Targeted Modified Recombinant Viral Particles for Targeted Delivery of Genetic Material into Human Cells and Their Use

Abstract

This invention provides a viral vector system that maintains the integrity of the modified viral structure while remaining adaptable to the targeted introduction of target nucleic acids into various target cells. [Solution] This invention describes a viral retargeting strategy that solves problems inherent in previous retargeting strategies by utilizing a first member and a second congener member of a specific binding pair, wherein the first member and the second congener member interact to form a specific chemical bond, preferably a covalent bond. When presented on a capsid protein, the first member functions as a scaffold for any target ligand fused to the second congener member, while upon binding of the first member and the second congener member, the isopeptide-bonded form and recombinant viral particle function as a one-component targeting vector.

Description

[Technical Field]

[0001] Sequence listing reference Submit as a text file via EFS Web. The sequence listing described in the file 10359WO01_ST25.txt is 183 kilobytes in size, was created on June 27, 2018, and is incorporated herein by reference.

[0002] The disclosure herein relates, in general terms, to directed modified recombinant virus particles and compositions containing them that are useful for the targeted delivery of genetic material into cells. [Background technology]

[0003] The delivery of genes to specific target cells has become one of the most important techniques in modern medicine for the potential treatment of various chronic and genetic diseases. Currently, the lack of an ideal gene delivery medium limits progress in the clinical application of gene therapy. To achieve therapeutic success, gene delivery mediums need to be able to transduce target cells while avoiding transduction of non-target cells. Specifically, if the innate targeting 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 innate targeting has been removed or reduced, and the desired targeting has been successfully manipulated. (Buchholz et al.)

[0004] In recent years, much of the progress in vector development has been achieved using non-enveloped viruses such as adeno-associated viruses (AAVs) and adenoviruses (Ad) (e.g., viruses containing a capsid formed by a viral capsid protein that does not have an envelope (e.g., a lipid bilayer)), as well as enveloped viruses such as retroviruses, lentiviruses, and herpes simplex viruses (e.g., viruses whose capsid is surrounded by a lipid bilayer). AAV vectors have been the focus of much research because they exhibit only modest immunogenicity and can transduce a wide range of species and tissues into vivo without any signs of toxicity.

[0005] AAV is a small, non-enveloped, single-stranded DNA virus. The AAV genome is 4.7 kb and is characterized by two inverted end sequences (ITRs) and two open reading frames that encode Rep and Cap proteins. The two ITRs are the only cis elements essential for AAV replication and capsid formation. 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 the 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 is VP3, and it is found in mature virions VP1, VP2, and VP3 in a relative abundance of approximately 1:1:10. In vivo, the three proteins spontaneously assemble into virion-like structures, such as viral capsids. Therefore, viral capsid formation in infected cells appears to proceed independently of viral DNA synthesis (verified by Kotin et al. (1994) Hum. Gene Ther. 5:793).

[0006] Of all known AAV serotypes, AAV2 is perhaps the best characterized serotype, as its infectious clone was the first to be constructed (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] Unlike other viral vectors, AAV is a promising vector for human gene therapy because it has not been shown to be associated with any known human disease and is generally not considered pathogenic (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Furthermore, AAV safely transduces postmittal tissue with relatively low immunogenicity, and although the virus may sometimes integrate into the host chromosome, integration occurs very frequently only when the Rep protein is supplied in trans to the safe harbor locus of human chromosome 19. The AAV genome rapidly circularizes and chains in infected cells and exists in a stable episomal state within the infected cell, providing long-term stable expression of the payload.

[0008] Some viruses, including AAV, which are infected cells via virus / ligand:cell / receptor interactions, ultimately lead to viral endocytosis by the infected cell. This ligand:receptor interaction has been the focus of much viral vector research and can be manipulated, for example, to reorient the innate targeting of viruses from cells that are naturally tolerant of infection by wild-type viruses into non-natural target cells via receptors expressed by the target cell.

[0009] Theoretically, retargeting a vector toward any cell surface protein or marker should result in infection, since most cell surface receptors are involved in the endocytosis pathway, either constitutively (e.g., for reusable use) or ligand-induced (e.g., receptor-mediated). These receptors clump together in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted, and are then sorted within the cell either for reusable use on the cell surface or degraded in lysosomes. Thus, platforms for retargeting viral vectors often aim to remove the innate targeting of the viral vector and reorient the viral vector to receptors or markers expressed on target cells alone or primarily by target cells. Many of the advances in targeted gene therapy using viral vectors can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral vectors, resulting in pseudotyping, expansion, and / or retargeting of the innate targeting of viral vectors. (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 the recombinant genetic modification of viral capsid proteins, and thus, it is on the surface of the viral capsid. On the other hand, in indirect recombinant approaches, the viral capsid is modified on a heterologous "scaffold" and then ligated to an adapter. The adapter binds to both the scaffold and the target cell. In direct recombinant targeting approaches, the targeting ligand is inserted directly into or ligated to the viral capsid, i.e., the protein viral capsid is modified to express a heterologous ligand. Next, the ligand reorients, e.g., binds to a receptor or marker that is preferentially expressed on or only on the target cell.

[0011] Each of the techniques has advantages and disadvantages. The ability to genetically modify the virus requires maintaining the structure of the capsid and placing the targeting ligand or scaffold at a position within the capsid protein that is durable for or appropriately presents the targeting ligand or scaffold. For example, the targeting ligand or scaffold introduced into the viral protein needs to meet size limitations such that it may not be able to accurately restrict the spectrum of natural molecules that the direct ligand or scaffold can be used as a target ligand or scaffold without disturbing the structure of the modified capsid. Further, the use of targeting ligands inserted directly into the viral capsid is not modular and needs to be re-engineered for each target. The scaffold platform is advantageous in the flexibility and modular nature of the adapter used, but the scaffolds on the viral particle and the adapter interact ionically and remain two separate entities, and the inherent instability of their interaction may limit their utility in vivo. In such a two-component system, optimal transduction efficiency may be difficult. Clearly, there remains a need for a viral vector system that maintains the integrity of the modified viral structure while remaining adaptable for the targeted introduction of the nucleic acid of interest to various target cells.

Prior Art Documents

[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 [Non-Patent Document 4] Gao et al. (2005) Curr. Gen. Ther. 5(3)285 - 97 [Non-Patent Document 5] Chiorini et al. (1997) J. Virol., 71:6823 - 6833 [Non-Patent Document 6] S. Muramatsu et al., (1996) Virol., 221:208 - 217 [Non-Patent Document 7] Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97 - 129 <s [Non-Patent Document 8] Nicklin and Baker(2002) Curr. Gene Ther. 2:273 - 93 [Non-Patent Document 9] Verheiji and Rottier(2012) Advances Virol 2012:1 - 15 [Summary of the Invention] [Means for Solving the Problems]

[0013] This specification describes a viral retargeting strategy that solves problems inherent in previous retargeting strategies by utilizing a first member and a second congener member of a specific binding pair, wherein the first member and the second congener member interact to form a specific chemical bond, preferably a covalent bond. When presented on a capsid protein, the first member functions as a scaffold for any target ligand fused to the second congener member, while upon binding of the first member and the second congener member, the isopeptide-bonded form and recombinant viral particle function as a one-component targeting vector.

[0014] This invention provides recombinant viral particles genetically modified to present a heterologous amino acid sequence including a specific binding pair, e.g., a recombinant viral capsid protein, a recombinant viral capsid containing the recombinant viral capsid protein, and / or a recombinant viral vector containing a recombinant viral capsid encapsulating a target nucleotide, wherein the amino acid sequence has an amino acid length of less than 50, and the recombinant viral capsid / particle protein reduces or disables innate directivity. The directivity of the recombinant viral capsid protein / capsid / vector may be restored and / or reoriented upon the formation of an isopeptide bond with a second homologous member of the specific binding pair, this second member being fused with a targeted ligand that specifically binds to target cells. Such binding results in a recombinant viral capsid protein / capsid / vector that presents a targeted ligand. When a targeted ligand is presented via a linker by a recombinant viral capsid / vector and / or on the surface of the viral capsid in a limited amount, surprisingly, the transduction efficiency and specificity are enhanced. Such viral particles, compositions containing them, and methods for producing and using them are provided herein.

[0015] Accordingly, recombinant viral capsid proteins comprising a peptide tag operably linked (e.g., covalently) to a capsid protein, wherein the viral capsid protein is derived from the 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 congener of the specific binding pair. In some embodiments, the recombinant capsid proteins described herein (which may be derived from the capsid gene of a virus that infects eukaryotic cells, e.g., 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 comprises a second congener of the specific binding pair, wherein the first and second members of the specific binding pair are linked by a covalent (isopeptide) bond, for example, the capsid protein comprises a first member operably linked to the capsid protein, and further comprises a second congener of the specific binding pair covalently linked to the first member. In some embodiments, the recombinant capsid protein described herein (which may be derived from the capsid gene of a virus that infects eukaryotic cells, e.g., a genetically modified capsid protein of a virus that infects eukaryotic cells) comprises a first member of a specific binding pair (i.e., a peptide tag) operably linked to the capsid protein, and further comprises a second congeneral member of the specific binding pair fused to a targeted ligand, wherein the first and second members of the specific binding pair are linked by a covalent (isopeptide) bond, for example, the capsid protein comprises a first member of a specific binding pair operably linked to the capsid protein, and further comprises a second congeneral member of the specific binding pair covalently linked to the first member, wherein the second congeneral member of the specific binding pair is operably linked to a targeted ligand that specifically binds to a cell surface marker (e.g., cell surface oligosaccharides, cell surface receptors, and / or cell surface markers) on a target cell.Furthermore, viral capsids containing recombinant viral capsid proteins and viral vectors containing target nucleotides encapsulated by the viral capsids described herein are also described. Compositions comprising recombinant viral particles (e.g., recombinant viral capsid proteins, recombinant viral capsids, and / or recombinant viral vectors) described herein, methods for using them for targeted delivery of target nucleotides, and methods for preparing them are also described.

[0016] In some embodiments, the peptide tag (first member of the specific binding pair) is operably linked to the capsid protein via a first or second linker, e.g., an amino acid spacer having at least one amino acid length (translated into a frame, chemically attached, and / or presented thereby). In some embodiments, the peptide tag (first member) is adjacent to the first and / or second linker, e.g., the first and / or second amino acid spacers, each of which has at least one amino acid 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 1 amino acid length or 2 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, or 3 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, or 4 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, or 5 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, or 5 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, or 6 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, or 7 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, or 8 amino acid length. In some embodiments, the first and / or second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, 8 amino acid length, or 9 amino acid length. In some embodiments, the first and second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, 8 amino acid length, or 9 amino acid length.In some embodiments, the first and second linkers are each independently 1 amino acid length, 2 amino acid length, 3 amino acid length, 4 amino acid length, 5 amino acid length, 6 amino acid length, 7 amino acid length, 8 amino acid length, or 9 amino acid length, or 10 amino acid length, or more.

[0018] In some embodiments, the first and second linkers are identical in sequence and / or length, each having a length of 1 amino acid. In some embodiments, the first and second linkers are identical in length, each having a length of 1 amino acid. In some embodiments, the first and second linkers are identical in length, each having a length of 2 amino acids. In some embodiments, the first and second linkers are identical in length, each having a length of 3 amino acids. In some embodiments, the first and second linkers are identical in length, each having a length of 4 amino acids, for example, the linker is GLSG (Sequence ID 40). In some embodiments, the first and second linkers are identical in length, each having a length of 5 amino acids. In some embodiments, the first and second linkers are identical in length, each having a length of 6 amino acids, for example, the first and second linkers each contain the sequence GLSGSG (Sequence ID 41). In some embodiments, the first and second linkers are identical in length, each having a length of 7 amino acids. In some embodiments, the first and second linkers are of the same length, each being 8 amino acids long, and for example, the first and second linkers each contain the sequence GLSGLSGS (SEQ ID NO: 42). In some embodiments, the first and second linkers are of the same length, each being 9 amino acids long. In some embodiments, the first and second linkers are of the same length, each being 10 amino acids long, and for example, the first and second linkers each contain the sequence GLSGLSGLSG (SEQ ID NO: 43) or GLSGGSGLSG (SEQ ID NO: 44). In some embodiments, the first and second linkers are of the same length, each being longer than 10 amino acids long.

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

[0020] Generally, the recombinant viral capsid proteins described herein may be derived from the capsid genes of non-enveloped viruses, for example, a cap gene modified to express a genetically modified capsid protein of a non-enveloped virus, where the non-enveloped virus infects human cells, or serotypes of non-enveloped viruses that commonly infect human cells, such as adenoviruses and adeno-associated viruses. In some embodiments, the recombinant viral capsid proteins described herein are derived from an AAV capsid gene encoding the VP1, VP2, and / or VP3 capsid proteins (or portions thereof) of AAV, and are encoded by a cap gene modified to encode a genetically modified adeno-associated virus (AAV) VP1, VP2, and / or VP3 capsid protein, for example, a genetically modified capsid protein of a human-infecting AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, 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, and are 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, recombinant viral capsid proteins are derived from AAV6 capsid genes, and are encoded by AAV6 cap genes modified to encode genetically modified AAV6 VP1, VP2, and / or VP3 capsid proteins, respectively, and the wild-type amino acid sequence of this AAV6 VP1 capsid protein is represented as Sequence ID No. 51, respectively.In some embodiments, the recombinant viral capsid protein is derived from the AAV2 capsid gene and encoded by an AAV2 cap gene modified to encode, for example, genetically modified AAV2 VP1, VP2, and / or VP3 capsid proteins, with the wild-type amino acid sequence of this AAV2 VP1 capsid protein represented as Sequence ID No. 9, respectively. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene and encoded by an AAV9 cap gene modified to encode, for example, genetically modified AAV9 VP1, VP2, and / or VP3 capsid proteins, with the wild-type amino acid sequence of this AAV9 VP1 capsid protein represented as Sequence ID No. 31, respectively.

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

[0022] In general, the recombinant viral capsid proteins described herein are modified to include a peptide tag (first member of a protein:protein binding pair) optionally linked to (e.g., inserted into and / or presented therein) the recombinant capsid protein via a linker, so that the peptide tag (first member of a protein:protein binding pair) and the optional linker themselves reduce and / or disable the innate targeting of the recombinant capsid protein or the capsid containing it compared to a reference capsid protein lacking the peptide tag (first member of a protein:protein binding pair) and the optional linker, respectively. In some embodiments, the peptide tag (first member of a protein:protein binding pair) is optionally linked to (e.g., inserted into and / or presented therein) a region of the capsid protein involved in the innate targeting of the wild-type reference capsid protein via a linker. In some embodiments, the peptide tag (first member of the protein:protein binding pair) and an optional linker are optionally operably linked to (e.g., inserted into and / or presented therein) a knob domain of the Ad fibril protein via the linker. In some embodiments, the peptide tag (first member of the protein:protein binding pair) is optionally operably linked to (e.g., inserted into and / or presented therein) an HI loop of the Ad fibril protein via the linker. In some embodiments, the peptide tag (first member of the protein:protein binding pair) is optionally operably linked to (e.g., inserted into and / or presented therein) an exposed variable loop in the AAV capsid protein via the linker.In some embodiments, the peptide tag (first member of the protein:protein binding pair) is optionally operably linked via a linker to an exposed variable loop of the AAV2 capsid protein (e.g., inserted into and / or presented therein). In some embodiments, the peptide tag (first member of the protein:protein binding pair) is optionally operably linked via a linker to an exposed variable loop of the AAV9 capsid protein (e.g., inserted into and / or presented therein).

[0023] In some embodiments, (i) the viral capsid protein is derived from the AAV2 capsid gene encoding the AAV2 VP1, VP2, and / or VP3 capsid proteins, and the peptide tag is optionally linked via a linker to an amino acid at position I453 or I587 of the AAV2 VP1 capsid protein (or the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acid of the VP1, VP2, and / or VP3 capsid proteins of different AAVs that infect humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9) (e.g., inserted therein and / or presented therein), or (ii) the viral capsid protein is derived from the AAV6 capsid gene, and the peptide tag (protein: first member of the protein-binding pair) is optionally linked via a linker to AAV6 (iii) The viral capsid protein is operably linked (e.g., inserted into and / or presented therein) to the amino acid at position I585 of the VP1 capsid protein (or the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acid of the VP1, VP2, and / or VP3 capsid proteins of different AAVs that infect humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9), or (iii) the viral capsid protein is derived from the AAV9 capsid gene encoding the AAV9 VP1, VP2, and / or VP3 capsid proteins, and the peptide tag is optionally linked via a linker to position I453 or I589 AAV9 It is manipulably ligated to (e.g., inserted into and / or presented therein) the amino acids of the VP1 capsid (or the corresponding positions of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acids of the VP1, VP2, and / or VP3 capsid proteins of different AAVs that infect humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8).

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

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

[0026] In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene and is, for example, a genetically modified AAV9 VP1, VP2, and / or VP3 capsid protein, comprising a peptide tag (first member of the protein:protein binding pair) inserted immediately after the amino acid at position 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) (e.g., fused to the C-terminus of said amino acid) and an optional linker, and further comprising the W503A mutation (or the corresponding mutation in the VP2 and / or VP3 capsid proteins encoded from the same AAV2 capsid gene). In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene and is, for example, a genetically modified AAV9 VP1, VP2, and / or VP3 capsid protein, comprising a peptide tag (first member of the protein:protein binding pair) inserted immediately after the amino acid at position 589 of the AAV9 VP1 protein (or the amino acid at the corresponding position in the AAV9 VP2 and / or VP3 capsid proteins encoded from the same AAV9 capsid gene) (e.g., fused to the C-terminus of said amino acid) and an optional linker, and further comprising the W503A mutation (or the corresponding mutation in the VP2 and / or VP3 capsid proteins encoded from 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 its biologically active portion), and the protein (second cognate member) is SpyCatcher (or its biologically active portion). In some embodiments, the peptide tag (first member) is SpyTag (or its biologically active portion), and the protein (second cognate member) is KTag (or its biologically active portion). In some embodiments, the peptide tag (first member) is KTag (or its biologically active portion), and the protein (second cognate member) is SpyTag (or its biologically active portion). In some embodiments, the peptide tag (first member) is SnoopTag (or its biologically active portion), and the protein (second congener member) is SnoopCatcher (or its biologically active portion). In some embodiments, the peptide tag (first member) is isopeptag (or its biologically active portion), and the protein (second congener member) is pyrin-C (or its biologically active portion). In some embodiments, the peptide tag (first member) is SpyTag002 (or its biologically active portion), and the protein (second congener member) is SpyCatcher002 (or its biologically active portion).

[0028] In some embodiments, the recombinant viral capsid protein includes a SpyTag. In some embodiments, the recombinant viral capsid, the recombinant viral vector containing the recombinant viral capsid, and / or the composition containing the recombinant viral capsid or viral vector includes an amino acid sequence represented as any SEQ ID NO: 13, as the amino acid sequence of the recombinant viral capsid protein. In some embodiments, the recombinant viral capsid, the viral vector containing the recombinant viral capsid, and / or the composition containing the recombinant viral capsid includes an amino acid sequence represented as SEQ ID NO: 15. In some embodiments, the recombinant viral capsid, the viral vector containing the recombinant viral capsid, and / or the composition containing the recombinant viral capsid includes an amino acid sequence represented as SEQ ID NO: 17. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 19. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 21. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 23. In some embodiments, a viral vector that is a recombinant viral capsid, including a recombinant viral capsid, a recombinant viral capsid, and / or a composition containing a recombinant viral capsid, includes an amino acid sequence represented as SEQ ID NO: 25. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 27.In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 29. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 35. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence represented as SEQ ID NO: 37. In some embodiments, a viral vector which is a recombinant viral capsid including a recombinant viral capsid, a recombinant viral capsid, and / or a composition containing a recombinant viral capsid includes an amino acid sequence represented as SEQ ID NO: 39.

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

[0030] In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a SpyTag covalently bound to a SpyCatcher fused to a targeted ligand. In some embodiments, recombinant viral capsids, recombinant viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a recombinant viral capsid protein and an amino acid sequence represented as any of the sequence numbers listed in Table 1 that encodes a polypeptide adapter representing the amino acid sequence represented as Sequence ID No. 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter representing the amino acid sequence represented as Sequence ID No. 13 and the amino acid sequence represented as Sequence ID No. 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter representing the amino acid sequence represented as Sequence ID No. 15 and the amino acid sequence represented as Sequence ID No. 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 17 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 19 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors, and compositions containing recombinant viral capsids and / or recombinant viral capsid products include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 21 and an amino acid sequence represented as SEQ ID NO: 46.In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 23 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 25 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 27 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 29 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 35 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors, and compositions containing recombinant viral capsid products include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 37 and an amino acid sequence represented as SEQ ID NO: 46. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include a polypeptide adapter comprising an amino acid sequence represented as SEQ ID NO: 39 and an amino acid sequence represented as SEQ ID NO: 46.

[0031] Generally, targeted ligands specifically bind to cell surface molecules, such as oligosaccharides, receptors, and cell surface markers, expressed on the surface of mammalian (e.g., human) eukaryotic cells, such as target cells. In some embodiments, targeted ligands bind to (human) hepatocytes, (human) brain cells, (human) T cells, (human) kidney cells, (human) intestinal cells, (human) lung cells, (human) cancer cells, or (human) cells infected with a heterologous pathogen.

[0032] In some embodiments, the targeted ligand binds to (human) hepatocytes, for example, to receptors expressed by asialoglycoprotein receptors, such as hASGR1. In some embodiments, the targeted ligand binds to molecules expressed by (human) nerve cells, for example, GABA, transferrin, etc. In some embodiments, the targeted ligand binds to molecules expressed by (human) T cells, for example, CD3, for example, CD3ε. In some embodiments, the targeted ligand binds to CD63. In some embodiments, the targeted ligand binds to molecules expressed by (human) hematopoietic stem cells, for example, CD34. In some embodiments, the targeted ligand binds to molecules expressed by (human) kidney cells. In some embodiments, the targeted ligand binds to molecules expressed by (human) muscle cells, for example, integrins. In some embodiments, the r-targeting ligand is a molecule expressed by (human) cancer cells, e.g., tumor-associated antigens, e.g., adipophyllin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetal protein ("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 carboxyesterase,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 acid enzyme, mammoglobin-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, pleomorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, Cesernin 1, 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, triose phosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptor, Melan A [MART1], gp 100, tyrosinase, TRP-1 / gp 75, and TRP-2 (in melanoma), MAGE-1 and MAGE-3 (in bladder cancer, head and neck cancer, and non-small cell carcinoma), HPV EG and E7 proteins (in cervical cancer), mucin [MUC-1] (in breast cancer, pancreatic cancer, colon cancer, and prostate cancer),It binds to prostate-specific antigen [PSA] (in prostate cancer), carcinoembryonic antigen [CEA] (in colorectal cancer, breast cancer, and gastrointestinal cancer), as well as common tumor-specific antigens such as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1, 2, 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 the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds to human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

[0033] In general, viral capsids containing recombinant viral capsid proteins described herein cannot infect target cells in the absence of a targeting ligand, for example, a second member operably linked to the target ligand. Generally, in the absence of a suitable targeting ligand, viral capsids containing recombinant viral capsid proteins described herein have reduced or neutralized innate targeting, reducing or neutralizing their ability to target or bind to reference cells that are naturally tolerant of transduction, compared to transduction of, for example, a reference viral capsid, for example, a capsid containing a reference viral capsid protein, for example, 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 / or protein:protein binding pairs. In some embodiments, the transduction efficiency of recombinant viral capsid proteins containing SpyTag is reduced or neutralized compared to a control wild-type viral capsid protein.

[0034] In some embodiments, when a suitable targeting ligand is absent, a viral capsid containing, for example, the recombinant viral capsid proteins listed herein as listed in Table 1, exhibits at least a 10% reduction in transduction efficiency compared to, for example, a suitable control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is absent, a viral capsid containing, for example, the recombinant viral capsid proteins listed herein as listed in Table 1, exhibits at least a 20% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is absent, a viral capsid containing, for example, the recombinant viral capsid proteins listed herein as listed in Table 1, exhibits at least a 30% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 40% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 50% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 60% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, viral capsids comprising, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibit at least a 70% reduction in transduction efficiency compared to, for example, the control wild-type viral capsids listed in Table 1.In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 75% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least an 80% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least an 85% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 90% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 95% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, when a suitable targeting ligand is not present, a viral capsid containing, for example, the recombinant viral capsid proteins listed in Table 1, as described herein, exhibits at least a 99% reduction in transduction efficiency compared to, for example, a control wild-type viral capsid listed in Table 1. In some embodiments, if a suitable targeting ligand is not present, the transduction of control cells by a viral capsid containing the recombinant viral capsid protein described herein is rendered ineffective, for example, by a method that measures the expression of a target nucleotide, such as a reporter assay, or is otherwise undetectable.

[0035] Conversely, viral capsids containing recombinant viral capsid proteins with peptide tags covalently bound to a suitable adapter polypeptide, such as a congener protein covalently bound to a targeting ligand, can infect target cells and, compared to transduction of a capsid containing, for example, a reference viral capsid protein, such as a wild-type control capsid protein, have a partially or completely restored ability to target and bind to reference cells that are naturally tolerant of transduction. In some embodiments, viral capsids containing recombinant viral capsid proteins listed herein, such as those listed in Table 1, covalently bound to a suitable adapter polypeptide, exhibit a transduction efficiency that is, for example, at least 10% of the transduction efficiency of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, viral capsids containing recombinant viral capsid proteins listed herein, such as those listed in Table 1, covalently bound to a suitable adapter polypeptide, exhibit a transduction efficiency that is, for example, at least 20% of the transduction efficiency of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, at least 30% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, at least 40% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, at least 50% of a suitable control wild-type viral capsid listed in Table 1.In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 60% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 70% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 75% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, at least 80% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, at least 85% of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, at least 90% of a suitable control wild-type viral capsid listed in Table 1.In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency that is, for example, at least 95% of the transduction efficiency of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency that is, for example, at least 99% of the transduction efficiency of a suitable control wild-type viral capsid listed in Table 1. In some embodiments, a viral capsid containing a recombinant viral capsid protein as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits the same transduction efficiency as, for example, a suitable control wild-type viral capsid covalently bonded in Table 1.

[0036] Similarly, recombinant viral capsid proteins containing peptide tags covalently linked to a suitable adapter polypeptide, for example, viral capsids containing a syngeneic protein operably linked to a targeting ligand, have an enhanced ability to target and bind to reference cells that are naturally tolerant of transduction, compared to transduction of a reference viral capsid that is identical to a recombinant viral capsid protein except that it contains a reference capsid protein, for example, lacking one or both of a protein:protein binding pair, and can infect target cells. In some embodiments, viral capsids containing recombinant viral capsid proteins covalently linked to a suitable adapter polypeptide, as described herein, for example, listed in Table 1, exhibit a transduction efficiency 10% greater than, for example, the transduction efficiency of a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency 20% greater than, for example, a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency 30% greater than, for example, a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency 40% greater than, for example, a suitable control reference viral capsid listed in Table 1. In some embodiments, viral capsids containing recombinant viral capsid proteins, such as those listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibit a transduction efficiency 50% greater than, for example, that of a suitable control reference viral capsid, also listed in Table 1.In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 60% greater than that of a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 70% greater than that of a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 75% greater than that of a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 80% greater than that of a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 85% greater than that of a suitable control reference viral capsid listed in Table 1. In some embodiments, a viral capsid containing recombinant viral capsid proteins as described herein, for example, listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibits a transduction efficiency of, for example, 90% greater than that of a suitable control reference viral capsid listed in Table 1. In some embodiments, viral capsids containing recombinant viral capsid proteins, such as those listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibit a transduction efficiency of, for example, 95% greater than that of suitable control reference viral capsids, also listed in Table 1.In some embodiments, viral capsids containing recombinant viral capsid proteins, such as those listed in Table 1, covalently bonded to a suitable adapter polypeptide, exhibit a transduction efficiency 99% greater than, for example, that of a suitable control reference viral capsid, also listed in Table 1.

[0037] In some embodiments, the viral capsid comprising the recombinant viral capsid protein described herein is a mosaic capsid and comprises, for example, at least two sets of VP1, VP2, and / or VP3 proteins, each set thereof encoded by a different cap gene, for example, a peptide tag, and a recombinant viral capsid protein comprising a reference capsid protein that does not contain the peptide tag in a particular ratio. In some embodiments, the reference capsid protein is a wild-type reference capsid protein in that it contains 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 contains the amino acid sequence of a recombinant viral capsid protein, except that the control reference capsid protein lacks a 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, due to mutations that reduce the targeting of the wild-type capsid protein (e.g., amino acid sequence deletion, amino acid sequence insertion, chimerization, etc.). In some embodiments, the compositions described herein contain 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 recombinant viral capsid proteins listed in Table 1 and their appropriate reference capsid proteins (or combinations thereof) 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 recombinant viral capsid proteins listed in Table 1 and their appropriate reference capsid proteins (or combinations thereof) 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 (1) exemplary and unrestrictive recombinant viral capsid proteins containing the peptide tags described herein, (2) corresponding control (C) wild-type viral capsid proteins of exemplary and unrestrictive examples that can be optionally used as references to determine the reduction or inactivation of the transduction efficiency of recombinant capsid proteins containing covalent protein tags in the absence of a targeted vector, and (3) sequence identification numbers (SEQ ID NOs) expressed as amino acid sequences of exemplary and unrestrictive examples of corresponding reference viral capsid proteins for generating mosaic capsids, as well as their use as references to determine the restoration of the transduction efficiency of recombinant capsid proteins containing protein:protein binders and targeted ligands. [Table 1-1] [Table 1-2]

[0040] Generally, the recombinant viral vectors described herein include a viral capsid containing a mosaic viral capsid, which contains the recombinant viral capsid protein described herein, and the viral capsid encapsulates the target nucleotide. In some embodiments, the target nucleotide is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, a bird promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the target nucleotide 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 target nucleotide can be one or more genes that may encode a detectable marker, such as a reporter, or a therapeutic polypeptide. In some embodiments, the target nucleotide is a reporter gene. In some embodiments, the target nucleotide is a reporter gene encoding a detectable marker selected from the group consisting of green fluorescent protein, luciferase, β-galactosidase, etc. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the target nucleotide is selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or a fragment thereof, a nucleotide encoding the CRISPR / Cas system or a part(s) thereof, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, a secretory enzyme, a gene encoding a therapeutic protein, etc. In one embodiment, the target nucleotide encodes a protein having at least two domains that provide two distinct functions, for example.

[0042] The compositions described herein generally comprise a viral vector containing the recombinant viral capsid protein described herein, for example, a capsid containing the recombinant viral capsid protein (e.g., a mosaic capsid), the capsid encapsulating the target nucleotide. In some embodiments, the compositions described herein comprise (1) a viral vector having a capsid containing the recombinant viral capsid protein described herein, and (2) a pharmaceutically acceptable carrier.

[0043] Methods for preparing and using recombinant viral capsid proteins, viral vectors containing them, compositions, etc. are also described herein. In some embodiments, methods including reorienting viruses, such as adenoviruses and adeno-associated viruses, and delivering diagnostic / therapeutic cargo to target cells, include contacting a recombinant viral vector containing the recombinant viral capsid protein described herein with target cells (in vitro or in vivo, which may be in humans), wherein the viral capsid or viral vector contains a targeted ligand that specifically binds to a protein expressed on the surface of the target cells. Such methods may include, as a first step, generating a recombinant viral vector, for example, culturing packaging cells under conditions sufficient for generating a viral vector, wherein the packaging cells contain a plasmid encoding a capsid protein including a peptide tag (first member) in the absence or presence of a plasmid encoding a reference capsid protein, and incubating the recombinant capsid protein with a second homogeneous member operably linked to the targeted ligand. In some embodiments, the target cell is a (human) hepatocyte, and the (mosaic) recombinant viral vector contains a targeted ligand that specifically binds to an asialoglycoprotein receptor, such as (h)ASGR1. In some embodiments, the target cell is a (human) neuron, and the (mosaic) recombinant viral vector contains a targeted ligand that specifically binds to GABA, a transferrin receptor, etc. In some embodiments, the target cell is a (human) T cell, and the (mosaic) recombinant viral vector contains a targeted ligand that specifically binds to CD3, such as CD3ε. In some embodiments, the target cell is a (human) hematopoietic stem cell, and the (mosaic) recombinant viral vector contains a targeted 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 contains a targeted ligand that specifically binds to an integrin.In some embodiments, the target cells are (human) cancer cells, and the (mosaic) recombinant viral vector contains a targeted ligand that specifically binds to tumor-associated antigens, such as E6 and E7, Her2, etc. In some embodiments, the targeted ligand binds to the human glucagon receptor (hGCGR).

[0044] The present invention also describes a method for inactivating a viral capsid and / or generating a viral vector, which generally comprises (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 (in the absence or presence of a plasmid encoding a reference capsid protein) containing a peptide tag, and / or (b) culturing packaging cells under conditions sufficient for generating a viral vector, wherein the packaging cells contain a nucleotide sequence. In some embodiments, the packaging cells further comprise a helper plasmid and / or a transplasmid containing the target nucleotide. In some embodiments, the method further comprises isolating a 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 cell fragments, (b) processing the supernatant containing a viral vector having a nuclease, such as DNase I, in the presence of MgCl2, (c) concentrating the viral vector, (d) purifying the viral vector, and (e) any combination of (a) to (d). The method also provides herein viral vectors prepared according to the method described herein, and packaging cells useful for generating the viral vectors described herein, such as packaging cells containing plasmids encoding recombinant capsid proteins.

[0045] The patent or application documents shall include at least one drawing prepared in color. Copies of the published patent or patent application, accompanied by any color drawing(s), shall be provided to the authorities upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0046] [Figure 1] We provide scatter plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescent protein (GFP) expression in 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). The C6.5-SpyC particles were conjugated with anti-HER2 scFv (C6.5) fused to a SpyCatcher (SEQ ID NO: 3) via SpyTag. The viruses express GFP as a marker of transformation. [Figure 2] We provide scatter plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescent protein (GFP) expression in either "uninfected" HER2-positive (+) 293 hErbB2 or HER2-negative (-) 293 parental cells, or cells infected with "AAV2 N587-SpyTag" particles or cells infected with "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 viruses express GFP as a marker of transformation. [Figure 3]This document provides scatter plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescent protein (GFP) expression in HER2-positive (+) 293 hErbB2 or HER2-negative (-) 293 parental cells infected with "AAV2 wild-type" particles, or in cells infected with "AAV2 G453-SpyTag+SpyC-anti-HER2" particles. The "AAV2 wild-type" capsid is unmutated or modified (SEQ ID NO: 9), and the "AAV2 G453-SpyTag" capsid is a mosaic virus particle consisting of a 1:7 ratio of the "SpyTag" capsid protein inserted immediately after residue G453 adjacent to the 10-amino acid linker (SEQ ID NO: 29) and the "AAV2 HBM" capsid, which lacks SpyTag but contains mutations R585A and R588A (SEQ ID NO: 11). AAV2 G453-SpyTag mosaic particles were conjugated to an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) via the SpyTag. The virus expresses GFP as a marker of transformation. [Figure 4A] This Western blot analyzes the reaction between anti-HER2 scFv fused to SpyCatcher "SpyC-anti-Her2 scFv" and a panel of AAV2 particles consisting of capsids with the following mutations: R585A, delR588, and insertion of the SpyTag peptide immediately after residue N587 adjacent to amino acid linkers of varying 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]This provides the percentage of GFP-expressing HER2+ cells (gray, y-axis) versus GFP-expressing HER2- cells (black, y-axis) 5 days after infection with AAV2 virus particles consisting of capsids with the following mutations: R585A, delR588, and N587-SpyTag adjacent to amino acid linkers of indicated lengths (linker 1, linker 2, linker 4, linker 6, linker 8, and linker 10) (sequence numbers 13, 15, 17, 19, 21, 23, and 25, respectively) (x-axis). AAV2 N587-SpyTag particles were conjugated to anti-HER2 scFv fused to a SpyCatcher (sequence number 3). [Figure 5A] This Western blot analyzes the reaction between SpyCatcher "SpyC-anti-Her2 antibody fused to anti-HER2 antibody (HERCEPTIN®)" and a panel of AAV2 virus particles consisting 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, 25, respectively). [Figure 5B] This provides the percentage of HER2+ cells expressing GFP (gray, y-axis) versus the percentage of HER2- cells expressing GFP (black, y-axis) 5 days after infection with AAV2 virus particles consisting of wild-type (wt) AAV2 particles or capsids having the following mutations: R585A, delR588, and N587-SpyTag adjacent to amino acid linkers of indicated lengths (no linker, linker 1, linker 2, linker 4, linker 6, linker 8, and linker 10) (sequence numbers 13, 15, 17, 19, 21, 23, and 25, respectively) (x-axis). AAV2 N587 SpyTag particles were conjugated to an anti-HER2 antibody (HERCEPTIN®) fused to a SpyCatcher (sequence number 3). [Figure 6A] This Western blot analyzes the reaction between anti-HER2 scFv C6.5 fused to SpyCatcher (SEQ ID NO: 3) "SpyC-anti-HER2 scFv" and a panel of mosaic AAV2 virus particles using a B1 antibody that recognizes a linear epitope shared by AAV2 VP1, VP2, and VP3 capsid proteins, where the AAV2 virus particles consist of a mixture between "SpyTag" capsid proteins containing mutants R585A, delR588, and N587-linker10-SpyTag (SEQ ID NO: 25) and "HBM" capsid proteins containing mutants R585A and R588A but not 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] This document provides percentages of HER2+293 hErbB2 cells (gray bars) and GFP-expressing HER2-293 parental cells (black bars) (y-axis) 5 days after infection with mosaic AAV2 virus particles (x-axis) composed of a mixture of "Linker 10" capsid protein containing mutant R585A, delR588, and N587-Linker 10-SpyTag (SEQ ID NO: 25) and "HBM" capsid protein containing mutant R585A and R588A but without SpyTag (SEQ ID NO: 11). The "Linker 10" and "HBM" capsid proteins were mixed in various ratios (1:0, 3:1, 1:1, and 1:3) and conjugated to anti-HER2 scFv fused to SpyCatcher (SEQ ID NO: 3). [Figure 7A]This document provides Western blots analyzing the reaction between a panel of mosaic AAV2 virus particles and an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) “SpyC-anti-HER2 antibody,” using a B1 antibody that recognizes linear epitopes shared by AAV2 VP1, VP2, and VP3 capsid proteins, where the AAV2 virus particles consist of a mixture of “SpyTag” capsid proteins containing mutant R585A, delR588, and N587-linker10-SpyTag (SEQ ID NO: 25) and “HBM” capsid proteins containing mutant R585A and R588A but not SpyTag (SEQ ID NO: 11). The “SpyTag” and “HBM” capsid proteins were mixed in various ratios (1:0, 3:1, 1:1, and 1:3). [Figure 7B] This report provides percentages of HER2+293 hErbB2 cells (gray bars) and GFP-expressing HER2-293 parental cells (black bars) (y-axis) 5 days after infection with mosaic AAV2 virus particles (x-axis) composed of a mixture of "Linker 10" capsid protein containing mutant R585A, delR588, and N587-Linker 10-SpyTag (SEQ ID NO: 25) and "HBM" capsid protein containing mutant R585A and R588A but without SpyTag (SEQ ID NO: 11). The "Linker 10" and "HBM" capsid proteins were mixed in various ratios (1:0, 3:1, 1:1, and 1:3) and conjugated to anti-HER2 antibodies (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3). [Figure 8A]This document provides a Western blot analyzing the reaction between a panel of mosaic AAV2 virus particles and an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) “SpyC-anti-HER2 antibody,” using a B1 antibody that recognizes linear epitopes shared by AAV2 VP1, VP2, and VP3 capsid proteins, where the AAV2 virus particles consist of a mixture of “SpyTag” capsid protein and “HBM” capsid protein containing mutants R585A and R588A but not SpyTag (SEQ ID NO: 11). The "SpyTag" capsid protein includes "G453 SpyTag," which contains the insertion of the SpyTag peptide immediately following mutants R585A, R588A, and residue G453 (SEQ ID NO: 27), and "G453 Linker 10 SpyTag," which contains the insertion of the SpyTag peptide adjacent to either side by 10 linker amino acids (SEQ ID NO: 29) immediately following mutants R585A, R588A, and residue G453. 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 data provides percentages 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 virus particles (x axis), comprising a mixture of the following: a "G453 SpyTag" capsid protein containing mutations R585A, R588A, and an insertion of a SpyTag peptide immediately after residue G453 (SEQ ID NO: 27); a "G453 linker10SpyTag" capsid protein containing mutations R585A, R588A, and an insertion of a SpyTag peptide immediately after residue G453 and adjacent on either side by 10 linker amino acids (SEQ ID NO: 29); and an "HBM" capsid protein containing mutations R585A and R588A but not containing SpyTag (SEQ ID NO: 11). "G453 SpyTag" or "G453 Linker 10 SpyTag" and "HBM" capsid 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]This document provides scatter plots obtained from fluorescence-activated cell sorting (FACS) evaluating cell-positive (+) green fluorescent protein (GFP) expression for ASGR1 expression after infection of "AAV2 wt" particles, "AAV2 SpyTag antibody-free" particles, or "AAV2 SpyTag anti-ASGR1" particles. The "AAV2 wt" capsid is wild-type without 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. The report also shows scatter plots obtained from fluorescence-activated cell sorting (FACS) evaluating cell-positive (+) green fluorescent protein (GFP) expression for CD63 expression after infection in "AAV2 wt" particles, "AAV2 SpyTag antibody-free" 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. The report also shows scatter plots obtained from fluorescence-activated cell sorting (FACS) evaluating cell-positive (+) green fluorescent protein (GFP) expression for PTPRN expression after infection with "AAV9 wt" particles, "AAV2 SpyTag unrelated antibody" particles, or "AAV2 SpyTag anti-PTPRN" particles.The "AAV9 wt" capsid is wild-type (SEQ ID NO: 31) without mutations or modifications, and the "AAV2 SpyTag" capsid is a mosaic virus particle composed of "SpyTag" capsid proteins in which the SpyTag is inserted immediately after residue G453 adjacent to the 10-amino acid linker (SEQ ID NO: 29), and capsids containing a Myc tag amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 53), which does not have a SpyTag but reduces innate receptor binding, in a 1:7 ratio. "AAV2 SpyTag unrelated antibody" particles were conjugated to a SpyCatcher fusion antibody that does not bind to PTPRN. "AAV2 SpyTag anti-PTPRN" particles were conjugated to a SpyCatcher fusion antibody that specifically binds to PTPRN. The virus expresses GFP as a marker of transformation. The results of luciferase assays evaluating cell-positive (+) firefly luciferase expression against hENTPD3 after infection with "AAV2 wt" particles, "AAV2+ unrelated mAb" particles, "AAV2+ anti-ENTPD3" particles, and "AAV2+ anti-hCD20" particles are also shown. 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 the SpyTag peptide immediately after residue N587 (SEQ ID NO: 13). "AAV2+ unrelated mAb" particles were conjugated to a SpyCatcher fusion antibody that does not bind to hENTPD3. "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 a further negative control. "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. Results of luciferase assays evaluating firefly luciferase expression by cell positivity (+) to hCD20 after infection with "AAV2 wt" particles, "AAV2 + unrelated mAb" particles, "AAV2 + anti-ENTPD3" particles, and "AAV2 + anti-hCD20" particles are also shown.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 the SpyTag peptide immediately following residue N587 (SEQ ID NO: 13). "AAV2+ unrelated 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 a further 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] This document provides a Western blot analyzing the reaction between a panel of AAV9 virus particles and an anti-HER2 antibody (HERCEPTIN®) fused to SpyCatcher (SEQ ID NO: 3) "SpyC-Herceptin," using a B1 antibody that recognizes a linear epitope shared by AAV9 VP1, VP2, and VP3 capsid proteins. These AAV9 virus particles consist of capsids containing a mutant W503A that reduces receptor binding, and either no linker or an insertion of a SpyTag peptide immediately after residue A589 or G453 adjacent to a 10-amino acid linker, or mosaic AAV9 virus particles composed of a "SpyTag" capsid protein containing the mutant W503A and either A589-linker10-SpyTag or G453-linker10-SpyTag in a 1:7 ratio, where the "W503A" capsid protein contains the mutant W503A but lacks SpyTag. [Figure 10B]The graph provides percentages of HER2+293 hErbB2 cells (gray bars) and HER2-293 parental cells (black bars) (y-axis) expressing GFP 5 days after infection in AAV9 virus particles conjugated to an anti-HER2 antibody (HERCEPTIN®) fused to a SpyCatcher (x-axis). These AAV9 virus particles consist of a capsid containing the mutant W503A, which reduces receptor binding, and either lacking a linker or containing an insertion of a SpyTag peptide immediately after residue A589 (or G453) adjacent to a 10-amino acid linker, or a mosaic AAV9 virus particle composed of a "SpyTag" capsid protein containing the mutant W503A and either A589-linker10-SpyTag or G453-linker10-SpyTag in a 1:7 ratio, where the "W503A" capsid protein contains the mutant W503A but lacks SpyTag. The virus expresses GFP as a marker of transformation. [Figure 11]This document provides immunofluorescence microscopy images of liver samples collected from C57BL / 6 mice genetically modified to express human ASGR1 on hepatocytes. Samples were collected 10 days after intravenous injection of 2.5 x 10¹¹ viral genomes (vg) / animals of SpyTag-labeled AAV2 particles loaded with the target CAGG eGFP nucleotide and modified with (1) SpyCatcher-anti-human CD3 antibody (AAV SpyT-anti-hCD3 CAGG eGFP) or (2) SpyCatcher-anti-human ASGR1 antibody (AAV SpyT-antibody-hASGR1 CAGG eGFP). Mice were sacrificed with 4% PFA and perfused by genetic modification. Liver, kidney, and heart organs were collected, dehydrated in 15% sucrose, and then dehydrated in 30% sucrose. Next, organs were frozen-sectioned on 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 "SpyTag-attached AAV2" 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]The following provides fluorescence images of individual mice (controls) that do not express human ASGR1 on hepatocytes and genetically modified mice that express human ASGR1 on hepatocytes (ASGR1 humanized mice) 14 days after intravenous injection of wild-type (wt) AAV2 particles or 3.0 x 10¹¹ viral genomes (vg) / animals of wild-type (wt) AAV2 particles or SpyTag-attached AAV2 particles that are loaded with the target firefly luciferase nucleotide and modified with (1) SpyCatcher-anti-human CD63 antibody or (2) SpyCatcher-anti-human ASGR1 antibody. These "AAV2" virus particles are mosaic virus particles composed in a 1:7 ratio between "SpyTag" capsid proteins, where the SpyTag is inserted immediately after residue G453 adjacent to the 10-amino acid linker (SEQ ID NO: 29), and between capsids containing a Myc-tagged amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 53), which does not have a SpyTag but reduces innate receptor binding. The virus expresses firefly luciferase as a marker of transduction. Mice were anesthetized with isoflurane, injected with luciferin substrate, and imaged 10 minutes later using the IVIS spectral in vivo imaging system (PerkinElmer). [Figure 13]This document provides immunohistochemical images of liver and pancreatic specimens taken from C57BL / 6 mice four weeks after intravenous injection of phosphate-buffered saline (PBS) or 1.0 x 10¹² viral genome (vg) / animal of wild-type (wt) AAV9 particles, or 1.0 x 10¹³ viral genome (vg) / animal of SpyTag-supported AAV2 particles loaded with the target CMV eGFP nucleotide and modified by (1) SpyCatcher-anti-human ASGR1 antibody (AAV2 SpyTag + unrelated mAb) or (2) SpyCatcher-anti-human ENTPD3 antibody (AAV2 SpyTag + anti-ENTPD3). Mice were sacrificed, and the livers and pancreases were collected and fixed in 10% neutral buffered formalin. The organs were then embedded on slides, frozen-sectioned, and stained with anti-GFP antibody. Each image represents one mouse. These "AAV2" virus particles are mosaic virus particles composed of a 1:7 ratio between "SpyTag" capsid proteins in which the SpyTag is inserted immediately after residue G453, adjacent to the 10-amino acid linker (SEQ ID NO: 29), and between capsids containing a Myc-tagged amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 53), which does not have a SpyTag but reduces innate 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-equipped AAV2 particles were detached from the liver, and no eGFP expression was observed in the livers of mice injected with "AAV2 SpyTag + unrelated mAb" or "AAV2 SpyTag + anti-ENTPD3" particles. Positive staining was detected in the islets in a pancreatic sample from one of the mice injected with "AAV2 SpyTag + anti-ENTPD3" particles. [Figure 14]This paper provides immunohistochemical images of liver and tongue specimens taken from C57BL / 6 mice 14 days after intravenous injection of SpyTag-equipped AAV2 particles loaded with phosphate-buffered saline (PBS), or 2.0 x 10¹² viral genome (vg) / animal wild-type (wt) AAV9 particles, or the target CMV eGFP nucleic acid, and modified with (1) SpyCatcher-anti-human ASGR1 antibody (AAV2 SpyTag + unrelated mAb) or (2) SpyCatcher-anti-human ENTPD3 antibody conjugated 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, frozen-sectioned, and stained with anti-GFP antibody. Each image represents one mouse, and three mice were injected and analyzed from the "AAV2 SpyTag + unrelated mAb" and "AAV2 SpyTag + anti-ENTPD3" groups, all showing similar GFP expression patterns. These "AAV2" virus particles are mosaic virus particles composed of a 1:7 ratio between "SpyTag" capsid proteins where the SpyTag is inserted immediately after residue G453 adjacent to the 10-amino acid linker (SEQ ID NO: 29), and between capsids containing a Myc-tagged amino acid sequence inserted immediately after residue N587 (SEQ ID NO: 52), which does not have the SpyTag but reduces innate receptor binding. The virus expresses eGFP as a marker of transduction. ENTPD3 is expressed in the tongue of mice 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 livers of mice injected with "AAV2 SpyTag + unrelated mAb" or "AAV2 SpyTag + anti-ENTPD3" particles, but it was detected in the tongues of all three mice injected with "AAV2 SpyTag + anti-ENTPD3" particles. [Modes for carrying out the invention]

[0047] WO2016 / 11291 describes the use of a specific binding pair (SpyCatcher:SpyTag) for producing virus-like particles (VLPs) derived from modified bacteriophage AP205 that present immunogenic antigens at high density on the AP205 capsid for vaccination purposes. Theoretically, such a high degree of modification of the viral capsid may be desirable to ensure that the innate targeting of the viral vector is substantially reduced or neutralized for the purpose of retargeting the viral vector. However, such high-density presentation of the targeting ligand on the viral surface may impair transduction efficiency. See Examples 4 and 5. It has been found that optimal transduction efficiency is achieved when the degree of modification of the viral surface using the member is reduced. Thus, genetically modified viral particles, compositions containing them, and methods for producing and using them are provided herein.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0049] The singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated by the context. Therefore, for example, a reference to “method” includes one or more methods and / or processes of the type described herein and / or which would be apparent to those skilled in the art upon reading this disclosure.

[0050] The term "antibody" includes immunoglobulin molecules, which consist of four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds). Each heavy chain has a heavy chain variable domain (V H ) and heavy chain constant region (C H ) includes. The heavy chain constant region consists of at least three domains, C H 1. C H 2, C H3, and optionally contains CH4. Each light chain has a light chain variable domain (C H ) and a light chain constant region (C L ). The heavy chain variable domain and the light chain variable domain can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), with more conserved regions called framework regions (FRs) interspersed. Each heavy chain variable domain and light chain variable domain contains three CDRs and four FRs, and are arranged in the following order from the amino terminus to the carboxy terminus: 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 formed by the association of a V H domain and a V L domain, and each of which together with its respective C H and C L [[ID=十二]] domain forms the antibody Fv region. A single domain antibody contains a single antigen-binding domain, e.g., V H or V L . The part of the antibody that recognizes and binds to the antigen-binding domain of an antigen, e.g., the first member of a specific binding pair of the antigen, is also called the "paratope". It is a small region (5-10 amino acids) of the Fv region of the antibody, is part of the fragment antigen-binding (Fab region), and may contain parts of the heavy and / or light chains of the antibody. The paratope specifically binds to the first member of the 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 having a K D for the first member of its target specific binding pair of about 10 -9 M or less (e.g., about 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, or about 1×10 -12 M). In one embodiment, K DThis is measured by surface plasmon resonance, for example, BIACORE®, and in another embodiment, K D It is measured by ELISA.

[0051] The term “complementarity-determining region” or “CDR” refers to an amino acid sequence encoded by the nucleic acid sequence of an immunoglobulin gene in an organism, which is typically (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 rearranged or unrearranged sequences, for example, by naive cells or mature B cells or T cells. CDRs may be somatically mutant (e.g., unlike sequences encoded in animal germline cells), humanized, and / or modified by amino acid substitutions, additions, or deletions. Under certain conditions (for example, 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 unrearranged nucleic acid sequences) but are contiguous in B cell nucleic acid sequences as a result of sequence splicing or joining (e.g., VDJ rearrangement to form heavy chain CDR3).

[0052] The term "inverted end sequence" or "ITR" refers to a symmetric nucleic acid sequence in the adeno-associated virus genome necessary for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs serve as the origin of replication for viral DNA synthesis and are essential cis-components for generating the AAV vector.

[0053] The term "light chain" includes immunoglobulin light chain sequences derived from any organism, and unless otherwise specified, includes not only surrogate light chains but also human κ-light chains, human λ-light chains, and VpreB. Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes a variable domain consisting of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the direction from the amino terminus to the carboxyl terminus, and a light chain constant region. Light chain variable domains are encoded by light chain variable region gene sequences, which generally derive from a repertoire of V and J segments present in the germline. L Segments and J L This includes segments. 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 to either the first or second member of a specific binding pair, which is selectively bound by the first member of the specific binding pair binding protein on which the light chain appears. Light chains also include light chains that bind to and recognize or assist a heavy chain or another light chain by binding and recognition of one or more first members of a specific binding pair, which is selectively bound by the first member of the specific binding pair binding protein on which the light chain appears. General or generic light chains include light chains derived from the human Vκ1-39Jκ genes or the human Vκ3-20Jκ genes, including their somatic variants (e.g., affinity maturation) versions. Exemplary human V LThe segments include the human Vκ1-39 gene segments, human Vκ3-20 gene segments, human Vλ1-40 gene segments, human Vλ1-44 gene segments, human Vλ2-8 gene segments, human Vλ2-14 gene segments, and human Vλ3-21 gene segments, including their somatic variant (e.g., affinity maturation) versions. A light chain can be constructed containing variable domains from one organism (e.g., human or rodent, e.g., rat or mouse, or bird, e.g., chicken) and constant regions from the same or different organisms (e.g., human or rodent, e.g., rat or mouse, or bird, e.g., chicken).

[0054] The terms "about" or "approximately" include being within a statistically significant range of a given value. Such a range may be within 10 times a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the terms "about" or "approximately" depends on the specific system under study and can be readily understood by those skilled in the art.

[0055] The term “capsid protein” refers to the proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are commonly called VP1, VP2, and / or VP3, and they are encoded by a single cap gene. For AAV, three AAV capsid proteins are generated in a redundant manner from a cap-open reading frame (ORF) via alternative mRNA splicing and / or the use of an alternative translation start codon, although all three proteins use a common stop codon. Warrington et al. (2004) J. Virol. 78:6595 (the whole is incorporated herein by reference). AAV2 VP1 is generally translated from the ATG start codon (amino acid M1) on a 2.4kb mRNA, while AAV2 VP2 and VP3 arise from a smaller 2.3kb mRNA using read-through translation to a weaker ACG start codon (amino acid T138) for VP2 production and to the next available ATG codon (amino acid M203) for the production of VP3, the most abundant capsid methane protein. Warrington, supra; Rutledge et al. (1998) J. Virol. 72:309-19 (the whole is incorporated herein by reference). The amino acid sequences of adeno-associated virus capsid proteins are well known in the art and are generally conserved, particularly within dependoparvovirus. See Rutledge et al. above. For example, Rutledge et al. (1998) above provides amino acid sequence alignments for the VP1, VP2, and VP3 capsid proteins of AAV2, AAV3, AAV4, and AAV6 in Figure 4B, where the respective start sites of VP1, VP2, and VP3 are indicated by arrows, and the variable domains are indicated by boxes.Therefore, the amino acid positions provided herein may be provided in relation 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 coding protein VP1 represented as Sequence ID No. 1, but those skilled in the art will be able to easily determine the positions of the same amino acids in the VP2 and / or VP3 capsid proteins of AAV, and the corresponding positions of amino acids in different serotypes, respectively. Therefore, the amino acid positions provided herein may be provided in relation 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 coding protein VP1 represented as Sequence ID No. 9, but those skilled in the art will be able to easily determine the positions of the same amino acids in the VP2 and / or VP3 capsid proteins of AAV, and the corresponding amino acids and positions in different AAV serotypes, respectively. Furthermore, those skilled in the art will be able to exchange domains between capsid proteins of different AAV serotypes for the formation of a "chimeric capsid protein".

[0056] Domain exchange between two AAV capsid protein constructs for the generation of “chimeric AAV capsid proteins” has been described. See, for example, Shen et al. (2007) Mol. Therapy 15(11):1955-1962 (the entire work is incorporated herein by reference). “Chimeria AAV capsid proteins” include AAV capsid proteins that contain amino acid sequences, e.g., domains derived from two or more different AAV serotypes, and are capable of and / or form AAV-like viral capsids / viral particles. Chimeric AAV capsid proteins are encoded by chimeric AAV capsid genes, e.g., by nucleotides comprising multiple, 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 distinguishable AAV serotype, and together these multiples encode a functional chimeric AAV capsid protein. Referencing a chimeric capsid protein in relation to a specific AAV serotype indicates that the capsid protein contains one or more domains derived from the capsid protein of that serotype, and one or more domains derived from the capsid protein of a different serotype. For example, the AAV2 chimeric capsid protein contains one or more domains from the AAV2 VP1, VP2, and / or VP3 capsid proteins, as well as one or more domains from the VP1, VP2, and / or VP3 capsid proteins 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 capsid described herein comprises recombinant VP1, VP2, and / or VP3 proteins encoded by a cap gene that has been genetically modified by the insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprises VP1, VP2, and / or VP3 proteins encoded by a reference cap gene, e.g., a wild-type reference cap gene encoding 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 encoding VP1, VP2, and / or VP3 proteins that are identical to the recombinant VP1, VP2, and / or VP3 proteins but lack a heterologous epitope, and VP1, VP2, and / or VP3 proteins but have a mutation (e.g., introduction, substitution, deletion) that has the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins but encodes substantially wild-type VP1, VP2, and / or VP3 proteins, but has a mutation that preferably reduces the directivity to wild-type VP1, VP2, and / or VP3 proteins. In some embodiments, the reference cap gene encodes chimeric VP1, VP2, and / or VP3 proteins.

[0059] The terms "heavy chain" or "immunoglobulin heavy chain" include immunoglobulin heavy chain sequences that contain the constant region sequence of an immunoglobulin heavy chain from any organism. Unless otherwise specified, the heavy chain variable domain includes three light chain CDRs and four FR regions. Fragments of the heavy chain include CDRs, CDRs, and FRs, as well as combinations thereof. A typical heavy chain has a variable domain followed by (from the N-terminus to the C-terminus) C H 1 domain, hinge, C H 2 domains, and C H It has three domains. The functional fragment of the heavy chain specifically recognizes the first member of a specific binding pair (e.g., K at micromolar, nanomolar, or picomolar concentrations). DThe fragment contains a specific binding pair that can recognize the first member of the specific binding pair, is expressible in cells, can be secreted from cells, and contains at least one CDR. The heavy chain variable domain is encoded by a variable region nucleotide sequence, which is generally present in germline V H Segment, D H Segment, and J H V derived from the segment repertoire H Segment, D H Segment, and J H This includes segments. The sequences, locations, and nomenclature of V heavy chain segments, D heavy chain segments, and J heavy chain segments for various organisms can be found in the IMGT database, which is accessible globally via the internet at the URL "imgt.org" (www).

[0060] Terms such as "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single-domain antigen-binding protein," and "single-domain binding protein" refer to monomeric or homodimeric immunoglobulin molecules containing an immunoglobulin-like chain that includes a variable region operably bound to a heavy chain constant region, where the heavy chain constant region is typically functional C H Because it lacks one domain, it cannot associate with the light chain. Therefore, terms such as "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single-domain antigen-binding protein," and "single-domain binding protein" are (i) functional C H (ii) A monomeric single-domain antigen-binding protein comprising one immunoglobulin-like chain containing a variable domain operably linked to a heavy chain constant region lacking one domain, or (ii) a homodimer single-domain antigen-binding protein comprising two immunoglobulin-like chains, each of which is functionally C H It encompasses both homodimeric single-domain antigen-binding proteins, which include a variable domain operably linked to a heavy chain constant region lacking one domain. In various embodiments, the homodimeric single-domain antigen-binding protein contains two identical immunoglobulin-like chains, each of which is functionally C HIt contains identical variable domains operably linked to identical heavy chain constant regions lacking one domain. Furthermore, each immunoglobulin-like chain of a single-domain antigen-binding protein encodes a C12 sequence (and optionally, a hinge region) of a heavy chain constant region gene (e.g., IgG, IgA, IgE, IgD, or a combination thereof). H Heavy chain constant region containing deletion or inactivating mutation in 1 (C H ) Heavy chain variable region gene segment linked to the gene sequence (e.g., V H , D H , J H ), light chain gene segment (e.g., V L , J L ), or a combination thereof, contains variable domains. Single-domain antigen-binding proteins containing variable domains derived from heavy chain gene segments are "V H Single-domain antibody or "V H These are sometimes called "single-domain antibody-binding proteins," and should be seen, for example, U.S. Patent Publication No. 8,754,287, U.S. Patent Publication No. 2014 / 0289876; 2015 / 0197553; 2015 / 0197554; 2015 / 0197555; 2015 / 0196015; 2015 / 0197556; and 2015 / 0197557 (each of which is incorporated by reference as a whole). Single-domain antigen-binding proteins containing a variable domain derived from a light chain gene segment are called "V L These are sometimes called "single-domain antigen-binding proteins," see, for example, U.S. Patent Publication 2015 / 0289489 (which is incorporated in its entirety by reference).

[0061] The term "light chain" includes immunoglobulin light chain sequences derived from any organism, and unless otherwise specified, includes not only surrogate light chains but also human kappa (κ) and human lambda (λ) light chains, as well as VpreB. The light chain variable domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes a variable domain containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the direction from the amino terminus to the carboxyl terminus, 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 generally derives from a repertoire of light chain V and light chain J gene segments present in the germline. L Gene segments and light chain J L This includes gene segments. 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 at the URL "imgt.org" on the global web (www). Light chains include, for example, light chains that do not selectively bind to either the first or second member of a specific binding pair, which is selectively bound by the first member of the specific binding pair binding protein on which the light chain appears. Light chains also include light chains that bind to one or more first members of a specific binding pair, which is selectively bound by the first member of the specific binding pair binding protein on which the light chain appears, and thereby bind to and recognize the heavy chain, or assist the heavy chain. Light chains also include light chains that bind to one or more first members of a specific binding pair, which is selectively bound by the first member of the specific binding pair binding protein on which the light chain appears, and thereby bind to and recognize the heavy chain, or assist the heavy chain. Common or generic light chains include light chains derived from the human Vκ1-39Jκ5 genes or the human Vκ3-20Jκ1 genes, including their somatic variants (e.g., affinity-matured versions).

[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 one another, or otherwise cooperate with one another to participate in an ecological event, such juxtaposition achieving or enabling such interaction and / or cooperation. For example, a regulatory sequence in a nucleic acid (e.g., an expression regulatory sequence) is said to be “operably linked” to a coding sequence if its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, “operable linkage” includes covalent bonding of components or elements relating to each other. Those skilled in the art will readily understand that in some embodiments, covalent bonding is not necessary to achieve effective and operable linkage. For example, in some embodiments, nucleic acid regulatory sequences that are operablely linked to the coding sequence they control are contiguous with the target nucleotide. Alternatively or additionally, in some embodiments, one or more such regulatory sequences act at a distance to control the trans or target coding sequence. In some embodiments, the term “expression regulatory sequence,” as used herein, refers to a polynucleotide sequence necessary and / or sufficient to influence the expression and processing of the coding sequence to which they are linked. In some embodiments, expression regulatory sequences may be, or may include, appropriate transcription start, 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 regulatory sequences are preferentially or only active in certain host cells or organisms, or of those types. For example, in prokaryotes, regulatory sequences typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, in many embodiments, regulatory sequences typically include promoters, enhancers, and / or transcription termination sequences.Those skilled in the art will understand that in many embodiments, the term “control sequence” refers to a component essential for expression and processing, and in some embodiments, includes components whose presence is advantageous for expression (e.g., including a leader sequence, a targeting sequence, and / or a fusion partner sequence).

[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, the wild-type which may be a reference and / or control virus for comparative studies. A recombinant capsid protein includes a capsid protein containing a heterologous amino acid sequence that may be inserted into and / or presented thereby. “Heterologous” in this context means heterologous compared to a virus, from which the capsid protein derives. The inserted amino acid may simply be inserted between two given amino acids of the capsid protein. Amino acid insertion may also occur in conjunction with the deletion of a given amino acid of the capsid protein at the insertion site (for example, one or more capsid protein amino acids being replaced by five or more heterologous amino acids).

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

[0065] "Specific binding pairs," "protein:protein binding pairs," etc., include two proteins (e.g., a first member (e.g., a first polypeptide)) and a second congeneral member (e.g., a second polypeptide) that interact to form a covalent isopeptide bond, which is bound under conditions that enable or promote isopeptide bond formation. The term "congeneral" refers to components that function together, i.e., react together, to form an isopeptide bond. Thus, two proteins that react efficiently together to form a covalent isopeptide bond under conditions that enable 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 have been re-examined by Veggiani et al. (2014) Trends Biotechnol. 32:506, and these include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, isopeptigmat:pyrin C, and SnoopTag:SnoopCatcher. Generally, peptide tags refer to members of protein:protein binding pairs that are generally shorter than 30 amino acids in length and form a covalent isopeptide bond with a second congener protein, where the second congener protein is generally larger, but this can also be shorter than 30 amino acids, such as in the SpyTag:KTag system.

[0066] The term "isopeptide bond" refers to an amide bond between a carboxyl group or carboxamide group and an amino group, where at least one amino group is not derived from the protein backbone or, alternatively, is not considered to be part of the protein backbone. Isopeptide bonds can be formed within a single protein or between two peptides or between a peptide and a protein. Thus, isopeptide bonds can be formed within a molecule within a single protein, or within a molecule, i.e., between two peptide / protein molecules, for example, between two peptide linkers. Typically, isopeptide bonds can be formed between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or between a 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 preferred embodiments of the present invention, isopeptide bonds may be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, isopeptide bonds may 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 whole), and is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein FbaB. By splitting the domain, Zakeri et al. obtained the peptide "SpyTag" having the sequence AHIVMVDAYKPTK (SEQ ID NO: 1), which forms an amide bond to its congener protein "SpyCatcher," a 112-amino acid polypeptide having the amino acid sequence represented by SEQ ID NO: 3 (Zakeri (2012), supra). A further 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 manipulated by deleting the β-chain from SpyCatcher, which contains reactive lysine, resulting in KTag, a 10-residue peptide tag having the amino acid sequence ATHIKFSKRD (SEQ ID NO: 2). The SpyTag002:SpyCatcher002 system was developed by Keeble et al. It is described in al (2017) Angew Chem Int Ed Engl 56:16521-25 (the entire article is incorporated herein by reference). 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. Streptococcus The D4 Ig-like domain of RrgA, an adhesive derived from pneumoniae, was split to form SnoopTag (residues 734-745, SEQ ID NO: 5) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher induces specific innate isopeptide bonds between complementary proteins. See Veggiani (2016) above.

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

[0070] The term "peptide tag" includes polypeptides that (1) are heterogeneous to the tagged protein, (2) are specific proteins capable of forming isopeptide bonds: members of a protein-binding pair, and (3) have an amino acid length of 50 or less.

[0071] The term "target cell" includes any cell in which the expression of the target nucleotide is desired. Preferably, target cells exhibit receptors on their surface that enable targeting by the targeting ligand, as described below.

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

[0073] As used herein, the term “wild-type” includes entities having the structure and / or activity found in nature in a “normal” state or context (as opposed to mutant, diseased, altered, etc.). Those skilled in the art will understand that wild-type viral vectors, e.g., wild-type capsid proteins, can be used as reference viral vectors in comparative studies. Generally, a reference viral capsid protein / capsid / vector is identical to the test viral capsid protein / capsid / vector, but identical to the change whose effect is being tested. For example, to determine the effect on transduction efficiency, the transduction efficiency of the test viral vector (in the presence or absence of a suitable targeting ligand) can be compared to the transduction efficiency of a reference viral vector that is identical to the test viral vector in all examples (e.g., in the presence or absence of a suitable targeting ligand, e.g., in the presence or absence of a suitable targeting ligand, e.g., to determine the effect on transduction efficiency, e.g., by inserting the first member of a specific binding pair into the test viral vector.

[0074] The retargeting strategy described herein offers the advantages of both the scaffold and direct recombination methods described above, and solves many of the disadvantages inherent in both. This strategy utilizes specific binding pairs, in which a first member and a second congener member specifically bind to each other, and upon binding, form a covalent bond that permanently links the viral particle to any targeted ligand fused to the congener member. Using such genetically modified viral particles, directivity is maintained as long as the viral capsid remains intact, and one advantage of the system provided herein compared to other scaffold methods is the ability of the "adapter," e.g., the ability of a targeted ligand to bind to a recombinant viral particle similar to that of direct recombination methods. However, in contrast to direct recombination methods, the system described herein maintains the flexibility of scaffold adapter methods in that the recombinant viral particle can maintain a constant degree of variability seen in the adapter, for example, in that a congener member can be fused to indicate a targeted ligand, and then different fusion proteins can be linked to the viral particle according to the target cell.

[0075] Recombinant viral capsid proteins, viral vectors, and nucleic acids This invention provides recombinant viral particles (e.g., viral capsid proteins, as well as recombinant viral capsids and / or recombinant viral vectors comprising recombinant viral capsid proteins) genetically modified to present heterologous amino acid sequences including a first member of a specific binding pair, wherein the amino acid sequence is shorter than 50 amino acids in length, and the recombinant viral capsid / particle protein reduces or disables innate targeting. In some embodiments, the viral particles further include a second homologous member of the specific binding pair, the first and second members being covalently linked, and the second member being fused to a targeting ligand.

[0076] In some embodiments, the heterogeneous amino acid sequence includes a first member of a specific binding pair and one or more linkers. In some embodiments, the heterogeneous amino acid sequence includes a first member of a specific binding pair adjacent to a linker, for example, the heterogeneous amino acid sequence includes the N-terminus to 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 long. In some embodiments, the first and second linkers are identical.

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

[0078] In some embodiments, the specific binding pair is a SpyTag:SpyCatcher binding pair, where the first member is SpyTag and the second congener member is SpyCatcher. In some embodiments, the specific binding pair is a SpyTag:KTag, where the first member is SpyTag and the second congener member is KTag. In some embodiments, the specific binding pair is a SpyTag:KTag, where the first member is KTag and the second congener member is SpyTag. In some embodiments, the specific binding pair is an isopeptag:pyrin-C, where the first member is an isopeptag and the second congener member is pyrin-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 congener member is SnoopCatcher.

[0079] In some embodiments, the recombinant viral capsid protein described herein is an Ad-serotype capsid protein 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 includes a first member of a specific binding pair in the fibrous protein domain, for example, at the carboxyl terminus of the fibrous protein, the fibrous knob, and / or the HI loop of the fibrous knob.

[0080] In some embodiments, the recombinant viral capsid proteins described herein are derived from adeno-associated virus (AAV) capsid protein genes, for example, capsid genes of AAV serotypes 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 the AAV2 capsid gene or the AAV9 capsid gene. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV2 VP1 capsid protein, whose wild-type amino acid sequence is represented as SEQ ID NO: 9. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9 VP1 capsid protein, whose wild-type amino acid sequence is represented as SEQ ID NO: 31.

[0081] Generally, the recombinant viral capsid proteins described herein include a first member of a specific binding pair inserted into and / or presented in the capsid protein such that the first member of the specific binding pair reduces and / or disables the innate targeting of the capsid protein or the capsid containing it. In some embodiments, the first member of the specific binding pair is inserted into a region of the capsid protein that is involved in the innate targeting of the wild-type reference capsid protein, for example, a region of the capsid protein that is involved in cell receptors. In some embodiments, the first member of the specific binding pair is inserted into and / or presented in the knob domain of the Ad fibril protein. In some embodiments, the first member of the specific binding pair is inserted into and / or presented in the HI loop of the Ad fibril 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 AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the first member of the specific binding pair is inserted and / or presented between amino acids N587 and R588 of the AAV2 VP1 capsid. In some embodiments, the recombinant viral capsid, the viral vector containing the recombinant viral capsid, and / or the composition containing the recombinant viral capsid includes an amino acid sequence represented as SEQ ID NOs: 13, 15, 17, 19, 21, 23, 25, 27, 29, 35, 37, or 39. Further preferred insertion sites identified by 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 proteins described herein may be AAV2 capsid proteins comprising a first member of a specific binding pair inserted at a position selected from the group consisting of 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, I-716, and combinations thereof. Further preferred 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 may 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 nomenclature I-### refers to an insertion site that names the number of amino acids in the VP1 protein of the AAV capsid protein as ###, but such insertions may be located directly at the N-terminus or C-terminus, preferably at the C-terminus of one amino acid in the sequence of five amino acids at the N-terminus or C-terminus of a given amino acid, preferably at the N-terminus or C-terminus of three, more preferably two, and especially at one(s) amino acids at the N-terminus or C-terminus of a given amino acid. Additionally, the positions referred to herein relate to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and their variations) can be easily identified for the VP2 and VP3 capsid proteins encoded by the capsid gene by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the reference AAV capsid gene.

[0083] Therefore, an insertion of the coding nucleic acid at the corresponding position in one of these sites of the cap gene leads to an insertion into VP1, VP2, and / or VP3, since the capsid protein is encoded by a shifted start codon and a duplicate reading frame of the same gene. Thus, for example, in AAV2, according to this nomenclature, only the insertion of amino acids 1-138 is inserted into VP1, the insertion of 138-203 is inserted into VP1 and VP2, and the insertion of 203-C-terminus is inserted into VP1, VP2, and VP3, and this naturally also applies to insertion site I-587. Therefore, the present invention encompasses the structural gene of AAV having the corresponding insertion in the VP1, VP2, and / or VP3 proteins.

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

[0085] In some embodiments, the insertion (presentation) of a first member of a specific binding pair reduces or disables the innate targeting of the viral vector, for example, the transduction of cells that are naturally tolerant to infection by a wild-type reference viral vector and / or target cells is undetectable in the absence of covalent bonding with a second congener of the binding pair that fuses to a suitable targeting ligand. In some embodiments, the insertion (presentation) of a first member of a specific binding pair reduces the innate targeting of the viral vector compared to the transduction of cells that are naturally tolerant to infection by a wild-type reference viral vector. In some embodiments, the insertion (presentation) of a first member of a specific binding pair reduces the innate targeting of the viral vector by at least 5%. In some embodiments, the insertion (presentation) of a first member of a specific binding pair reduces the innate targeting of the viral vector by at least 5%. In some embodiments, the insertion (presentation) of a first member of a specific binding pair reduces the innate targeting of the viral vector by at least 10%. In some embodiments, the insertion (presentation) of a first member of a specific binding pair reduces the innate targeting of the viral vector by at least 20%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 30%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 40%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 50%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 60%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 70%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 80%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 90%.In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 95%. In some embodiments, insertion (presentation) of the first member of the specific binding pair reduces the innate targeting of the viral vector by at least 90%. In embodiments where insertion (presentation) of the first member of the specific binding pair does not completely neutralize the innate targeting of the recombinant viral capsid, the innate targeting of such recombinant viral capsid may be further reduced by a second different mutation. For example, in one embodiment, the recombinant viral capsid protein described herein may be derived from the AAV9 serotype, may include the first member of the specific binding pair, and may further include a mutation, such as the W503A mutation.

[0086] The detachment of this virus from its native host is important, especially when systemic versus topical or localized administration of the viral vector is intended, because the uptake of the viral vector by the native host cells limits the effective dose of the viral vector. For AAV2 and AAV6, HSPG has been reported to be the primary receptor for viral uptake in a large number of cells, particularly hepatocytes. For AAV2, HSPG binding activity depends 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, a preferred point mutation, in the case of HSPG as the primary receptor for viral vector binding to target cells, is one that reduces the transduction activity of the viral vector to a given target cell mediated by the native receptor by at least 50%, preferably at least 80%, and particularly at least 95%.

[0087] As a result, preferred further mutations for HSPG-binding viral vectors are those that remove or substitute R or K, which is involved in HSPG binding of each virus, by basic amino acids such as R, K, or H, preferably non-basic amino acids such as A, D, G, Q, S, and T, preferably A, or amino acids present in the corresponding positions of different but highly conserved AAV serotypes that lack such an amino acid at this position. Consequently, preferred amino acid substitutions are R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A, or R588T, in particular R585A and / or R588A for AAV2, and K531A or K531E for AAV6. In one particularly preferred embodiment of the present invention, the AAV2 capsid protein mutant additionally contains two point mutations, R585A and R588A, such that these two point mutations are sufficient to result in significantly increased HSPG binding activity. These point mutations enable efficient detachment from HSPG-expressing cells and increase the specificity of each mutant virus to its new target cells for targeting.

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

[0089] There are many suitable cell surface proteins, such as cell surface receptors, that can be targeted by targeted ligands, for which targeted ligands, such as antibodies or parts thereof, are already available. Such structures include major histocompatibility antigens of class I and class II, 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 sexually transmitted hormones, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), colony-stimulated growth factor, endothelial growth factor, epidermal growth factor, fibroblast growth factor, glial-derived neurotrophic factor, glial cell growth factor, and gro-beta / mip 2. Hepatocyte growth factor, insulin-like growth factor, interferon (α-IFN, β-IFN, γIFN, consensus IFN), interleukin (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 suppressor factor, macrophage / monocyte chemoattractant activator, nerve growth factor, neutrophil-activating protein 2, platelet-derived growth factor, stem cell factor, transforming growth factor, tumor necrosis factor, intravascular non-growth factor, lipoprotein (further type 1 transmembrane receptors such as PRLR, G protein-binding receptors such as GCGR) This includes, but is not limited to, ion channels such as Nav1.7, ASIC1, or ASIC2, cell adhesion molecules, transport molecules for metabolites such as amino acids, light source receptors for B lymphocytes or T lymphocytes (e.g., B cell receptors and associated proteins (e.g., CD19, CD20, etc.), as well as T cell receptors and associated proteins (e.g., CD3, CD4, CD8, etc.)), and tetraspanin proteins (e.g., CD63). The recombinant viral capsids described herein enable cell type-specific infection by using targeted ligands that bind to differentiated cell surface antigens as targets for viral vector complexes.

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

[0091] In some embodiments, the targeted ligand binds to receptors expressed by (human) hepatocytes, for example, asial glycoprotein receptors, such as hASGR1. In some embodiments, the targeted ligand binds to receptors expressed by (human) brain cells. In some embodiments, the targeted ligand binds to receptors expressed by (human) T cells, for example, CD3, such as CD3ε. In some embodiments, the targeted ligand binds to receptors expressed by (human) kidney cells, for example. In some embodiments, the targeted ligand binds to receptors expressed by (human) muscle cells, such as integrins. In some embodiments, the targeted ligand binds to receptors expressed by (human) cancer cells, for example, tumor-associated antigens, such as E6 and E7. In some embodiments, the targeted ligand binds to the human glucagon receptor (hGCGR).

[0092] In some embodiments, the targeted ligand binds to tumor-associated antigens expressed by tumor cells. 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, Examples include MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART-1, mesoserine, 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 targeted ligand binds to CD markers related to the immune response, such as CD3, CD4, CD8, CD19, CD20, etc.

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

[0095] One embodiment of the present invention is a nucleic acid encoding the capsid protein described above. The nucleic acid is preferably a vector containing the nucleic acid of the claims. The nucleic acid, and in particular the vector, is necessary for recombinant expression of the capsid protein of the present invention.

[0096] Further embodiments of the present invention involve the use of at least one recombinant viral capsid protein and / or nucleic acid encoding them, preferably at least one multimeric structure (e.g., a viral vector) for the production of a gene transfer vector and its use as a gene transfer vector.

[0097] Method of use and preparation Further embodiments of the recombinant viral capsid proteins described herein are their use for delivering a target nucleotide, e.g., a reporter gene or therapeutic gene, to a target cell. Generally, the target nucleotide may be a transplasmid that typically contains a reporter gene(s) or therapeutic gene(s) and adjacent 5' and 3' inverted terminal (ITR) sequences (which, if contained within an AAV vector, may be under the control of a viral or non-viral promoter). In one embodiment, the target nucleotide is a transplasmid containing a 5'~3':5'ITR, a promoter, a gene (e.g., a reporter and / or therapeutic gene), and a 3'ITR.

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

[0099] Various reporter genes (or detectable regions) can be encapsulated in a multimeric structure containing 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. While the methods described herein demonstrate the construction of a targeted vector using a reporter gene encoding a green fluorescent protein, a person skilled in the art who has read this disclosure will understand that the non-human animals described herein may be produced in the absence of a reporter gene or using any reporter gene known in the art.

[0100] Various therapeutic genes can also be encapsulated, for example, as part of an introduction vector, in a multimeric structure containing the recombinant viral capsid protein described herein. Non-limiting examples of therapeutic genes include therapeutic genes encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, the CRISPR / Cas system or parts thereof, antisense RNA, siRNA, shRNA, etc.

[0101] Further embodiments of the present invention are processes for the preparation of recombinant capsid proteins, the method being described below. a) A step of expressing nucleic acid encoding a recombinant cap system protein under suitable conditions, b) The process includes isolating the capsid protein expressed in step a).

[0102] In some embodiments, the viral particles described herein include a mosaic capsid, for example, a genetically modified capsid protein described herein, in a specific ratio with respect to a reference capsid protein (e.g., in or without covalent bonding with a targeted ligand). Methods for producing such mosaic viral particles are: a) Expressing nucleic acids encoding recombinant capsid protein and nucleotides encoding reference capsid protein in a ratio of 1:1 to 10:1 (wt / wt) under suitable conditions, b) The process includes isolating the capsid protein expressed in step a).

[0103] In general, mosaic capsids formed according to this method are considered to have a modified capsid protein:reference capsid protein ratio similar to the ratio (wt:wt) of nucleic acids encoding the mosaic capsid used to generate the mosaic capsid. Therefore, in some embodiments, the compositions described herein contain recombinant viral capsid protein and 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 recombinant viral capsid protein and 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 directivity 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 containing a heterologous amino acid sequence; and / or (b) culturing packaging cells under conditions sufficient for the production of a viral vector, wherein the packaging cells contain nucleic acid. Further embodiments of the present invention are methods for presenting a targeted ligand on the surface of a capsid protein, comprising: (a) expressing a nucleic acid encoding a recombinant viral capsid protein described herein (and optionally, a nucleotide encoding a reference capsid protein) under preferred conditions in which the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair; (b) isolating the expressed capsid protein or a capsid comprising the first member of the specific binding pair from step (a); and (c) incubating the capsid protein or capsid with a second congener of the specific binding pair under conditions preferred to enable the formation of an isopeptide bond between the first member and the second member, wherein the second congener of the specific binding pair is fused with a targeted ligand.

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

[0106] Packaging cells useful for generating viral vectors as described herein include, for example, animal cells that are tolerant to viruses, or cells that have been modified to be tolerant to viruses, or packaging cell constructs obtained by using a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for generating viral vectors as described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., ATCC number CRL-1573), SV40 Large HEK-293 cells containing T-antigen (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Large (CCL-86), epithelial glioblastoma-astrocytoma-like cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC numbers CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC number CCL-2), Vero cells, NIH Examples include 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RATI cells, mouse L cells (ATCC number CCLI.3), HLHepG2 cells, CAP cells, and CAP-T cells.

[0107] L929 cells, the FLY virus packaging cell line outlined 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 and Pichia pastoris), plant cells (including but not limited to tobacco NTl and 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 further information on packaging cells and systems, and packaging techniques and vectors for packaging nucleic acid genomes into pseudotyped viral vectors, see, for example, Polo, et al, Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express the viral component, or transiently transfecting cells with plasmids.

[0109] Further embodiments include methods for reorienting a virus and / or delivering a reporter or therapeutic gene to target cells, the methods including methods for transducing cells in vitro or in vivo, the methods including the step of contacting target cells with a viral vector containing a capsid described herein, the capsid containing a targeted ligand that specifically binds to a receptor expressed by the target cells. In some embodiments, the target cells are in vitro. In other embodiments, the target cells are 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 the delivery of target nucleotides. Target cells will generally be selected based on the target nucleotide and the desired effect.

[0111] In some embodiments, the target nucleotide may be delivered to target cells to produce proteins that restore biological deficiencies, such as enzyme deficiencies and immunodeficiency, including X-linked severe combined immunodeficiency. Therefore, in some embodiments, cells that typically produce animal proteins are targeted. In other embodiments, cells within a region where the protein is most beneficial are targeted.

[0112] In other embodiments, a target nucleotide, such as a gene encoding an siRNA, may inhibit the expression of a specific gene within the target cell. For example, the target nucleotide may inhibit the expression of a gene involved in the pathogen life cycle. Therefore, cells that are susceptible to or infected by a pathogen can be targeted. In other embodiments, the target nucleotide may inhibit the expression of a gene involved in toxin production within the target cell.

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

[0114] In yet another embodiment, a target nucleotide encoding a therapeutic protein.

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

[0116] As discussed above, the target receptor can be any receptor from which a targeted 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 a binding partner (e.g., 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 targeted ligand.

[0117] Therefore, target cells can be selected based on a variety of factors, including, for example, (1) a specific use (e.g., therapy, expression of a recovered protein, and conferral of disease resistance) and (2) expression of a marker with a desired level of specificity.

[0118] The target cells are not limited in any way and include both germline cells and cell lines, as well as somatic cells and cell lines. The target cells may be stem cells derived from any of these origins. If the target cells are germline cells, they are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).

[0119] Pharmaceutical compositions, dosage forms, and administration Further embodiments provide a pharmaceutical product comprising at least one recombinant viral capsid protein and a suitable targeted ligand according to the present invention, and / or a nucleic acid according to the present invention. Preferably, such a pharmaceutical product is useful for gene transfer particles.

[0120] Pharmaceutical compositions comprising the virus particles described herein and pharmaceutically acceptable carriers and / or excipients are also disclosed herein. In addition, pharmaceutical dosage forms comprising the virus particles described herein are disclosed 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] The viral particle-based pharmaceutical compositions disclosed herein may be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The viral particles may be formulated for administration, for example, by injection, inhalation, or isolation (through either the mouth or nose), or by oral, intraoral, 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. Technologies and formulations can be found, for example, in Remrnington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous injections. For injection, pharmaceutical compositions can preferably be formulated in a liquid solution with 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 preferred.

[0124] For oral administration, pharmaceutical compositions are prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, crystalline 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), and may take the form of tablets or capsules. Tablets may also be coated by methods well known in the art. Liquid preparations for oral administration may take the form of solutions, syrups, or suspensions, or may be presented as dried products for compositions containing water or other suitable media before use. Such liquid preparations may be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifiers (e.g., lecithin or acacia), non-aqueous media (e.g., ionized oils, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoic acid or sorbic acid). The preparations may also optionally contain buffer salts, flavorings, colorants, and sweeteners.

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

[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. For example, compounds can be formulated with suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion-exchange resins, or as sparingly soluble derivatives, such as sparingly soluble salts. Other suitable delivery systems include microspheres, which offer the possibility of long-term, local, non-invasive delivery of drugs. This technique may include microspheres having a precapillary size that can be injected into any selected portion 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 is also possible by transmucosal or transdermal means. For transmucosal or transdermal administration, a suitable penetrating agent is used in the formulation to create a barrier for penetration. Such penetrating agents are generally known in the art and include, for example, transmucosal agents, bile salts, and fusidic acid derivatives. In addition, detergents may be used to promote penetration. Transmucosal administration may be achieved by using intranasal sprays or suppositories. For topical administration, the viral particles described herein can be formulated into ointments, plasters, gels, or creams, which are generally known in the art. To accelerate healing, a cleansing solution may also be used topically to treat the injury or inflammation.

[0128] Suitable pharmaceutical forms 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 immediate preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical form must be sterile and fluid. It must also be stable under manufacturing conditions and specific storage parameters (e.g., cooling and freezing) and must be protected from microbial contamination such as bacteria and fungi.

[0129] When the formulations disclosed herein are used as therapeutic agents to promote an immune response in a subject, the therapeutic agents may be formulated into neutral or salt-form compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups may 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, and procaine.

[0130] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents known in the art. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of the injectable composition can be achieved by the use of absorption-delaying agent compositions, such as aluminum monostearate and gelatin.

[0131] Sterile, injectable solutions can be prepared by incorporating the active compound or construct in the required amount into a suitable solvent, using various other components listed above as needed, and then sterilizing by filtration.

[0132] When prescribed, the solution may be administered in a manner compatible with the formulation and in a therapeutically effective amount. The formulation can be readily administered in various dosage forms, such as the injectable solution types described above, but sustained-release capsules or microparticles and microspheres may also be used.

[0133] For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration. In this context, the sterile aqueous media that can be used will be apparent to those skilled in the art in light of this disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid, or injected into the proposed injection site.

[0134] The person responsible for administration will, in any case, determine the appropriate dose for each individual subject. For example, a subject may be administered the viral particles described herein daily or weekly, or monthly, twice a year, or once a year for a period of time, depending on the subject's need or exposure to pathogenic microorganisms or to 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 forms currently in use.

[0136] In addition, intranasal or inhalable solutions or sprays, aerosols, or inhalants may be used. Nasal solutions may be aqueous solutions designed to be administered into the nasal passages in drops or sprays. Nasal solutions may be prepared to resemble nasal secretions in many respects. Therefore, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5–7.5. In addition, antimicrobial antiseptics similar to those used in ophthalmic preparations and appropriate drug stabilizers may be included in the formulation as needed. Various commercially available nasal preparations are known, and for example, they may contain antibiotics and antihistamines and are used for asthma prevention.

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

[0138] Tablets, lozenges, pills, capsules, etc., may also contain: binders such as tragacanth gum, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint oil, cornflower oil, or cherry flavoring. If the dosage unit form is a capsule, a liquid carrier may be included in addition to the above types of materials. Various other materials may be present as coatings or otherwise to alter the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Elixir syrups may contain sucrose as a sweetener, methyl and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavoring.

[0139] Further embodiments disclosed herein may relate to kits for use in conjunction with methods and compositions. The kits may also include suitable containers, such as vials, tubes, mini-tubes or microtubes, test tubes, flasks, bottles, syringes, or other containers. Where further components or agents are provided, the kits may include one or more further containers in which these agents or components may be placed. The kits herein will also typically include means for containing viral particles and any other reagent containers in sealed containment for commercial purposes. Such containers may include injection or blow-molded plastic containers in which the desired vials are held. Optionally, one or more further active agents may be required for the compositions described, such as anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antitumor agents.

[0140] The compositions disclosed herein may be administered by any means known in the art. For example, the compositions may be administered to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intrainjured, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreously, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucosa, 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 may be used for the large-scale production of the viral particles, packaging cells, and particle constructs described herein. For example, master and working seed stocks may be prepared under GMP conditions in a suitable major CEF or by other means. Packaging cells may be plated in a large surface area flask and grown to approximately the confluence point to purify the viral particles. The cells may be harvested and the viral particles released into isolated and purified culture medium, or intracellular viral particles may be released by mechanical breakage (cell fragments may be removed by large-pore depth filtration and digestion of host cell DNA with endonucleases). The viral particles may then be purified, concentrated by tangential flow filtration, and then dialyzed. The resulting concentrated bulk may be formulated by diluting with a buffer containing a stabilizer, packed into vials, and lyophilized. Compositions and formulations may be stored for later use. For use, the lyophilized viral particles may be reconstituted by adding a diluent.

[0142] Certain additional agents used in 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, tensorergic agents, bacterial derivatives, media, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants may stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy. [Examples]

[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 parent 293 cell lines using vectors expressing the corresponding cDNA. All cell lines were obtained from the Regeneron TC core facility. The B1 antibody recognizes linear epitopes shared by AAV VP1, VP2, and VP3.

[0146] AAV Capsid Protein Construct GeneBlocks encoding the desired SpyTag insertion, adjacent linker amino acids, and further mutations were purchased from IDT and cloned into pAAV2-CAP wt or pAAV9-CAP wt digested with BsiWI and XcmI using Gibson Assembly according to the manufacturer's protocol (NEB).

[0147] Fusion of antibodies to SpyCatcher We purchased a GeneBlock encoding SpyCatcher from IDT and used Gibson Assembly to clone the encoding sequence frame by frame into the scFv or antibody heavy chain expression plasmid at the C-terminus of each construct, which was isolated by the flexible amino acid linker GSGESG (SEQ ID NO: 48).

[0148] Preparation of AAV virus vectors Viruses were produced by transfecting 293T packaging cells with PEI Pro using the following plasmids: a genomic plasmid containing the pAd helper and reporter protein, and a pAAV-CAP plasmid encoding the AAV Rep and Cap genes, with or without further plasmids encoding either scFv or the heavy and / or light chain of the antibody. All scFv and antibody heavy chain constructs were fused to the SpyCatcher at the C-terminus described above. Transfection was 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 the virus was recovered from the cell lysates using a standard freeze-thaw protocol. Briefly, the packaging cells were lifted by degrading and pelletizing. 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 consecutive freeze-thaw cycles consisting of shutting the cell suspension with vigorous agitation between a dry ice / ethanol bath and a 37°C water bath. The viscosity was reduced by treating the lysates with EMD Millipore Benzonase (50 U / ml of cell lysates) at 37°C for 60 minutes with occasional agitation. Next, the cell fragments were pelleted by centrifugal isolation, and the resulting supernatant was filtered directly into the upper chamber of an Amicon Ultra-15 centrifugal filter unit with an Ultracel-100 membrane (100 kDa MWCO) filter cartridge through a 0.22 μm PVDF Millex-GV filter. The filter unit was centrifuged in the upper chamber at 5-10 minute intervals until the desired volume was reached, and then the concentrated crude virus was pipetteed into low-protein bound tubes and stored at 4C. Titer (viral genome vg / mL per milliliter) was determined by qPCR using standard curves for known viral concentrations.

[0150] Cell infection / transduction and flow cytometry analysis To infect the cells, virus particles were directly added to the culture medium of the cells, 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, the cells were trypsinized, resuspended in PBS with 2% FBS, and the percentage of GFP+ cells was recovered on a BD FACSCanto flow cytometry system and analyzed using FlowJo software.

[0151] Western blot analysis The reaction between SpyTag-tagged proteins VP1, VP2, and VP3 and SpyCatcher-tagged antibodies or scFv was observed by Western blotting. Novex® Tris-glycine SDS sample buffer containing a reducing agent was added to an equal volume of crude virus preparation, the sample was heated to 85°C for 5 minutes, then cooled to room temperature, and loaded onto precast 4-12% Tris-glycine gels (Invitrogen). Proteins were separated by reduced SDS-PAGE and blotted onto PVDF via wet introduction. The membrane was blocked with Li-Cor Odyssey TBS blocking buffer, probed with mouse monoclonal B1 antibody (ARP American Research Products, Inc.), and diluted 1:100 in TBST overnight at 4°C. The blot was 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 is oriented towards antigen-specific targeting. Each virus was produced as described above by transfecting 293T packaging cells in one 15 cm plate with the following plasmids and quantities. [Table A-1] [Table A-2]

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

[0154] AAV2 cells carrying heparin-binding mutations (HBM) R585A and R588A, along with a SpyTag peptide at capsid position N587, were generated in the presence or absence of C6.5-SpyCatcher and HER2-binding scFv, and fused to the SpyCatcher at their C-terminus. AAV2 cells fused to HER2-targeted scFv specifically infect 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 is oriented towards antigen-specific targeting. Each virus was produced as described above by transfecting 293T packaging cells in one 15 cm plate with the following plasmids and quantities. [Table B]

[0156] In the presence or absence of the heavy and light chains of an antibody encoding SpyCatcher-Herceptin, which binds to HER2, wild-type AAV2, AAV2 possessing heparin-binding mutations (HBM) R585A and R588A, and a SpyTag peptide at capsid position N587 were generated and 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 the HER2-targeted antibody specifically infected HER2+ cells, with very little background infection of HER2- cells. Figure 2

[0157] Example 3: Antibody conjugation to a peptide inserted into the AAV2 capsid at residue G453 is oriented towards antigen-specific targeting. Residues N587 and G453 are located on exposed regions of the AAV2 capsid, respectively, that form protein spikes extending away from the virion surface. Because the residues are 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 293T packaging cells in one 15 cm plate with the following plasmids and quantities. [Table C]

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

[0159] Example 4: Increasing the modification of AAV virions by scFv reduces their infectivity. In an attempt to optimize the efficiency of the SpyTag-SpyCatcher reaction, the deliverability of SpyTag on the viral surface was improved by aligning the peptide tag with the flexible linker amino acids on each side. A panel of N587 SpyTag insertion mutants adjacent to increased linker length was generated, and viruses were prepared using these AAV2 Rep-Cap constructs in the presence or absence of C6.5-SpyCatcher, a scFv that binds to HER2, and fused to SpyCatcher at their C-terminus. The reaction between SpyTag-tagged AAV2 proteins VP1, VP2, and VP3 and SpyCatcher-tagged C6.5 was observed by Western blotting, and SpyTag-tagged capsid proteins that reacted with SpyCatcher-tagged scFv showed an increase in size by SDS-PAGE. Cells infected with the above-described viral particles were evaluated by flow cytometry to measure transduction.

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

[0161] The pAAV2-CAP N587 linker SpyTag construct includes the following: 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 the SpyTag was not adjacent to the 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 (1–6 amino acids), the VP-SpyTag-SpyCatcher-scFv complex began to become detectable via Western blotting, and the efficiency of viral transduction increased. Figure 4. However, when the SpyTag was adjacent to the two longest linkers (8–10 amino acids), almost all VP proteins reacted with SpyCatcher-Vh by Western blotting, but these fully modified viruses no longer efficiently transduce cells. Figure 4. Therefore, it was found that excessive modification of AAV particles by scFv is detrimental to their ability to transduce target cells, and that only a small number of conjugated scFv are necessary for the virus to retarget target cells.

[0163] Example 5: Increased modification of AAV virions by antibodies reduces their infectivity. Using a panel of N587 SpyTag insertion mutations adjacent to linker length increases, viruses were prepared using these AAV2 Rep-Cap constructs in the presence or absence of the heavy and light chains of an antibody encoding SpyCatcher-Herceptin, which binds to HER2, and fused to SpyCatcher at the C-terminus of the heavy chain. The reaction between SpyTag-tagged AAV proteins VP1, VP2, and VP3 and the SpyCatcher-tagged Herceptin heavy chain (Vh) was observed by Western blotting, and the SpyTag-tagged capsid protein reacted with the SpyCatcher-tagged antibody would show size changes by SDS-PAGE.

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

[0165] The pAAV2-CAP N587 linker SpyTag construct includes the following: 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 the SpyTag was not adjacent to a linker amino acid, or was adjacent to a very short amino acid, the VP-SpyTag-SpyCatcher-Vh complex was not detected by Western blotting, but the virus specifically infected HER2+ cells with near wild-type efficiency (Figure 5). Conversely, when the SpyTag was adjacent to a longer linker (six or more amino acids), almost all VP proteins reacted with SpyCatcher-Vh by Western blotting, but these fully modified viruses no longer infected (Figure 5). Increased modification of AAV particles by antibodies is detrimental to their ability to transduce target cells, and only a small number of conjugated antibodies are required to retarget the virus to target cells.

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

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

[0169] Figure 8 shows the reactions of G453 SpyTag HBM and G453 Linker 10 SpyTag HBM with SpyCatcher-anti-HER2 antibody. Both SpyTag insertions at G453 reacted very efficiently with SpyCatcher-tagged Herceptin, as measured by Western blotting, either as a SpyTag alone or adjacent to Linker 10. This suggests that the G453 insertion site is more naturally deliverable than N587, which does not react readily unless adjacent to a linker amino acid. When the virus was severely modified by the SpyCatcher-Herceptin antibody, as observed in the N587 linker panel, the virus no longer infected. Therefore, high levels of modification of AAV particles by the antibody 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 target pairs. We investigated the ability of the SpyTag-SpyCatcher method to retarget AAV to targets other than HER2. We cloned SpyCatcher-tagged antibodies that target additional cell surface proteins and investigated the ability of these antibodies to retarget SpyTag-tagged AAV to cell types expressing these additional targets. In experiments targeting ASGR1 and CD63, each virus was produced as described above by transfecting 293T packaging cells in one 15 cm plate with the plasmids and quantities listed below. [Table G]

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

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

[0173] SpyCatcher-Vh and Vk plasmids encoding antibody heavy and light chains recognizing human proteins ASGR1, CD63, PTPRN, ENTPD3, and CD20 were tested. To produce mosaic AAV particles with a small number of exposed SpyTags, SpyTag and non-SpyTag plasmids were present in the transfection mixture in a 1:7 ratio, which had previously been determined to be the ideal ratio of SpyTag to non-SpyTag capsid for retargeting AAV 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 could specifically infect congeneral target cells expressing ASGR1, CD63, and PTPRN, respectively, and presented 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 a luciferase assay using a standard protocol. AAV2 conjugated to ENTPD3 and CD20-specific antibodies was able to specifically infect congeneral target cells expressing ENTPD3 and CD20, respectively, and showed very low background infection in the absence of antibodies. Figure 9

[0174] Example 8: The SpyTag-SpyCatcher system can be adapted for the retargeting of AAV9s. The compatibility of the SpyTag-SpyCatcher system with other AAV serotypes was tested. AAV9 is a widely used serotype that produces high-titer viruses and is highly efficient in transducing mouse tissues. Since AAV2 binds to heparin sulfate proteoglycans and AAV9 binds to galactose, the residues crucial for receptor binding differ between AAV2 and AAV9. Residues known to be important for 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 to be used when generating mutant constructs because this single amino acid mutation significantly reduced receptor binding. A region of the AAV9 capsid that is orthologous to the two protrusions (variable loops) containing AAV2 N587 and G453 was also identified, with the corresponding residues in AAV9 being A589 and G453. SpyTag was inserted in combination with the receptor-binding mutation W503A into these two sites in AAV9, one with and one without adjacent linker amino acids.

[0175] Each virus was produced as described above by transfecting 293T packaging cells in one 15 cm plate 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 viruses, 3.5 ug of pAAV9-CAP W503A and either 0.5 ug of pAAV9-CAP A589 linker 10SpyT_W503A or pAAV9-CAP G453 linker 10SpyT_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] SpyTag insertions in AAV9 RC A589 and G453 facilitated the reaction with SpyCatcher-Herceptin and mediated specific transduction of HER2+ cells. Figure 10. Similar to AAV2, SpyTag insertions in AAV9 RC A589 without adjacent linkers were very poorly delivered and did not react well with SpyCatcher. Figure 10. Conversely, adding an amino acid linker on either side of the SpyTag allowed for more robust reactivity with SpyCatcher, and mosaic particles with several highly reactive SpyTag were very efficient in transducing target cells. Figure 10.

[0180] Example 9: In vivo retargeting of SpyTag-equipped AAV particle-SpyCatcher-Vh complex. To determine whether VP-SpyTag-SpyCatcher-Vh can be retargeted to hepatocytes expressing hASGR1 in vivo, mice genetically modified to express hASGR1 against a C57BL / 6 background, and control wild-type mice, were intraperitoneally injected with wild-type AAV alone or VP-SpyTag-SpyCatcher-Vh virus particles (as pure or mosaic particles, with or without an amino acid linker) carrying a reporter gene, e.g., green fluorescent protein or firefly luciferase. Controls included mice injected with phosphate-buffered saline (PBS). To determine whether VP-SpyTag-SpyCatcher-Vh can be detached from the liver and retargeted to other organs, wild-type AAV alone or VP-SpyTag-SpyCatcher-Vh virus particles (as mosaic particles) carrying a reporter gene, e.g., green fluorescent protein, were injected intravascularly. To demonstrate detachment from the liver and retargeting to other organs, we investigated the expression of a gene 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 data from publicly available databases (GenePaint.org) http: / / www.informatics.jax.org / assay / MGI:5423021 We selected the protein ENTPD3 because it is expressed in other organs (such as those from the Riken FANTOM5 project, and 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 mouse proteins) were cloned, and the ability of these antibodies to retarget SpyTag-tagged AAV2 carrying either eGFP or firefly luciferase in vivo was tested. Each virus was produced as described above by transfecting 293T packaging cells in 15 cm plates with the following plasmids and quantities. [Table K-1] [Table K-2]

[0182] SpyCatcher-Vh and Vk plasmids encoding antibody heavy and light chains that recognize human proteins ASGR1, CD3, or CD63 were tested in genetically modified mice whose liver cells expressed hASGR1 on a C57BL / 6 background. SpyCatcher-Vh and Vk plasmids encoding antibodies that recognize human protein ASGR1 (as a non-targeted control) or mouse and human protein ENTPD3 were tested in wild-type mice. Ten days after infection, mice were sacrificed, reporter gene expression was tested, and the liver, spleen, 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 the bioluminescence of the animals, live animals were anesthetized with isoflurane 14 days after infection and imaged 10 minutes later using the IVIS spectral 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, and not in the livers of wild-type mice that do not express hASGR1. Positive EGF was not 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 with 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 collected 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 detached from the liver of wild-type mice, and all mice injected with AAV conjugated to antibodies targeting ENTPD3 and hASGR1 showed similar deficiencies in eGFP expression in the liver. Mice injected with AAV conjugated to antibodies targeting ENTPD3 had cells expressing eGFP 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 with AAV conjugated to antibodies targeting ENTPD3 or hASGR1, as hASGR1 is not expressed in wild-type mice, serving as a non-targeting control. Fourteen days after infection, organs were collected 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 detached from the liver of wild-type mice, and all mice injected with AAV conjugated to antibodies targeting ENTPD3 and hASGR1 showed similar deficiencies in eGFP expression in the liver. All three mice injected with AAV conjugated to a non-targeting, unrelated antibody (anti-ASGR1) showed no staining in the tongue, while all three mice conjugated to anti-ENTPD3 showed eGFP-expressing cells in the tongue, suggesting that ENTPD3 is expressed here.

[0185] Example 10 Delivery of suicide gene to cells expressing targeted 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, and 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 use a xenograft nude mouse model of HER2+ breast cancer described by Wang et al. ((2010) Cancer Gene Therapy 17:559-570).

[0187] The VP-SpyTag-SpyCatcher-Vh complex carrying the suicide gene (SG) is produced in the same manner as described in the Materials and Methods section.

[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 cancer 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 the medium recommended by ATCC.

[0189] Female nude mice 6-8 weeks old are obtained and housed under conditions free of specific pathogens. On day 0, the mice are given (1)10 7 (1) BT474, SK-BR-3, Calu-3, A-673, or HeLa were subcutaneously injected into the right flank, and (2) intravenously treated with a reporter (e.g., EGFP) or a VP-SpyTag-SpyCatcher-Vh complex carrying a suicide gene. Untreated animals (injected with tumor cells only), animals injected with wild-type AAV particles carrying the reporter or suicide gene, and animals injected with SpyTag virus particles only served as controls. All animals were treated with an appropriate prodrug one day after injection and treatment. The size of each tumor was measured twice weekly using calipers, and tumor volume was defined as length × width 2 Calculate using a multiplier of 0.52. (Condition: diseased state, tumor ulceration, tumor diameter 15mm, or tumor volume 1000mm) 3 At this point, the mice are slaughtered, and the date of slaughter is recorded as the date of death. The liver, spleen, kidneys, and tumors of animals injected with viral particles carrying the reporter gene are fixed, and reporter gene expression is visualized.

[0190] Targeted delivery of suicide-inducing genes has been described (Zarogoulidis P., et al. (2013) J. Genet. Syndr. Gene Ther. 4:16849), but this example describes the delivery of a suicide gene to cells expressing a targeted HER2 ligand using the viral particles described herein. In additional experiments, the suicide gene is delivered to other cell types expressing one or more other target ligands using the viral particles described herein. Exemplary and non-limiting examples of reporters suitable for targeting include viral particles, e.g., a reporter that mediates endocytosis of calcinin embryonic antigen (CEA) (Qiu Y, et al. (2012) Cancer Lett. 316:31-38) and a vascular endothelial growth factor reporter (VEGFR) (Leng A, et al. This includes al. (2013) Tumour Biol. 32:1103-1111 and Liu T, et al. (2011) Exp Mol Pathol. 91:745-752). Further 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), surface antigen classification 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 surface antigen classification 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.) This includes 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 is specifically shown and described with reference to several embodiments, it will be understood by those skilled in the art that modifications in form and detail can be made to 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] Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but some preferred methods and materials are described herein. All publications cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. The present invention provides, for example, the following items. (Item 1) Recombinant viral capsid protein comprising a protein:a first member of a protein-binding pair operably linked to the capsid protein, wherein optionally the first member is a peptide tag, and optionally the mutation of the first member and / or disables the innate directivity of the capsid protein. (Item 2) The protein: The recombinant viral capsid protein according to item 1, further comprising a second congener member of a protein-binding pair, wherein the first and second members are covalently linked. (Item 3) The recombinant viral capsid protein described in item 2, wherein the aforementioned covalent bond is an isopeptide bond. (Item 4) The recombinant viral capsid protein according to item 2 or 3, wherein the second member is operably linked to a targeting ligand, and optionally the targeting ligand is the binding site. (Item 5) The recombinant viral capsid protein according to any one of items 1 to 4, wherein the first member is adjacent to first and / or second linkers that link the first member to the capsid protein, and the first and / or second linkers are each independently at least 1 amino acid long. (Item 6) Recombinant viral capsid proteins as described in item 5, wherein the first and second linkers are not identical. (Item 7) The recombinant viral capsid protein described in item 5, wherein the first and second linkers are identical and have a length of 10 amino acids. (Item 8) A recombinant viral capsid protein according to any one of items 1 to 7, further comprising mutations at amino acid positions involved in the binding of the viral capsid protein to its innate receptor, wherein the mutations include insertion of a heterologous peptide into the capsid protein, substitution of one or more amino acids of the capsid protein with a heterologous peptide, 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) It will be reduced by 10%, (ii) It will be reduced by 20%, (iii) Reduced by 30%, (iv) Reduced by 40%, (v) Reduced by 50%, (vi) Reduced by 60%, (vii) Will it be reduced by 70%? (viii) Reduced by 80%, (ix) Reduced by 90%, or (x) Recombinant viral capsid proteins described in any one of items 1-8 that are to be disabled. (Item 10) The recombinant viral capsid protein according to any one of items 1 to 9, wherein the viral capsid protein is derived from the capsid gene of adeno-associated virus (AAV), the capsid gene encodes the AAV VP1, VP2, and / or VP3 capsid proteins, and optionally further comprises mutations at amino acid positions involved in the binding of the capsid. (Item 11) The recombinant viral capsid protein described in item 10, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 12) The recombinant viral capsid protein described in item 10, wherein the adeno-associated virus is AAV2. (Item 13) The recombinant viral capsid protein according to item 10, wherein the adeno-associated virus is AAV9. (Item 14) The protein: the protein binding pair is (i) SpyTag: SpyCatcher, (ii) SpyTag: KTag, (iii) Isopeptag: Pyrin-C, (iv) SnoopTag: SnoopCatcher, or (v) SpyTag002: SpyCatcher002, the recombinant viral capsid protein according to any one of items 1 to 13. (Item 15) The recombinant viral capsid protein according to any one of items 1 to 14, wherein the first member and any linker together have an amino acid length of about 50 or less. (Item 16) The recombinant viral capsid protein according to any one of items 1 to 15, wherein the first member is SpyTag. (Item 17) The recombinant viral capsid protein according to any one of items 2 to 16, wherein the second cognate member is SpyCatcher. (Item 18) The recombinant viral capsid protein according to any one of items 2 to 16, wherein the second cognate member is KTag. (Item 19) The recombinant viral capsid protein according to any one of items 2 to 15, wherein the first member is KTag and the second cognate member is SpyTag. (Item 20) The recombinant viral capsid protein according to any one of items 2 to 15, wherein the first member is SnoopTag and the second cognate member is SnoopCatcher, Item 2 to 15 of the recombinant viral capsid protein according to any one of the items. (Item 21) A recombinant viral capsid protein according to any one of items 2 to 15, wherein the first member is an isopeptag and the second congeneral member is pyrin-C. (Item 22) A recombinant viral capsid protein according to any one of items 2 to 15, wherein the first member is SpyTag002 and the second congeneral member is SpyCatcher002. (Item 23) The recombinant viral capsid protein according to any one of items 1 to 22, wherein the recombinant viral capsid protein or the viral capsid comprising the recombinant viral capsid protein is reduced or ineffective in infecting target cells in the absence of the appropriate targeting ligand, compared to the viral capsid protein or capsid comprising the appropriate targeting ligand. (Item 24) The recombinant viral capsid protein according to any one of items 4 to 23, wherein the binding portion is an antibody or a portion thereof. (Item 25) The recombinant viral capsid protein described in item 24, wherein the antibody or a portion thereof is fused to SpyCatcher. (Item 26) The recombinant viral capsid protein according to item 25, wherein the antibody or a portion thereof is fused to a linker at its C-terminus, and the linker is fused to a SpyCatcher at its C-terminus. (Item 27) The recombinant viral capsid protein described in item 26, wherein the linker contains the sequence represented as sequence number 48 (GSGESG). (Item 28) The recombinant viral capsid protein described in any one of items 4 to 27, wherein the targeted ligand comprises the amino acid sequence represented as Sequence ID No. 46. (Item 29) The targeted ligand specifically binds to cell surface molecules, and selectively, the cell surface marker is... (i) Asialoglycoprotein 1 (ASGR1), (ii) ENTPD3, (iii) PTPRN, (iv) CD20, (v)CD63, or (vi) Her2 recombinant viral capsid protein as described in any one of items 4-28. (Item 30) The recombinant viral capsid protein described in item 29, wherein the cell surface molecule is asialoglycoglycoprotein 1 (ASGR1). (Item 31) The recombinant viral capsid protein described in item 29, wherein the cell surface molecule is CD63. (Item 32) The recombinant viral capsid protein described in item 29, wherein the cell surface molecule is ENTDP3. (Item 33) A recombinant viral capsid containing a recombinant viral capsid protein as described in any one of items 1 to 32. (Item 34) The recombinant viral capsid according to item 33, further comprising a reference viral capsid protein lacking any member of the aforementioned specific binding pair. (Item 35) The recombinant viral capsid according to 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 the binding of the viral particle to its native ligand. (Item 36) A 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) A recombinant viral vector containing a target nucleotide encapsulated by a recombinant viral capsid as described in any one of items 33-36. (Item 38) Recombinant virus particles as described in item 37, wherein the target nucleotide is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, a bird promoter, a fish promoter, an insect promoter, and any combination thereof. (Item 39) Recombinant viral particles as described in item 37, wherein the target nucleotide is under the control of a human promoter. (Item 40) Recombinant viral particles as described in item 37, wherein the target nucleotide is under the control of a non-human promoter. (Item 41) A recombinant virus particle as described in any one of items 37 to 40, wherein the target nucleotide is a reporter gene. (Item 42) A recombinant virus particle as described in item 41, wherein the reporter gene encodes a green fluorescent protein. (Item 43) Recombinant virus particles as described in 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) The recombinant viral particle according to any one of items 37 to 40, wherein the target nucleotide is selected from the group consisting of a nucleotide encoding a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a nucleotide encoding a CRISPR / Cas system or a part (s) thereof, a nucleotide encoding an antisense RNA, and a nucleotide encoding an shRNA. (Item 45) (a) A viral capsid according to any one of items 33 to 36, or a viral vector according to any one of items 37 to 44, and (b) a pharmaceutically acceptable carrier. (Item 46) A method for delivering a target nucleotide to a target cell, comprising contacting the target cell with the viral particle according to any one of items 37 to 44 or the composition according to 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) The method according to item 46, wherein the target cell is in vitro. (Item 48) The method according to item 46, wherein the target cell is in the body of a subject. (Item 49) The method according to item 48, wherein the subject is a human. (Item 50) The method according to any one of items 46 to 49, wherein the target cell is a human target cell. (Item 51) The method according to item 50, wherein the target cell is a human hepatocyte and the targeting ligand binds to the human asialoglycoprotein receptor (ASGR1). (Item 52) The method according to item 50, wherein the target cell is a human nerve cell and the targeting ligand binds to GABA. (Item 53) The method according to item 50, wherein the target cell is a human T cell and the targeting ligand binds to CD3, optionally CD3ε. (Item 54) The method according to item 50, wherein the targeted ligand binds to PTPRN. (Item 55) The method according to item 50, wherein the target cells are human hematopoietic cells and the targeting ligand binds to CD34. (Item 56) The method according to item 50, wherein the target cells are human kidney cells. (Item 57) The method according to item 50, wherein the target cells are human cancer cells and the targeting ligand binds to a tumor-associated antigen. (Item 58) The method according to item 57, wherein the tumor antigen is E6, E7, or Her2. (Item 59) The method according to item 50, wherein the targeted ligand binds to CD20. (Item 60) The method according to item 50, wherein the targeted ligand binds to a human glucagon receptor. (Item 61) The method according to any one of items 46 to 50, wherein the targeted ligand specifically binds to CD63. (Item 62) The method according to any one of items 46 to 50, wherein the targeted ligand specifically binds to human extracellular nucleoside triphosphate diphosphohydrolase 3 (hENTPD3). (Item 63) A method for providing a viral capsid protein having a scaffold and / or adapter, (a) Specific protein: Inserting a nucleic acid encoding the first member of a 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 containing the first member of the specific binding pair, and optionally the linker, and inserting, (b) A method comprising culturing packaging cells under conditions sufficient for the generation of viral particles, wherein the packaging cells contain the nucleic acid. (Item 64) A method for generating viral particles, comprising culturing packaging cells under conditions sufficient for generating the viral particles, wherein the packaging cells comprise a nucleotide sequence encoding a genetically modified capsid protein comprising a first member of a specific protein:protein binding pair, and an amino acid linker for optionally linking the first member to the capsid protein. (Item 65) The method according to item 63 or item 64, wherein the packaging cells further comprise a helper plasmid and / or transducer plasmid containing the target nucleotide. (Item 66) The method according to any one of items 63 to 65, wherein the packaging cells further comprise a plasmid encoding a reference mosaic capsid. (Item 67) The method according to 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. Removal of cell fragments, b. Treating the supernatant containing virus particles with a nuclease, c. Concentrating virus particles, d. Purifying the aforementioned virus particles, The method described in any one of items 63 to 67, further including any combination of ea to d. (Item 69) The method according to any one of items 63 to 68, wherein the nucleotide sequence encoding the genetically modified capsid protein further includes amino acid mutations at positions involved in the natural directivity of the capsid protein, the mutations include insertion of a heterologous peptide into the capsid protein, substitution of one or more amino acids of the capsid protein with a heterologous peptide, deletion of one or more amino acids of the capsid protein, or a combination thereof. (Item 70) Virus particles prepared according to the method described in any one of items 63-69. (Item 71) Packaging cells for generating viral particles containing a plasmid encoding the recombinant viral capsid protein described in any one of items 1 to 32. (Item 72) A recombinant vector encoding a recombinant viral capsid protein as described in any one of items 1 to 32.

Claims

[Claim 1] The protein or method described in the specification.

Images