capsid comprises a targeting ligand that specifically binds to a protein expressed on the surface of the target cell. (Item 47) 47. The method of claim 46, wherein the target cell is in vitro. (Item 48) 47. The method of claim 46, wherein the target cell is in vivo in a subject. (Item 49) 49. The method of claim 48, wherein the subject is a human. (Item 50) Item 51. The method according to any one of Items 46 to 49, wherein the target cells are human target cells. 51. The method of claim 50, wherein the target cells are human hepatocytes and the targeting ligand binds to the human asialoglycoprotein receptor (ASGR1). (Item 52) 51. The method of claim 50, wherein the target cell is a human neuronal cell and the targeting ligand binds to GABA. (Item 53) 51. The method of claim 50, wherein the target cell is a human T cell and the targeting ligand binds to CD3, optionally CD3ε. (Item 54) 51. The method of claim 50, wherein the targeting ligand binds to PTPRN. (Item 55) 51. The method of claim 50, wherein the target cells are human hematopoietic cells and the targeting ligand binds to CD34. (Item 56) 51. The method of claim 50, wherein the target cells are human kidney cells. (Item 57) 51. The method of claim 50, wherein the target cell is a human cancer cell and the targeting ligand binds to a tumor-associated antigen. (Item 58) 58. The method of item 57, wherein the tumor antigen is E6, E7, or Her2. (Item 59) 51. The method of claim 50, wherein the targeting ligand binds to CD20. (Item 60) 51. The method of claim 50, wherein the targeting ligand binds to the human glucagon receptor. (Item 61) 51. The method of any one of items 46 to 50, wherein the targeting ligand specifically binds to CD63. (Item 62) 51. The method of any one of items 46 to 50, wherein the targeting ligand specifically binds to human extracellular nucleoside triphosphate diphosphohydrolase 3 (hENTPD3). (Item 63) 1. A method for providing a viral capsid protein having a scaffold and / or adaptor, comprising: (a) inserting a nucleic acid encoding a first member of a specific protein:protein binding pair, and optionally a linker, into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising said first member of said specific binding pair, and optionally said linker; (b) culturing packaging cells under conditions sufficient for the production of viral particles, wherein said packaging cells contain said nucleic acid. (Item 64) A method for producing viral particles, comprising culturing packaging cells under conditions sufficient for the production of said viral particles, said packaging cells comprising a nucleotide sequence encoding a genetically modified capsid protein comprising a first member of a specific protein:protein binding pair, and optionally an amino acid linker linking said first member to said capsid protein. (Item 65) 65. The method of claim 63 or 64, wherein the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleotide of interest. (Item 66) 66. The method of any one of items 63 to 65, wherein the packaging cells further contain a plasmid encoding a reference mosaic capsid. (Item 67) 67. The method of any one of items 63 to 66, further comprising lysing the packaging cells and isolating the adeno-associated virus vector from the cell lysate. (Item 68) a. Removing cellular debris; b. treating the supernatant containing the viral particles with a nuclease; c. Concentrating the viral particles; d. purifying the viral particles; 68. The method according to any one of items 63 to 67, further comprising any combination of e a to d. (Item 69) 69. The method of any one of items 63 to 68, wherein the nucleotide sequence encoding the genetically modified capsid protein further comprises an amino acid mutation at a position responsible for the native tropism of the capsid protein, wherein the mutation comprises an insertion of a heterologous peptide into the capsid protein, a substitution of one or more amino acids of the capsid protein with a heterologous peptide, a deletion of one or more amino acids of the capsid protein, or a combination thereof. (Item 70) 70. A virus particle produced according to the method of any one of items 63 to 69. (Item 71) 33. A packaging cell for producing viral particles, comprising a plasmid encoding the recombinant viral capsid protein according to any one of items 1 to 32. (Item 72) A recombinant vector encoding the recombinant viral capsid protein according to any one of items 1 to 32.

Claims

[Claim 1] A composition or method as described in the specification.

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