Targeted modified recombinant viral vectors for introducing genetic material into human cells and their use

Abstract

Provided are tropic modified recombinant viral vectors and compositions comprising same that are useful for targeted transfer of genetic material into cells and / or tissues. [Solution] The present invention provides a recombinant viral capsid protein comprising an epitope, the epitope being heterologous to the capsid protein, the heterologous epitope or a portion thereof specifically binding to an antibody paratope, and the viral capsid protein forming a recombinant viral capsid with reduced or abolished native tropism. In one embodiment, the recombinant viral capsid protein comprises a substitution, insertion, or deletion at an amino acid position responsible for the native tropism of the viral capsid, such that the recombinant viral capsid protein forms a viral capsid with reduced or abolished native tropism.

Description

Technical Field

[0001] Reference to Sequence Listing Submitted as a text file via EFS-Web The sequence listing described in the file 10335WO01_ST25.txt is 88 kilobytes, was created on June 27, 2018, and is incorporated herein by reference.

[0002] The disclosure herein generally relates to targeted recombinant viral vectors and compositions containing the same that are useful for targeting and introducing genetic material into cells and / or tissues.

Background Art

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

[0004] I In recent years, much of the progress in vector development has been achieved using not only enveloped viruses such as retroviruses, lentiviruses, and herpes simplex viruses (e.g., viruses whose capsids are surrounded by a lipid bilayer), but also naked viruses such as adeno-associated viruses (AAVs) and adenoviruses (Ads) (e.g., viruses whose capsids are formed by viral capsid proteins that do not have an envelope (e.g., a lipid bilayer)). AAVs are non-enveloped viruses that exhibit only mild immunogenicity but can transduce a wide range of species and tissues into vivo without apparent toxicity, making AAV vectors a focus of much research.

[0005] AAV is a small, non-enveloped, single-stranded DNA virus. The AAV genome is 4.7 kb and features two inverted end repeats (ITRs) and two open reading frames that encode Rep and Cap proteins, respectively. The two ITRs are the only cis elements essential for AAV replication, packaging, and integration. The Rep reading frame encodes four proteins with molecular weights of 78 kD, 68 kD, 52 kD, and 40 kD. These proteins primarily regulate AAV replication and function in the integration of AAV into the host cell's chromosomes. 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 composed of VP3, and in mature virions, VP1, VP2, and VP3 are found in a relative abundance of approximately 1:1:10. In vitro, 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 AAV3A, AAV3B, AAV4, and AAV6 have also been determined (Rutledge et al. (1998) J. Virol. 72:309-319, Chiorini et al. (1997) J. Virol. 71:6823-6833, S. Muramatsu et al. (1996) Virol. 221:208-217). Generally, all AAVs share more than 80% identity in their nucleotide sequences.

[0007] Unlike other viral vectors, AAV has not been shown to be associated with any known human disease and is generally not considered pathogenic, making it a promising vector for human gene therapy (Muzyczka et al. (1992) Current Topics in Microbiology and Immunology 158:97-129). Furthermore, AAV can safely transduce postmittal tissue with relatively low immunogenicity and integrate into host chromosomes in a site-specific manner, and into tissue culture cells at chromosome 19 when the Rep protein is supplied in trans. (Kotin et al. (1990) Proc. Natl. Acad. Sci. USA87:2211-2215, Samulski et al. (1991) EMBO J.10(12):3941-3950, Balague et al. (1997) J. Virol.71:3299-3306, Surosky et al. al. (1997) J. Virol. 71:7951-7959). The integrated genome of AAV has been shown to enable long-term gene expression in numerous tissues, including muscle, liver, and brain (Fisher (1997) Nature Med. 3(3):306-312, Snyder et al. (1997) Nature Genetics 16:270-276, Xiao et al. (1997) Experimental Neurology 144:113-124, Xiao et al. (1996) J. Virol. 70(11):8098-8108).

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

[0009] Since most cell surface receptors or markers are involved in the endocytosis pathway either constitutively (e.g., for reuse) or ligand-induced (e.g., receptor-mediated), theoretically, retargeting a vector toward any cell surface protein or marker should result in infection by the target cell. These receptors clump together in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are classified, and are then either reused on the cell surface, stored within the cell, 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 the target cell alone or primarily by the target cell. 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 viral vector's innate targeting. (This has been verified in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93 and Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[0010] Non-genetic methods typically utilize adapters that recognize both wild-type (unmodified) viral surface proteins and target cells. Soluble pseudoreceptors (wild-type virus), polymers such as polyethylene glycol, and antibodies or portions thereof have been used as the virus-binding domain of the adapter, while natural peptides or vitamin ligands, as well as antibodies or portions thereof, have been used as the cell-binding domain of the aforementioned adapter. In this method, retargeting of the viral vector to target cells can be achieved when the vector:adapter complex binds to proteins expressed on the surface of the target cells (e.g., cell surface proteins).

[0011] Such methods are used for AAV (Bartlett et al. (1999) Nat. Biotechnol. 74:2777-2785), adenovirus (Hemminki et al. (2001) Cancer Res. 61:6377-81, van Beusechem et al. (2003) Gene Therapy 10:1982-1991, Einfeld, et al. (2001) J. Virol. 75:11284-91, Glasgow et al. (2009) PLOS One 4:e8355), herpesvirus (Nakano et al. (2005) Mol. Ther. 11:617-24), and paramyxovirus (Bian et al. (2005) Cancer Gene Ther. 12:295-303, Bian et It has been used for coronaviruses (Haijema et al. (2005) Int. J. Oncol. 29:1359-69), and for coronaviruses (Haijema et al. (2003) J. Virol. 77:4528-43)8, Wurdinger et al. (2005) Gene Therapy 12:1394-1404).

[0012] A more common method is recombinant gene modification of the viral capsid protein, and therefore the surface of the viral capsid. In indirect recombination methods, the viral capsid is modified using a heterologous "scaffold" which is then ligated to an adapter. The adapter binds to the scaffold and target cells. (See also Arnold et al. (2006) Mol.Ther. 5:125-132, Ponnazhagen et al. (2002) J.Virol. 76:12900-907, WO97 / 05266). Ad incorporates scaffolds such as (1) Fc-binding molecules that bind to the Fc of the antibody adapter (e.g., Fc receptor, protein A, etc.), (2) (strept)avidin that binds to the biotin-labeled adapter, (3) biotin that binds to the adapter that fuses with (strept)avidin, and (4) proteins that form isopropyl peptide bonds, such as SpyCatcher that binds to the Spy-labeled adapter:protein binding pairs (Pereboeva et al. (2007) Gene Therapy 14:627-637, Park et al. (2008) Biochemical and Biophysical Research Communications 366:769-774, Henning et al. (2002) Human Gene Therapy 13:1427-1439, Banerjee et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988), AAV (Gigout et al. al. (2005) Molecular Therapy11:856-865, Stachler et al. (2008) Molecular Therapy16:1467-1473), and togavirus (Quetglas et al. (2010) Virus Research153:179-196, Ohno et al. (1997) Nature Biotechnology15:763-767, Klimstra et al. al.(2005) Virology338:9-21).

[0013] In direct recombinant targeting methods, the target ligand is either directly inserted into or ligated to the viral capsid, i.e., the protein viral capsid is modified to express the heterologous target ligand. The ligand is then reoriented, for example, to bind to a receptor or marker that is preferentially or exclusively expressed on the target cell. (Stachler et al. (2006) Gene Ther. 13:926-931, White et al. (2004) Circulation 109:513-519.) Direct recombinant methods include AAV (Park et al. (2007) Frontiers in Bioscience13:2653-59, Girod et al. (1999) Nature Medicine5:1052-56, Grifman et al. (2001) Molecular Therapy3:964-75, Shi et al. (2001) Human Gene Therapy12:1697-1711, Shi and Bartlett(2003)Molecular Therapy7:515-525), retroviruses (Dalba et al.Current Gene Therapy5:655-667, Tai and Kasahara(2008)Frontiers in Bioscience13:3083-3095, Russell and Cosset(1999)Journal of Gene Medicine1:300-311, Erlwein et al. (2002) Virology302:333-341, Chadwick et al. (1999) Journal of Molecular Biology285:485-494, Pizzato et al. (2001) Gene Therapy8:1088-1096), poxvirus (Guse et al. (2011) Expert Opinion on Biological Therapy11:595-608, Galmiche et al. (1997) Journal of General Virology78:3019-3027, Paul et al.(2007) Viral Immunology 20:664-671), paramyxovirus (Nakamura and Russell (2004) Expert Opinion on Biological Therapy 4:1685-1692, Hammond et al. (2001) Journal of Virology 75:2087-2096, Galanis (2010) Clinical Pharmacology and Therapeutics 88:620-625, Blechacz and Russell (2008) Current Gene Therapy 8:162-175, Russell and Peng (2009) Current Topics in Microbiology and Immunology 330:213-241), and herpesvirus (Shah and Breakefield (2006) Current Gene Therapy 6:361-370, Campadelli-Fiume et al. (2011) Reviews in It is used in Medical Virology 21:213-226. Each of the three methods has its advantages and disadvantages. The main advantage of direct recombination is that the specificity of the viral vector is inherent to the viral genome and is maintained during replication. However, both this and indirect recombination methods require the maintenance of the capsid structure and the placement of the targeted ligand or scaffold in a position that will accommodate and properly present the targeted ligand or scaffold, thus limiting the repertoire of suitable ligands or scaffolds that can be used. Thus, recombinant retargeting methods are limited by naturally occurring molecules useful as targeted ligands, which can lead to the incorporation of other binding ligands, such as antibodies or parts thereof. Both non-recombinant and recombinant adapter platforms have advantages in terms of the flexibility of the adapter used. However, achieving optimal transduction efficiency is difficult with these two component systems. This specification provides a viral retargeting strategy that solves problems inherent in conventional recombinant retargeting strategies by inserting heterologous epitopes into viral capsids. When this recombinant viral capsid is combined with multispecific, or optionally bispecific, binding molecules, particularly within a certain ratio of viral vector:multispecific binding molecules, including an antibody paratope such as Fv that specifically binds to a heterologous epitope and a retargeting ligand that specifically binds to target cells, it exhibits reduced or ineffective intrinsic targeting that is restored and reoriented. [Prior art documents] [Patent Documents]

[0014] [Patent Document 1] International Publication No. 97 / 05266 [Non-patent literature]

[0015] [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. USA79:2077-2081 [Non-Patent Document 3] Rutledge et al. (1998) J. Virol. 72:309-319 [Non-Patent Document 4] Chiorini et al. (1997) J. Virol. 71:6823-6833 [Non-Patent Document 5] S. Muramatsu et al. (1996) Virol. 221:208-217 [Overview of the project] [Means for solving the problem]

[0016] Disclosed herein is a recombinant viral capsid protein comprising a viral capsid comprising a recombinant viral capsid protein and a viral vector containing a target nucleotide encapsulated by the recombinant viral capsid, wherein the capsid protein, capsid, and viral vector are genetically modified to contain (present) a heterologous epitope, and the heterologous epitope (part thereof or combined with the viral capsid protein) forms a binding pair with an antibody paratope, and the recombinant viral capsid protein or the viral capsid containing the recombinant viral capsid protein is low Recombinant viral capsid proteins / capsids / vectors may further include mutations, insertions, or deletions of the (natural) directivity of the viral capsid protein / capsid / vector at amino acid positions involved in receptor binding, for example (e.g., having a transduction efficiency lower than that of a standard viral capsid protein / capsid / vector lacking a heterologous epitope in the absence of a bispecific, or a transduction efficiency that is undetectable in the absence of a bispecific, or optionally multispecific, bispecific binding molecule). Such reduced or inactivated (natural) directivity of recombinant viral capsid proteins / capsids / vectors can be enhanced or restored in the presence of appropriate bispecific, or optionally multispecific, bispecific binding moieties. Accordingly, the Viral Vector:Composition comprising a composition containing a specific ratio of multispecific binding molecules, comprising (1) a recombinant viral vector having a capsid containing the recombinant capsid protein described herein, and (2) a multispecific, optionally bispecific binding molecule containing an antibody paratope and a targeting ligand, and its use for oriented and / or introducing genetic material into target cells is also described herein.Furthermore, the book also describes, for example, a method for retargeting a recombinant viral vector to deliver target nucleotides to target cells, which includes contacting the recombinant viral vector with a bispecificity-dependent, orally selective, binding molecule, and also describes a method for producing recombinant viral vectors.

[0017] Described herein are recombinant viral capsid proteins containing an epitope, wherein the epitope is heterogeneous to the capsid protein, the epitope or a portion thereof specifically binds to an antibody paratope, and the recombinant viral capsid protein, or viral capsid containing the recombinant viral capsid, has, for example, multispecificity, or intrinsic targeting which is selectively reduced or inactivated in the absence of a bispecific binding moiety.

[0018] In some embodiments, insertion and / or presentation of a heterologous epitope reduces or neutralizes the (innate) directivity of the viral capsid compared to a standard viral capsid lacking the heterologous epitope. For example, the heterologous epitope is inserted (presented) into the recombinant viral capsid protein such that the viral capsid includes mutations involving the insertion of an epitope at an amino acid position and / or the substitution of an amino acid with an epitope at an amino acid position, and the mutation reduces or neutralizes the (innate) directivity of the capsid protein, for example, in the absence of a bispecific binding site, or selectively. In some embodiments, the insertion and / or presentation of a heterologous epitope is inserted (presented) into the viral capsid protein such that, for example, multispecifically and optionally in the absence of a bispecific binding site, the heterologous epitope partially reduces the (innate) targeting of the recombinant viral capsid compared to a standard viral capsid lacking the heterologous epitope, and the viral capsid further includes further mutations (e.g., substitutions, deletions, insertions other than insertion of a heterologous epitope) that further reduce and / or neutralize the (innate) targeting of the recombinant viral capsid or recombinant viral vector containing it, for example, multispecifically and optionally in the absence of a bispecific binding site, compared to a standard viral capsid lacking the mutation.

[0019] Generally, the recombinant viral capsid proteins described herein may be derived from a capsid gene, encoded by a capsid gene modified to express an epitope, and / or genetically modified non-enveloped viruses that generally infect human cells, such as adenoviruses and adeno-associated viruses, or serotypes of non-enveloped viruses that generally infect human cells. In some embodiments, the recombinant viral capsid proteins described herein are AAV serotypes of genetically modified adeno-associated virus (AAV) capsid proteins derived from an AAV capsid gene, encoded by a capsid gene modified to express an epitope, and / or infect primates, wherein AAV is optionally selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant viral capsid protein is derived from the AAV2, AAV6, AAV8, or AAV9 capsid gene, for example, a genetically modified AAV2 capsid protein, a genetically modified AAV6 capsid protein, a genetically modified AAV8 capsid protein, or a genetically modified AAV9 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from the AAV2 capsid gene, for example, a genetically modified AAV2 VP1, VP2, and / or VP3 capsid protein encoded by an AAV2 capsid gene modified to express an epitope, and / or the amino acid sequence of the wild-type AAV2 VP1 protein is represented as Sequence ID No. 1, respectively. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV6 VP1, VP2, and / or VP3 capsid protein derived from the AAV6 capsid gene, for example, encoded by an AAV6 capsid gene modified to express an epitope, and / or having the amino acid sequence of wild-type AAV6 VP1 represented as Sequence ID No. 3.In some embodiments, the recombinant viral capsid protein is a genetically modified AAV8 VP1, VP2, and / or VP3 capsid protein derived from the AAV8 capsid gene, for example, encoded by an AAV8 capsid gene modified to express an epitope, and / or the amino acid sequence of the wild-type AAV VP1 protein is represented as SEQ ID NO: 21, respectively. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9 VP1, VP2, or VP3 capsid protein derived from the AAV9 capsid gene, for example, encoded by an AAV9 capsid gene modified to express an epitope, and / or the wild-type amino acid sequence of AAV9 VP1 is represented as SEQ ID NO: 5, respectively. In some embodiments, the recombinant viral capsid protein is derived from the AAV2 capsid gene, for example, encoded by an AAV2 capsid gene modified to express an epitope, and / or is a genetically modified VP1, VP2, and / or VP3 capsid protein of AAV2. In some embodiments, the recombinant viral capsid protein is derived from the AAV6 capsid gene, for example, encoded by an AAV6 capsid gene modified to express an epitope, and / or is a genetically modified VP1, VP2, and / or VP3 capsid protein of AAV6. In some embodiments, the recombinant viral capsid protein is derived from the AAV8 capsid gene, for example, encoded by an AAV8 capsid gene modified to express an epitope, and / or is a genetically modified VP1, VP2, and / or VP3 capsid protein of AAV8. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene and is encoded by, for example, an AAV9 capsid gene modified to express an epitope, and / or is a genetically modified VP1, VP2, and / or VP3 capsid protein of AAV9.

[0020] In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV capsid gene modified to express an epitope, for example, encoded by a modified chimeric AAV capsid gene, the chimeric AAV capsid gene comprising multiple nucleic acid sequences, each of which encodes 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 viral capsid protein is derived from the chimeric AAV8 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from the chimeric AAV9 capsid gene.

[0021] Generally, the recombinant viral capsid proteins described herein include heterologous epitopes inserted into and / or presented by the recombinant capsid protein, compared to a standard capsid containing a heterologous epitope or a standard capsid lacking a capsid, such that the heterologous epitope itself reduces and / or inactivates the innate targeting of the recombinant capsid protein or the capsid containing it. In some embodiments, the heterologous epitope is inserted (presented) into a region of the capsid protein that is involved in the innate targeting of the wild-type standard capsid protein, e.g., a region of the capsid protein involved in cell targeting. In some embodiments, the heterologous epitope is inserted into and / or presented by the knob domain of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the HI loop of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the heparin-binding site of the AAV capsid protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the heparin-binding site of the AAV2 capsid protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the heparin-binding site of the AAV6 capsid protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the heparin-binding site of the AAV8 capsid protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the heparin-binding site of the AAV9 capsid protein. In some embodiments, (i) the viral capsid protein is derived from the AAV2 capsid gene, and the epitope is inserted after and / or replaces the amino acid at the corresponding position in the AAV2 VP1 capsid protein, i.e., the amino acid at position I453 or I587, and / or the amino acid at the corresponding position in the AAV2 VP2 and / or VP3 capsid proteins.(ii) The viral capsid protein is derived from the AAV6 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position I585 of the AAV6 VP1 capsid protein, and / or the amino acid at the corresponding position of the AAV6 VP2 and / or VP3 capsid proteins; (iii) The viral capsid is derived from the AAV8 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position I590 of the AAV8 VP1 capsid protein, and / or the amino acid at the corresponding position of the AAV8 VP2 and / or VP3 capsid proteins; or (iv) The viral capsid protein is derived from the AAV9 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position I453 or I589 of the AAV9 VP1 capsid protein, and / or AAV9 The heterologous epitope is inserted after the amino acid at the corresponding position in the VP2 and / or VP3 capsid protein and / or replaces that amino acid. In some embodiments, the heterologous epitope is G453 of the AAV2 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or the corresponding amino acid in 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), N587 of the AAV2 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded from the same capsid gene) The corresponding amino acids in 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, at the corresponding positions of the protein, Q585 of the AAV6 capsid protein VP1 (or the corresponding positions of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, e.g., VP1, VP2, of different AAVs that infect humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9,(and / or the corresponding amino acid of the VP3 capsid protein), N590 of the AAV8 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 AAV9), G453 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 different AAVs that infect humans, e.g., It is inserted immediately after an amino acid selected from the group consisting of the corresponding amino acids of the VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8 (or A589 of the VP1 of the AAV9 capsid protein) (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) (e.g., fused at the C-terminus). In some embodiments, the heterologous epitope is inserted immediately after G453 of the AAV2 capsid protein VP1 (or at the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or immediately after 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, AAV8, and AAV9) (e.g., fused at the C-terminus). In some embodiments, the heterologous epitope is inserted immediately after N587 of the AAV2 capsid protein VP1 (or at the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or immediately after 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,The heterologous epitope is inserted immediately after (e.g., fused to the C-terminus) the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of AAV7, AAV8, and AAV9. In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus) the Q585 of the AAV6 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or immediately after (e.g., fused to the C-terminus) the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9. In some embodiments, the heterologous epitope is inserted (e.g., fused to the C-terminus) immediately after N590 of the AAV8 capsid protein VP1 (or at the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or immediately after 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 AAV9). In some embodiments, the heterologous epitope is inserted immediately after G453 of the AAV9 capsid protein VP1 (or at the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or at 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 at the C-terminus). In some embodiments, the heterologous epitope is inserted immediately after A589 of the AAV9 capsid protein VP1 (or at the corresponding position of the VP2 and / or VP3 capsid proteins encoded from the same capsid gene, or at the corresponding position 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 at the C-terminus).It is inserted immediately after (e.g., fused to the C-terminus) the corresponding amino acid of the VP3 capsid protein (and / or the VP3 capsid protein). In some embodiments, the heterologous epitope is inserted between amino acids N587 and R588 of the AAV2 VP1 capsid protein (or at the corresponding positions of the VP2 and / or VP3 capsids encoded from the same capsid gene) and / or presented by the AAV2 VP1 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: 2. 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: 4. 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 encoded by a nucleic 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 encoded by a nucleic acid sequence represented as SEQ ID NO: 26. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence encoded by a nucleic acid sequence represented as SEQ ID NO: 27.

[0022] In some embodiments, the recombinant capsid proteins described herein include a second distinct mutation in addition to a heterologous epitope. For example, in some embodiments, the recombinant viral capsid proteins described herein may be genetically modified AAV2 capsid proteins that include a heterologous epitope and may further include mutations, e.g., R585A and / or R588A mutations. In some embodiments, the recombinant viral capsid proteins are derived from the AAV2 capsid gene, e.g., genetically modified AAV2 VP1 capsid proteins that include a heterologous epitope inserted immediately after G453 of the AAV2 VP1 protein (e.g., fused to the C-terminus of G453) and further include mutations selected from the group consisting of R585A and / or R5889A. In some embodiments, the recombinant viral capsid protein is derived from the AAV2 capsid gene, for example, a genetically modified AAV2 VP1 capsid protein, and includes a heterologous epitope inserted immediately after N587 of the AAV2 VP1 protein (e.g., fused to the C-terminus of N587), and further includes a mutation selected from the group consisting of R585A and / or R588A. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein, and includes a heterologous epitope inserted immediately after G453 of the AAV9 VP1 protein (e.g., fused to the C-terminus of G453), and further includes a W503A mutation. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein, and includes a heterologous epitope inserted immediately after A589 in the AAV9 VP1 protein (for example, fused to the C-terminus of A589), and further includes the W503A mutation.

[0023] Generally, a viral vector containing recombinant viral capsid protein and / or recombinant viral capsid contains a heterologous epitope that is at least 1 amino acid long. In some embodiments, the heterologous epitope may be about 5 to 35 amino acids long and form a binding pair with an antibody paratope (e.g., an immunoglobulin variable domain). In some embodiments, the heterologous epitope contains at least 10 amino acids long. In some embodiments, the heterologous epitope contains an affinity tag. In some embodiments, the heterologous epitope and / or affinity tag do not form a binding pair with an immunoglobulin constant domain. In some embodiments, the heterologous epitope and / or affinity tag contain a metal ion, e.g., Ni 2+ Co 2+ Cu 2+ Zn 2+ Fe 3+ It does not form binding pairs with such as. In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A. In some embodiments, the affinity tag is selected from the group consisting of FLAG (SEQ ID NO: 7), HA (SEQ ID NO: 8), and c-myc (EQKLISEEDL, SEQ ID NO: 6). In some embodiments, the heterologous epitope includes c-myc (EQKLISEEDL, SEQ ID NO: 6).

[0024] In some embodiments, the recombinant viral capsid protein is a genetically modified AAV2 VP1 capsid protein and includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after G453 of the AAV2 VP1 capsid protein (e.g., fused to the C-terminus of G453). In some embodiments, the recombinant viral capsid protein (i) is derived from the AAV2 capsid gene and is, for example, a genetically modified AAV2 VP1 capsid protein; (ii) includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and inserted immediately after G453 of the AAV2 VP1 capsid protein (e.g., fused to the C-terminus of G453); and (iii) further includes a mutation selected from the group consisting of R585A and / or R5889A. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV2 VP1 capsid protein and includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after N587 of the AAV2 VP1 capsid protein (e.g., fused to the C-terminus of N587). In some embodiments, the recombinant viral capsid protein (i) is derived from the AAV2 capsid gene and is, for example, a genetically modified AAV2 VP1 capsid protein; (ii) includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and inserted immediately after N587 of the AAV2 VP1 capsid protein (e.g., fused to the C-terminus of N587); and (iii) further includes a mutation selected from the group consisting of R585A and / or R588A. In some embodiments, the recombinant viral capsid protein is (i) derived from the AAV6 capsid gene, for example, a genetically modified AAV6 VP1 capsid protein, and (ii) includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) which is inserted immediately after Q585 of the AAV6 VP1 capsid protein (for example, fused to the C-terminus of Q585).In some embodiments, the recombinant viral capsid protein is a genetically modified AAV8 VP1 capsid protein and includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after N590 of the AAV8 VP1 capsid protein (e.g., fused to the C-terminus of N590). In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9 VP1 capsid protein and includes a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after G453 of the AAV9 VP1 capsid protein (e.g., fused to the C-terminus of G453). In some embodiments, the recombinant viral capsid protein is (i) derived from the AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein, (ii) comprising a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after G453 of the AAV9 VP1 capsid protein (for example, fused to the C-terminus of G453), and (iii) further comprising the W503A mutation. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9 VP1 capsid protein, comprising a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after A589 of the AAV9 VP1 capsid protein (for example, fused to the C-terminus of A589). In some embodiments, the recombinant viral capsid protein (i) is derived from the AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) which is inserted immediately after A589 in the AAV9 VP1 capsid protein (for example, fused to the C-terminus of A589), and (iii) further comprises the W503A mutation.

[0025] In some embodiments, the recombinant viral capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I587 of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein, and includes, for example, an amino acid sequence represented as SEQ ID NO: 2.

[0026] In some embodiments, the recombinant viral capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV6 VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I585 of the AAV6 VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted between Q585 and S586 of the AAV6 VP1 capsid protein, and includes, for example, an amino acid sequence represented as SEQ ID NO: 4.

[0027] In some embodiments, the recombinant viral capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV8 VP1 capsid. In some embodiments, the recombinant viral capsid includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I590 of the AAV8 VP1 capsid protein. In some embodiments, the recombinant viral capsid includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted between N590 and T591 of the AAV8 VP1 capsid protein, and includes, for example, an amino acid sequence represented as SEQ ID NO: 25.

[0028] In some embodiments, the recombinant viral capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I453 of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted between G453 and S454 of the AAV9 VP1 capsid protein, and includes an amino acid sequence represented, for example, SEQ ID NO: 26. In some embodiments, the recombinant viral capsid includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I589 of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted between A589 and Q590 of the AAV9 VP1 capsid protein, and includes, for example, the amino acid sequence represented as SEQ ID NO: 27.

[0029] In some embodiments, the heterologous epitope comprises an affinity tag and one or more linkers. In some embodiments, the heterologous epitope comprises an affinity tag adjacent to a linker, for example, the heterologous epitope comprises a first linker, an affinity tag, and a second linker, from the N-terminus to the C-terminus. In some embodiments, the first and second linkers are each independent polypeptides with at least one amino acid length. In some embodiments, the heterologous epitopes described herein, for example, the affinity tag itself, or the affinity tag combined with one or more linkers, are about 5 to about 35 amino acid lengths. In some embodiments, the first and second linkers are of the same length and / or contain the same amino acid sequence.

[0030] In general, recombinant viral capsids containing recombinant viral capsid proteins as described herein have reduced or neutralized intrinsic targeting, and their ability to target and bind to standard cells that are naturally tolerant to transduction is reduced or impossible, compared to standard viral capsids, e.g., capsids containing standard viral capsid proteins, e.g., viral capsid proteins identical to recombinant viral capsid proteins but lacking heterologous epitopes. In some embodiments, and with appropriate multispecificity, and in the absence of optional bispecificity binding molecules, recombinant viral capsids containing recombinant viral capsid proteins as described herein exhibit transduction efficiency reduced by at least 10% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optional bispecificity binding molecules, recombinant viral capsids containing recombinant viral capsid proteins as described herein exhibit transduction efficiency reduced by at least 20% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 30% lower than that of standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 40% lower than that of standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 50% lower than that of standard viral capsids.In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids containing the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 60% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids containing the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 70% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids containing the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 75% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids containing the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 80% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 85% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 90% compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and in the absence of optionally bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit a transduction efficiency of at least 95% compared to standard viral capsids.In some embodiments, and with appropriate multispecificity, and optionally in the absence of bispecificity binding molecules, recombinant viral capsids comprising the recombinant viral capsid protein described herein exhibit at least 99% lower transduction efficiency compared to standard viral capsids. In some embodiments, and with appropriate multispecificity, and optionally in the absence of bispecificity binding molecules, transduction of control cells by recombinant viral capsids comprising the recombinant viral capsid protein described herein is ineffective, for example, undetectable.

[0031] In some embodiments, the viral capsid comprising the recombinant viral capsid protein described herein is a mosaic capsid comprising, for example, a recombinant viral capsid protein containing heterologous epitopes and a standard capsid protein not containing heterologous epitopes in a certain ratio. In some embodiments, the standard capsid protein is a wild-type standard 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 standard capsid protein is a control standard capsid protein in that it contains the amino acid sequence of the recombinant viral capsid protein except that the control standard capsid protein lacks heterologous epitopes. In some embodiments, the standard capsid protein is a mutant wild-type standard protein in that it contains substantially the same amino acid sequence as that of a wild-type capsid protein having the same serotype as the recombinant viral capsid protein, except that it contains mutations that reduce the directivity of the wild-type capsid protein (e.g., amino acid sequence insertions, chimerization, etc.). In some embodiments, the compositions described herein contain recombinant viral capsid protein and standard capsid protein in a ratio ranging from 1:1 to 1:15, or the methods described herein combine them in this way. 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.

[0032] Also disclosed herein are nucleic acids encoding recombinant viral capsid proteins disclosed herein (for example, which can be used in methods for producing recombinant viral capsids) and / or compositions comprising recombinant viral capsid proteins (for example, compositions essentially consisting of recombinant viral capsid proteins, compositions comprising only viral vectors encapsulated by a capsid containing the viral capsid proteins described herein, compositions comprising such viral vectors with multispecificity, optionally in a specific ratio of bispecificity binding molecules (molecule:molecule), compositions comprising such viral vectors with multispecificity, optionally bispecificity binding molecules, compositions comprising such viral vectors, retargeting molecules, and pharmaceutically acceptable carriers). In some embodiments, the nucleic acids described herein comprise a nucleotide sequence encoding the amino acid sequence of EQKLISEEDL (SEQ ID NO: 6) and a nucleotide sequence encoding at least five consecutive amino acids of an adenovirus or adeno-associated viral capsid protein.

[0033] In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV2 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I587 of the AAV2 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein, for example, in some embodiments, the nucleic acids described herein encode an amino acid sequence including an amino acid sequence represented as SEQ ID NO: 2.

[0034] In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV6 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I585 of the AAV6 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted between Q585 and S586 of the AAV6 VP1 capsid protein, for example, in some embodiments, the nucleic acids described herein encode an amino acid sequence including an amino acid sequence represented as SEQ ID NO: 4.

[0035] In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV8 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I590 of the AAV8 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted between N590 and T591 of the AAV8 VP1 capsid protein, for example, in some embodiments, the nucleic acids described herein encode an amino acid sequence including an amino acid sequence represented as SEQ ID NO: 25.

[0036] In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV9 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I453 of the AAV9 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted between G453 and S454 of the AAV9 VP1 capsid protein, for example, in some embodiments, the nucleic acids described herein encode an amino acid sequence including an amino acid sequence represented as SEQ ID NO: 26. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6) inserted at I589 of the AAV9 VP1 capsid protein. In some embodiments, the nucleic acids described herein include a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO: 6), which is inserted between A589 and Q590 of the AAV9 VP1 capsid protein. For example, in some embodiments, the nucleic acids described herein encode an amino acid sequence including an amino acid sequence represented as SEQ ID NO: 27.

[0037] Generally, the recombinant viral vectors described herein comprise a viral capsid containing the recombinant viral capsid protein described herein, the viral capsid encapsulating 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.

[0038] Generally, the target nucleotide may be one or more genes, which may encode a detectable marker, such as a reporter or 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, etc. In one embodiment, the target nucleotide encodes a protein for multi-domain therapy, for example, a protein having at least two domains that provide two distinct functions.

[0039] The compositions described herein generally comprise a viral vector comprising a recombinant viral capsid protein as described herein, for example, a capsid comprising a recombinant viral capsid protein in which the capsid encapsulates a target nucleotide. In some embodiments, the compositions described herein comprise (1) a viral vector having a capsid comprising a recombinant viral capsid protein genetically modified to contain a heterologous epitope, (2) a multispecific, optionally bispecific binding molecule comprising (i) an antibody paratope that specifically binds to the epitope, and (ii) a retargeting ligand that specifically binds to a receptor, and optionally, (3) a pharmaceutically acceptable carrier.

[0040] The antibody paratopes described herein generally contain, at a minimum, a complementarity-determining region (CDR) that specifically recognizes a heterogeneous epitope, such as a CDR3 region of the heavy chain and / or light chain variable domain. In some embodiments, the multispecific, optionally bispecific binding molecule comprises an antibody (or a portion thereof) containing an antibody paratope that specifically binds to a heterogeneous epitope. For example, the multispecific, optionally bispecific binding molecule may contain a single-domain heavy chain variable region or a single-domain light chain variable region, and the single-domain heavy chain variable region or single-domain light chain variable region contains an antibody paratope that specifically binds to a heterogeneous epitope. In some embodiments, the multispecific, optionally bispecific binding molecule may contain an Fv region, for example, the multispecific, optionally bispecific binding molecule may contain an scFv containing an antibody paratope that specifically binds to a heterogeneous epitope. In some embodiments, the multispecific, or optionally bispecific binding molecule comprises an antibody (or a portion thereof) containing an antibody paratope that specifically binds to a heterogeneous epitope, wherein the antibody (or a portion thereof) further comprises one or more antibody constant domains (e.g., heavy chain constant domains (e.g., CH1, hinge, CH2, CH3, CH4, etc.) and / or light chain constant domains (e.g., CL)), and one or more antibody constant domains that do not bind to the heterogeneous epitope.

[0041] The multispecific, or optionally bispecific binding molecules described herein further comprise a retargeting ligand in addition to a paratope (e.g., an antibody or portion thereof containing a paratope) that specifically binds to a heterologous epitope inserted / presented on a recombinant viral capsid protein. In some embodiments, the retargeting ligand specifically binds to a receptor on the surface of beads (e.g., for isolation and / or purification of the recombinant viral capsid protein described herein). In some embodiments, the retargeting ligand specifically binds to cell surface proteins, e.g., receptors, cell surface markers, etc., expressed on the surface of mammalian (e.g., human) eukaryotic cells, e.g., target cells. In some embodiments, the retargeting ligand binds to (human) hepatocytes, (human) brain cells, (human) T cells, (human) kidney cells, (human) intestinal cells, (human) pancreatic cells, (human) cancer cells, and / or (human) cells infected with a heterologous pathogen. In some embodiments, the retargeting ligand binds to (human) hepatocyte-specific markers, (human) brain cell-specific markers, (human) T cell-specific markers, (human) kidney cell-specific markers, (human) intestinal cell-specific markers, (human) pancreatic cell-specific markers, (human) tumor cell-specific markers, and / or pathogenic epitopes.

[0042] In some embodiments, the retargeting ligand binds to receptors expressed by (human) hepatocytes, such as the asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the retargeting ligand binds to receptors expressed by (human) nerve cells, such as GABA, transferrin, etc. In some embodiments, the retargeting ligand binds to receptors expressed by (human) T cells, such as CD3, e.g., CD3ε. In some embodiments, the retargeting ligand binds to receptors expressed by (human) hematopoietic stem cells, such as CD34. In some embodiments, the retargeting ligand binds to receptors expressed by (human) kidney cells. In some embodiments, the retargeting ligand binds to receptors expressed by (human) muscle cells, such as integrin, etc. In some embodiments, the retargeting ligand is a receptor expressed by (human) cancer cells, e.g., tumor-associated antigens, e.g., adipophyllin, AIM-2, ALDH1A1, alpha-actin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin A1, dek-can Fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH(hMena), Ep-CAM, EpCAM, EphA3, Epithelial carcinoma 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, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A,KMHN1, also known as KK-LC-1, KKLC1, KM-HN-1, 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, malate enzyme, mammurobin - A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, Mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin Class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secretin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4 STEAP1, Survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, telomerase, TGF-beta-RII, 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], gp100, tyrosinase, TRP-1 / gp75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); mucin [MUC-1] (in breast cancer, pancreatic cancer, colon cancer,It binds to shared 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, TRP2-INT2, etc. In some embodiments, the retargeting ligand binds to E6 and / or E7. In some embodiments, the retargeting ligand binds to Her2. In some embodiments, the retargeting ligand binds to the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds to human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

[0043] In some embodiments, the paratope (e.g., an antibody or a portion thereof) and the retargeting ligand are directly fused to each other. In some embodiments, the paratope (e.g., an antibody or a portion thereof) that specifically binds to a heterologous epitope and the retargeting ligand are covalently bonded to each other.

[0044] In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., an antibody comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain comprises a paratope that specifically binds to a heterologous epitope inserted / presented in a recombinant viral capsid protein, and the second antigen-binding domain specifically binds to a cell surface protein expressed by a target cell. In some embodiments, the bispecific binding molecule is a bispecific antibody comprising a first and a second antigen-binding domain, wherein the first antibody-binding domain comprises a paratope that specifically binds to a heterologous epitope inserted / presented in a recombinant viral capsid protein, the second antigen-binding domain specifically binds to a receptor expressed by a target cell, the first antigen-binding domain is operably linked to a first heavy chain region comprising a first CH3 domain, the second antibody-binding domain is operably linked to a second heavy chain region comprising a second CH3 domain, and the first and second Ig C H 3 domains are different from each other by at least one amino acid, and at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A, and the second Ig C H 3 domain contains a mutation that reduces or abolishes protein A binding, such as the H95R modification (H435R in EU numbering according to IMGT exon numbering). The second C H 3 domain may further comprise a Y96F modification (Y436F in EU according to IMGT). The second C HFurther modifications that may be found within the three domains include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in the EU); for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT; N384S, K392N, and V422I in the EU); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in the EU).

[0045] In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecific antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to an affinity tag presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to a receptor expressed on the surface of a target cell. In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecific antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to a receptor expressed on the surface of a target cell. In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecific antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to hASGR1. In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecificity antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to a CD3 protein, for example, CD3ε. In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecificity antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to an integrin.In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecificity antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to an integrin, for example, hGCGR. In some embodiments, the multispecificity conjugate molecule is a bispecificity conjugate molecule, for example, a bispecificity antibody comprising first and second antigen-binding domains, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) presented by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to ENTPD3.

[0046] Furthermore, methods for preparing and using recombinant viral capsid proteins, viral vectors containing them, compositions, etc., are described herein. In some embodiments, for example, a method for reorienting viruses such as adenoviruses and adeno-associated viruses to deliver diagnostic / therapeutic cargo to target cells comprises combining a recombinant viral vector containing the recombinant viral capsid protein described herein, for example, a viral vector containing a capsid containing a recombinant viral capsid that presents a heterologous epitope, with a bispecificity binding molecule, the bispecificity binding molecule comprising (i) an antibody paratope that specifically binds to the epitope, and (ii) a retargeting ligand that specifically binds to a receptor. Such a method may include, as a first step in generating the recombinant viral vector, for example, culturing packaging cells under conditions sufficient for generating the viral vector, the packaging cells containing a plasmid encoding a capsid protein containing the epitope. When delivering a diagnostic / therapeutic cargo to target cells, the methods described herein may involve contacting target cells with a combination of a viral vector comprising a capsid containing a recombinant viral capsid that presents a heterologous epitope and a multispecific binding molecule, wherein the multispecific binding molecule comprises (i) an antibody paratope that specifically binds to the epitope, and (ii) a retargeting ligand that specifically binds to a receptor expressed by the target cell. 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.

[0047] In some embodiments, the composition described herein includes, or the method described herein combines, a recombinant viral vector containing a target nucleotide encapsulated in a capsid containing the recombinant capsid protein described herein, and a multispecific binding molecule, in a molecule-to-molecule ratio that restores the transduction efficiency of the viral vector to that of a standard viral vector. In some embodiments, the recombinant viral vector to multispecific binding molecule ratio (molecule:molecule) is in the range of 1:0.5 to 1:100. In some embodiments, the recombinant viral vector to multispecific binding molecule ratio (molecule:molecule) is in the range of 1:4 to 1:20. In some embodiments, the recombinant viral vector to bispecific binding molecule ratio (molecule:molecule) is in the range of 1:8 to 1:15. In some embodiments, the recombinant viral vector to multispecific binding molecule ratio (molecule:molecule) is 1:4. In some embodiments, the recombinant viral vector to multispecific binding molecule ratio (molecule:molecule) is 1:8. In some embodiments, the recombinant viral vector to multispecific binding molecule ratio (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is 1:20.

[0048] Also described herein are methods for inactivating a viral capsid and / or generating a viral vector, the methods generally comprising (a) inserting a nucleic acid encoding a heterologous protein into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein containing the heterologous protein, and / or (b) culturing packaging cells under conditions sufficient to generate a viral vector, wherein the packaging cells contain the nucleotide sequence. In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid containing a 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) treating the supernatant containing the viral vector with DNase I and MgCl2, (c) concentrating the viral vector, (d) purifying the viral vector, and (e) any combination of (a) to (d). Also provided herein are 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 the recombinant capsid protein described herein.

[0049] In certain embodiments, the following items are provided, for example: (Item 1) A recombinant viral capsid protein containing an epitope, wherein the epitope is heterogeneous to the capsid protein. The heterologous epitope or a portion thereof specifically binds to the antibody paratope. Recombinant viral capsid protein, wherein the viral capsid protein forms a recombinant viral capsid with reduced or inactivated natural targeting. (Item 2) The recombinant viral capsid protein described in item 1, wherein the epitope is at least one amino acid long. (Item 3) The recombinant viral capsid protein according to item 1 or 2, comprising substitutions, insertions, or deletions at amino acid positions involved in the innate directivity of the viral capsid, such that the recombinant viral capsid protein forms a reduced or inactivated viral capsid. (Item 4) The recombinant viral capsid protein described in items 1 to 3, wherein the viral capsid protein is derived from an adeno-associated virus (AAV) capsid gene, and the heterologous epitope is optionally inserted at a position selected from the group consisting of I587 of AAV2, I585 of AAV6, I590 of AAV8, I453 of AAV9, I589 of AAV9, and any corresponding amino acid of an AAV serotype that infects primates, and the AAV serotype that infects primates is optionally selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 5) The recombinant viral capsid protein described in item 4, wherein the adeno-associated virus is AAV2. (Item 6) The recombinant viral capsid protein described in item 4, wherein the adeno-associated virus is AAV6. (Item 7) The recombinant viral capsid protein described in item 4, wherein the adeno-associated virus is AAV8. (Item 8) The recombinant viral capsid protein described in item 4, wherein the adeno-associated virus is AAV9. (Item 9) (i) The viral capsid protein is a genetically modified AAV2 VP1 capsid protein, and the epitope is inserted after the amino acid at position I453 or I587 and / or replaces that amino acid. (ii) The viral capsid protein is a genetically modified AAV6 VP1 capsid protein, and the epitope is inserted after the amino acid at position I585 and / or replaces that amino acid. (iii) The viral capsid is a genetically modified AAV8 VP1 capsid protein, and the epitope is inserted after the amino acid at position I590 and / or replaces that amino acid. (iv) The recombinant viral capsid protein according to any one of items 1 to 8, wherein the viral capsid protein is a genetically modified AAV9 VP1 capsid protein, and the epitope is inserted after and / or replaces the amino acid at position I453 or I589. (Item 10) The recombinant viral capsid protein according to any one of items 1 to 9, wherein the viral capsid protein is encoded by an AAV2 capsid gene modified to express the epitope located between amino acids N587 and R588 of the AAV2 VP1 capsid protein. (Item 11) The recombinant viral capsid protein according to any one of items 1 to 9, wherein the viral capsid protein is derived from the AAV6 capsid gene and the epitope is inserted immediately after amino acid Q585. (Item 12) A recombinant viral capsid protein according to any one of items 1 to 9, wherein the viral capsid protein is derived from the AAV8 capsid gene, and the epitope is inserted immediately after amino acid N590 of the AAV8 VP1 capsid protein. (Item 13) The recombinant viral capsid protein according to any one of items 1 to 9, wherein the viral capsid protein is derived from the AAV9 capsid gene, the epitope is inserted into the capsid protein after the amino acid at position G453 or A589 of the AAV9 VP1 capsid protein and / or replaces that amino acid, and the capsid protein further comprises additional mutations. (Item 14) The aforementioned additional mutation is W503A, which is the recombinant viral capsid protein described in item 13. (Item 15) The recombinant viral capsid protein according to any one of items 1 to 14, wherein the epitope comprises the amino acid sequence EQKLISEEDL, and the amino acid sequence or a portion thereof specifically and optionally binds to the antibody paratope. (Item 16) The epitope comprises the amino acid sequence EQKLISEEDL, and the amino acid sequence or a portion thereof specifically binds to the antibody paratope. A recombinant viral capsid protein according to any one of items 1 to 15, wherein the amino acid sequence EQKLISEEDL is adjacent to and operably linked to at least five consecutive amino acids of the AAV capsid protein. (Item 17) The recombinant viral capsid protein according to item 16, wherein the viral capsid protein comprises the amino acid sequence represented as SEQ ID NO: 2, the amino acid sequence represented as SEQ ID NO: 4, the amino acid sequence represented as SEQ ID NO: 25, the amino acid sequence represented as SEQ ID NO: 26, or the amino acid sequence represented as SEQ ID NO: 27. (Item 18) The recombinant viral capsid protein, or the viral capsid containing the recombinant viral capsid protein, is unable to infect target cells in the absence of the multispecific binding molecule including the antibody paratope, and selectively, in the absence of the multispecific binding molecule including the antibody paratope, the transduction efficiency of the viral capsid protein, or the viral capsid containing the recombinant viral capsid protein, is (i) Reduced by at least 10%, (ii) Reduced by at least 20%, (iii) Reduced by at least 30%, (iv) Reduced by at least 40%, (v) Reduced by at least 50%, (vi) Reduced by at least 60%, (vii) Reduced by at least 70%, (viii) Reduced by at least 80%, (ix) Reduced by at least 90%, or (x) Recombinant viral capsid proteins described in any one of items 1-17 that are deactivated. (Item 19) An isolated nucleic acid containing a nucleotide sequence encoding a recombinant viral capsid protein as described in any one of items 1 through 18. (Item 20) The isolated nucleic acid described in item 19, which encodes a recombinant viral capsid protein, wherein the nucleotide sequence comprises the amino acid sequence represented as SEQ ID NO: 2, the amino acid sequence represented as SEQ ID NO: 4, the amino acid sequence represented as SEQ ID NO: 25, the amino acid sequence represented as SEQ ID NO: 26, or the amino acid sequence represented as SEQ ID NO: 27. (Item 21) A recombinant viral vector comprising a target nucleotide encapsulated by a recombinant viral capsid described in item 20, wherein the recombinant viral capsid is optionally a mosaic capsid. (Item 22) The recombinant viral vector described in item 21, 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 23) The recombinant viral vector described in item 22, wherein the target nucleotide is under the control of a non-human promoter. (Item 24) The recombinant viral vector described in item 23, wherein the target nucleotide is adjacent to the AAV ITR sequence. (Item 25) A recombinant viral vector as described in any one of items 21 to 24, wherein the target nucleotide is a reporter gene. (Item 26) The recombinant viral vector described in item 25, wherein the reporter gene encodes a green fluorescent protein. (Item 27) A recombinant viral vector according to any one of items 21 to 24, wherein the target nucleotide is selected from the group consisting of a suicide gene, an antibody or a fragment thereof, a nucleotide that codes for the CRISPR / Cas system or a part(s) of it, an antisense RNA, an siRNA, and combinations thereof. (Item 28) A composition comprising (a) a recombinant viral vector as described in any one of items 21 to 27, and (b) a multispecificity binding molecule comprising an antibody paratope that specifically binds to an epitope, wherein the multispecificity binding molecule optionally further comprises a retargeting ligand that specifically binds to a receptor expressed on a target cell, and the multispecificity binding molecule optionally is a bispecificity binding molecule. (Item 29) The composition according to item 28, wherein the viral vector and the multispecific binding molecule are present in a ratio of 1:4. (Item 30) The composition according to item 28 or 29, wherein the antibody paratope is an Fv domain. (Item 31) The composition according to item 30, wherein the Fv domain is directly fused to the heavy chain constant domain. (Item 32) The composition according to any one of items 28 to 31, wherein the retargeting ligand is an antibody or a portion thereof. (Item 33) The multispecific binding molecule is a bispecific antibody. The composition according to any one of items 28 to 32, wherein the paratope and the retargeting ligand each comprise separate Fv domains fused to first and second heavy chain constant domains. (Item 34) The composition according to item 33, wherein the first and second heavy chains bind to protein A with different binding affinities. (Item 35) The multispecific binding molecule is a bispecific antibody. The retargeting ligand comprises a tetrameric antibody structure containing two identical immunoglobulin heavy chains and two identical light chains. The composition according to any one of items 28 to 32, wherein the paratope bound to a heterologous epitope is attached to the C-terminus or N-terminus of one or both heavy chains, and / or attached to the C-terminus or N-terminus of one or both heavy chains. (Item 36) The composition according to item 35, wherein the paratope is scFv, and optionally, the scFv comprises the amino acid sequence represented as SEQ ID NO: 37. (Item 37) The composition according to any one of items 28 to 36, wherein the heterologous epitope comprises the amino acid sequence EQKLISEEDL or a portion thereof, and the antibody paratope specifically binds to the amino acid sequence EQKLISEEDL or a portion thereof. (Item 38) The composition according to any one of items 28 to 37, wherein the retargeting ligand specifically binds to a cell surface protein that is a cell surface marker. (Item 39) The composition according to item 38, wherein the cell surface marker is asialoclycoprotein 1 (ASGR1). (Item 40) The composition according to item 38, wherein the cell surface marker is CD3. (Item 41) The composition according to item 38, wherein the cell surface marker is ENTPD3. (Item 42) A composition according to any one of items 28 to 41, further comprising a pharmaceutically acceptable carrier. (Item 43) A method for orienting a recombinant viral vector described in any one of items 21 to 27 into target cells, This includes contacting the recombinant viral vector with a multispecific binding molecule, A method wherein the multispecific binding molecule comprises (i) an antibody paratope that specifically binds to an epitope, and (ii) a retargeting ligand that specifically binds to a protein expressed on the surface of the target cell. (Item 44) The method described in item 43, wherein the target cells are present in vivo. (Item 45) The method described above, which involves ex vivo, is performed as described in item 43 or item 44. (Item 46) A method for delivering a target nucleotide to a target cell expressing a cell surface protein, comprising contacting the target cell with a composition described in any one of items 28 to 42, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein. (Item 47) The method described in item 46, wherein the target cells are located outside the body. (Item 48) The method described in item 46, wherein the target cells are present in the organism of the subject. (Item 49) The method described in 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 cell. (Item 51) The method according to any one of items 46 to 50, wherein the target cells are selected from the group consisting of hepatocytes, brain cells, T cells, kidney cells, intestinal cells, pancreatic cells, cancer cells, and cells infected with a different pathogen. (Item 52) The method according to any one of items 46 to 51, wherein the target cells are human liver cells. (Item 53) The method according to any one of items 46 to 52, wherein the cell surface protein is human asialoglycoprotein receptor 1 (hASGR1). (Item 54) The method according to any one of items 46 to 51, wherein the target cell is a human T cell. (Item 55) The method according to item 54, wherein the cell surface protein is CD3. (Item 56) The method according to any one of items 46 to 51, wherein the cell surface protein is human glucagon receptor (hGCGR). (Item 57) The method according to any one of items 46 to 50, wherein the target cell is an intestinal cell. (Item 58) The method according to any one of items 46 to 50, wherein the target cells are pancreatic cells. (Item 59) The method according to items 57 and 58, wherein the cell surface protein is ENTPD3. (Item 60) A method for inactivating viral capsid proteins, (a) Inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein containing the heterologous epitope, (b) A method comprising culturing packaging cells under conditions sufficient for the production of a viral vector, wherein the packaging cells contain the nucleotide sequence. (Item 61) A method for generating a viral vector, comprising culturing packaging cells under conditions sufficient for generating a viral vector, wherein the packaging cells comprise a plasmid encoding a recombinant capsid protein as described in any one of items 1 to 18. (Item 62) The method according to item 60 or item 61, wherein the packaging cells further comprise a transfer plasmid containing a helper plasmid and / or a target nucleotide. (Item 63) The method according to any one of items 60 to 62, further comprising isolating a self-complementary adeno-associated virus vector from the culture supernatant. (Item 64) The method according to any one of items 60 to 63, further comprising lysing the packaging cells and isolating a single-stranded adeno-associated virus vector from the cell lysate. (Item 65) a. Removal of cell fragments, b. Processing the supernatant containing the viral vector with DNase I and MgCl2, c. Enriching the viral vector, d. Purifying the aforementioned viral vector, The method described in any one of items 60 to 64, further including any combination of ea to d. (Item 66) A viral vector prepared according to the method described in any one of items 60-65. (Item 67) Packaging cells for generating a viral vector containing a plasmid encoding the capsid protein described in any one of items 1 through 18. (Item 68) A binding molecule comprising a paratope bound to Sequence ID No. 6, wherein the paratope comprises the scFv of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 sequence, which is encoded by the nucleic acid sequence represented as Sequence ID No. 28. (Item 69) The binding molecule described in item 68, wherein the antibody paratope is an Fv domain. (Item 70) The binding molecule described in item 69, wherein the Fv domain is directly fused to the heavy chain constant domain. (Item 71) The aforementioned binding molecule is a bispecific antibody and further contains a retargeting ligand. The binding molecule according to any one of items 68 to 70, wherein the paratope and the retargeting ligand each comprise separate Fv domains fused to the first and second heavy chain constant domains, respectively. (Item 72) The binding protein described in item 71, wherein the first and second heavy chains bind to protein A with different binding affinities. (Item 73) The aforementioned binding molecule is a bispecific antibody and further contains a retargeting ligand. The retargeting ligand comprises a tetrameric antibody structure containing two identical immunoglobulin heavy chains and two identical light chains. The binding protein according to either item 68 or 69, wherein the paratope is attached to the C-terminus or N-terminus of one or both heavy chains, and / or to the C-terminus or N-terminus of one or both heavy chains. (Item 74) The binding protein according to item 73, wherein the paratope is scFv, and optionally, the scFv comprises the amino acid sequence represented as SEQ ID NO: 37. (Item 75) A binding protein according to any one of items 69 to 72, further comprising an antibody or a retargeting ligand which is a portion thereof. (Item 76) The binding protein described in item 75, wherein the retargeting ligand specifically binds to a cell surface protein that is a cell surface marker. (Item 77) The binding protein described in item 76, wherein the cell surface marker is asialoclycoglycoprotein 1 (ASGR1). (Item 78) The binding protein described in item 76, wherein the cell surface marker is CD3. (Item 79) The binding protein described in item 76, wherein the cell surface marker is GCGR. (Item 80) The binding protein described in item 76, wherein the cell surface marker is ENTPD3. (Item 81) A composition comprising (a) a recombinant viral vector as described in any one of items 21 to 27, and (b) a binding protein as described in any one of items 68 to 81. (Item 82) The composition according to item 81, wherein the viral vector and the multispecific binding molecule are present in a ratio of 1:4. (Item 83) The composition according to item 81 or item 82, further comprising a pharmaceutically acceptable carrier. This patent or application includes at least one drawing prepared in color. Copies of this patent or publication of this patent application, accompanied by the color drawing(s), will be provided to the authorities upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0050] [Figure 1-1] The following images are provided: (A) wild-type scAAV2-CMV-eGFP viral vector only, (B) scAAV2-N587Myc-CMV-hrGFP viral vector only, and scAAV2-N587Myc viral vector mixed with a bispecific anti-Myc-ASGR1 antibody in the following ratios: (C) 1:0.5, (D) 1:1, (E) 1:2, (F) 1:4, (G) 1:8, (H) 1:15, (I) 1:20, (J) 1:50, or (K) 1:100, (L), or scAAV-N587Myc viral vector mixed with a monospecific anti-Myc antibody in a ratio of 1:8. These images show the evaluation of green fluorescence (GFP) expression in HepG2 cells cultured with these vectors. Immunofluorescence microscopy images (upper panel) or histograms (lower panel) obtained from fluorescence-activated cell sorting (FACS). [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2A-1] We provide dot plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescence (GFP) expression in 29T3-hASGR1 cells cultured with wild-type scAAV2 virus vector alone (i), scAAV2-N587Myc-CMV-eGFP virus vector alone (ii), scAAV2-N587Myc-CMV-eGFP virus vector mixed with bispecific anti-Myc-ASGR1 antibody in the following ratios: 1:0.5 (iii), 1:1 (iv), 1:2 (v), 1:4 (vi), 1:8 (vii), 1:15 (viii), 1:20 (ix), or 1:100 (x), or scAAV2-N587Myc-CMV-eGFP virus vector mixed with an unrelated bispecific anti-Myc-GCGR antibody in a ratio of 1:8 (xii). GFP expression (xi) by 29T3 cells cultured with scAAV-N587Myc viral vector mixed with bispecific anti-myc-ASGR1 antibody in a 1:8 ratio has also been shown. Each experiment used 2 x 10⁵ cells and 5 x 10⁹ viral vectors. [Figure 2A-2] Same as above. [Figure 2B-1] We provide histograms obtained from fluorescence-activated cell sorting (FACS) evaluations of green fluorescence (GFP) expression in 29T3-hASGR1 cells cultured with the following ratios of AAV9-A589Myc-CAGG-eGFP viral vector (i) alone, AAV9-A589Myc-CAGG-eGFP viral vector (ii) alone, and AAV9-A589Myc-CAGG-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody in the following ratios: 1:1 (iii), 1:2 (iv), 1:4 (v), 1:8 (vi), 1:20 (vii), 1:50 (viii), or 1:100 (ix). Each experiment used 2 x 10⁵ cells and 1 x 10¹⁰ viral vector (tittached by qPCR). [Figure 2B-2] Same as above. [Figure 3-1] We provide dot plots obtained from fluorescence-activated cell sorting (FACS) to evaluate green fluorescence (GFP) expression in 29T3-hASGR1 cells pre-cultured with bivalent anti-ASGR1 antibody at concentrations of 0 nM (C), 50 nM (D), 10 nM (E), 2 nM (F), 0.4 nM (G), 0.08 nM (H), 0.016 nM (I), or 0.0032 nM (J), and in (L) 29T3-hASGR1 cells subsequently infected with a scAAV2-N587Myc-CMV-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody in a 1:8 ratio. 293T3-hASGR1 cells cultured with wild-type scAAV viral vector alone (A) or scAAV2-N587Myc-CMV-eGFP viral vector alone (B) serve as controls. Each experiment used 2 x 10⁵ cells and 5 x 10⁹ viral vectors (tittached by qPCR). [Figure 3-2] Same as above. [Figure 4]This document provides immunofluorescence microscopy images evaluating green fluorescence (GFP) expression in 293T-hASGR1 cells cultured with wild-type scAAV virus vector alone (A), scAAV2-N587Myc-CMV-eGFP virus vector alone (B), or subsequently with 1x10⁹ (C), 2x10⁹ (D), 4x10⁹ (E), 8x10⁹ (F), 2x10¹⁰ (G), 1x10¹¹ (H), and 1x10¹² (I) bispecific anti-Myc-ASGR1 antibodies, followed by 1x10⁹ scAAV2-N587Myc-CMV-eGFP virus vector. Also shown are immunofluorescence microscopy images of 293T cells (J) cultured sequentially with a 1x10¹¹ anti-myc-ASGR1 antibody molecule, followed by a 1x10⁹ scAAV2-N587Myc-CMV-eGFP viral vector, and 293T-hASGR1 cells (K) cultured sequentially with a 1x10¹¹ unrelated bispecific anti-Myc-GCGR antibody molecule, followed by a 1x10⁹ scAAV2-N587Myc-CMV-eGFP viral vector. [Figure 5-1] We provide dot plots obtained from fluorescence-activated cell sorting (FACS) to evaluate green fluorescence (GFP) expression in 29T3-hASGR1 cells cultured with wild-type ssAAV virus vector alone (A), ssAAV2-N587Myc-CMV-hrGFP virus vector alone (B), ssAAV2-N587Myc-CMV-hrGFP virus vector mixed with bispecific anti-Myc-ASGR1 antibody in the following ratios: 1:1 (C), 1:2 (D), 1:4 (E), 1:8 (F), 1:20 (G), 1:100 (H), 1:1000 (I), or ssAAV-N587Myc virus vector mixed with an unrelated bispecific anti-myc-GCGR antibody in a ratio of 1:8 (K). GFP expression (J) by 29T3 cells cultured with ssAAV-N587Myc viral vector mixed with bispecific anti-Myc-ASGR1 antibody in a 1:8 ratio is also shown. Each experiment used 2 x 10⁵ cells and 5 x 10⁹ viral vectors. [Figure 5-2] Same as above. [Figure 6-1]We provide dot plots obtained from fluorescence-activated cell sorting (FACS) to evaluate green fluorescence (GFP) expression in 29T3-hGCGR cells cultured with the following: wild-type ssAAVP virus vector only (A), scAAV2-N587Myc-CMV-eGFP virus vector only (B), scAAV2-N587Myc-CMV-eGFP virus vector mixed with bispecific anti-Myc-GCGR antibody in the following ratios: 1:0.5 (C), 1:1 (D), 1:2 (E), 1:4 (F), 1:8 (G), 1:15 (H), 1:20 (I), 1:50 (J), or 1:100 (K), or scAAV2-N587Myc-CMV-eGFP virus vector mixed with an unrelated monospecific anti-Myc antibody (Regeneron Pharmaceuticals, Tarrytown, NY) in a ratio of 1:8 (L). Each experiment used 2 x 10⁵ cells and a 5 x 10⁹ viral vector. [Figure 6-2] Same as above. [Figure 7-1] We provide dot plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescence (GFP) expression in Jurkat cells cultured with Jurkat cells alone (A), or with only wild-type scAAV6-EF1-eGFP viral vector (B), only AAV6-Q585Myc-EF1a-eGFP viral vector (C), or AAV6-Q585Myc-EF1a-eGFP viral vector mixed with bispecific anti-Myc-CD3 antibody in the following ratios: 1:1 (D), 1:5 (E), 1:10 (F), 1:100, or (G), 1:1000 (H). Each experiment used 2 x 10⁵ cells and 1 x 10⁹ viral vector. [Figure 7-2] Same as above. [Figure 8A] Immunofluorescence microscopy images of the liver are provided. [Figure 8B] Immunofluorescence microscopy images of the spleen are provided. [Figure 8C]Immunofluorescence microscopy images of the kidney are provided. All samples are collected from C57BL / 6 mice (i-iv) genetically transformed to express human ASGR1 by hepatocytes, or wild-type C57BL / 6 mice (v-viii), 10 days after intravenous injection of 1x10¹¹ wild-type scAAV2-CMV-eGFP (i, v), physiological saline (ii, vi), 1x10¹¹ scAAV2-N587myc-CMV-eGFP viral vector alone (iii, vii), or scAAV2-N587myc-CMV-eGFP viral vector containing bispecific anti-myc-ASGR1 antibody (iv, vii). [Figure 9-1] This document provides immunofluorescence microscopy images of liver samples taken from C57BL / 6 mice (D-F, J-L, P-R) genetically transformed to express human ASGR1 on hepatocytes, or from wild-type C57BL / 6 mice (A-C, G-I, M-O), four weeks after intravenous injection of 2.18x10¹¹ wild-type ssAAV2-CAGG-eGFP (B, C, E, F), physiological saline (A, D), 2.18x10¹¹ ssAAV2-N587myc-CAGG-eGFP viral vector alone (G-I, J-L), or ssAAV2-N587myc-CAGG-eGFP viral vector containing bispecific anti-myc-ASGR1 antibody (M-O, P-R). Each image represents one mouse. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 10] We provide immunofluorescence microscopy images of liver samples taken from C57BL / 6 mice that were genetically transformed to express human ASGR1 in hepatocytes 10 days after intravenous injection of (A) wild-type AAV9, (B) 250 nM NaCl, (C) AAV9-A589myc-CAGG-eGFP virus particles combined with a bispecific anti-myc-hCD3 antibody, or (D) AAV9-A589myc-CAGG-eGFP virus particles combined with a bispecific anti-myc-ASGR1 antibody. [Figure 11]This invention provides a graphical, non-scalable, and non-limiting format of multispecificity-binding molecules, useful in some embodiments of the present invention. [Figure 12-1] We provide dot plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescence (GFP) expression in 29T3-hASGR1 cells cultured with (A) wild-type AAV8 virus vector alone, (C) AA8-N590-myc virus vector alone, or pAAV RC8 N590myc virus vector mixed with the bispecific anti-hASGR1-IgG4-Fc / anti-myc bispecific molecule in the following ratios: (D) 1:1, (E) 1:2, (F) 1:4, (G) 1:8, (H) 1:12, (I) 1:15, (J) 1:50, or (K) 1:100. GFP expression in simulated transfected 29T3-hASGR1 cells (B) is also shown. Each experiment used 2 x 10⁵ cells and 1 x 10⁹ virus vector. [Figure 12-2] Same as above. [Figure 13] (A)-(C) wild-type AAV8, (D)-(F) AA8-N590-myc viral vector and control bispecificity conjugate molecule, or (G)-(I) anti-hASGR1-IgG4-Fc / anti-myc bispecificity conjugate molecule. Immunofluorescence microscopy images are provided of liver samples taken from C57BL / 6 mice genetically transformed and modified to express human ASGR1 in hepatocytes 10 days after intravenous injection. [Figure 14-1]We provide dot plots obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescence (GFP) expression in 29T3-h ENTPD3 cells cultured with (A) wild-type AAV2 virus vector alone, (B) AAV2-N587Myc-CAGG-eGFP virus vector alone, or AAV2-N587Myc-CAGG-eGFP virus vector mixed with the following bispecific anti-hENTPD3-IgG4-Fc / anti-myc bispecific molecules in the following ratios: (C) 1:1, (D) 1:2, (E) 1:4, (F) 1:8, (G) 1:20, (H) 1:50, (I) 1:100, or (K) 1:200. Each experiment used 2 x 10⁵ cells and 1 x 10⁹ virus vector. [Figure 14-2] Same as above. [Figure 15A] Immunofluorescence microscopy images of liver samples are provided. [Figure 15B] Immunofluorescence microscopy images of intestinal samples are provided. [Figure 15C] Fluorescence microscopy images of pancreatic specimens are provided. All specimens are collected from wild-type C57BL / 6 mice 10 days after intravenous injection of PBS (15A(i), 15B(i), and 15C(i)), 5x10¹¹ wild-type AAV9 (15A(ii), 15B(ii), and 15C(ii)), 5x10¹¹ AAV2-N587myc-CAGG-eGFP viral vector (15A(iii), 15B(iii), and 15C(iii)) containing 1x10³ unrelated bispecific IgG4-Fc / anti-myc binding protein, or 5x10¹¹ AAV2-N587myc-CAGG-eGFP viral vector (15A(iv), 15B(iv), and 15C(iv)) containing 1x10¹³ bispecific hENTPD3-IgG4-Fc / anti-myc binding protein. [Modes for carrying out the invention]

[0051] A common problem with adapter techniques using unmodified or scaffold-modified viral capsids is the suboptimal transduction efficiency of the modified capsid (Grifman et al. (2001) Mol. Ther. 3:964-75). For example, Curiel et al. described the generation and characterization of recombinant adenovirus vectors containing a fiber in which the RGD-4C sequence was genetically incorporated within the HI loop of the carboxy-terminal knob domain, demonstrating the usefulness of the fiber knob's HI loop as an optimal site for incorporating short peptide ligands. See, for example, U.S. Patent No. 7,297,542, and also Beatty and Curiel (2012) Adv. Cancer Res. 115:39-67. Similarly, insertion of ligand peptides into AAV capsid proteins allows for the presentation of ligands on the capsid surface, mediating transduction through the interaction between the receptor and the ligand, thereby resulting in capsids that reorient the virus-directivity of genetically modified capsids (Girod et al. (1999) Nat. Med. 5(9):1052-6, 1438 (errata included) (1999), Grifman et al. (2001) Mol. Ther. 3(6):964-75, Nicklin et al. (2001) Mol. Ther. 4(3):174-81, Shi et al. (2001) Hum Gene Ther. 17(3):353-61 (2006), Wu et al. (2000) J. Virol. 74(18):8635-47). Specifically, by inserting the integrin-binding Arg-Gly-Asp (RGD) motif into the insertion site I-587 of the AAV capsid protein VP1, α vIt has been demonstrated that β1 integrin enables the transduction of AAV virus vectors into cells (Girod et al. (1999) above). In contrast, insertion of the 14-amino acid peptide L14 after amino acid R447 (I-447) resulted in a capsid still recognizable by the conformation-sensitive antibody A20, but such recombinant viral vectors could not be transduced into cells expressing the L-14 receptor (Girod et al. 1999, see Wu et al. (2000) (successful transduction into cells expressing the HA peptide and insertion of hemagglutinin (HA) peptide at position I-447 has been reported)). Insertion of the Myc epitope between T448 and N449 was recognized by an anti-myc antibody and therefore present on the capsid surface, but resulted in an inactivated viral vector (Grifman et al., 2001). In contrast, successful retargeting by insertion of the NGR motif after N587 was again reported, but not with c-myc insertion after N587 (Grifman et al. (al., 2001). U.S. Patent No. 9,624,274 describes I-453 of the AAV capsid protein as a preferred insertion site for heterologous epitopes. While these studies demonstrate the successful insertion and presentation of heterologous peptides, such as epitopes, by the AAV capsid protein, none of these studies provide the prospect that multispecific binding molecules, such as bispecific antibodies, which specifically bind to heterologous peptides and cell surface proteins, can retarget modified viral vectors to cells expressing cell surface proteins and restore their transduction efficiency.

[0052] Disclosed herein are heterologously modified recombinant viral capsid proteins that can be used in conjunction with a multispecific binding molecule comprising an Fv domain that specifically binds to a paratope, such as an epitope, and a ligand that binds to a receptor expressed on the surface of a target cell. As shown herein, by contacting the multispecific binding molecule with a viral vector having a capsid formed of the capsid proteins described herein in a specific ratio, the transduction efficiency of the viral capsid can be restored to a level comparable to that of wild-type viruses (see, e.g., Example 2). Generally, the heterologously modified capsid proteins described herein may be derived from non-enveloped viruses such as adenoviruses (Ad) and adeno-associated viruses (AAV), but are not limited to these.

[0053] While the present invention is specifically illustrated and described with reference to numerous embodiments, those skilled in the art will understand that the form and details of the various embodiments disclosed herein may be modified without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to function to limit the claims.

[0054] Any methods and materials similar to 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 below. 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.

[0055] Definition of Terms 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.

[0056] The singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated by the context. Therefore, for example, a reference to “one method” includes one or more methods and / or processes of the kind described herein and / or which would be apparent to those skilled in the art by reading this disclosure.

[0057] 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 H 3, and optionally including CH4. Each light chain contains a light chain variable domain (C H ) and light chain constant region (C L ) is included. The heavy and light chain variable domains can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are inserted into more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure contains two identical antigen-binding domains, each of which is V H and V L It was formed through a meeting with each of the following, and each of them is a C H and C L It forms the antibody Fv region together with the other domains. A single-domain antibody has a single antigen-binding domain, for example, V H or V LThe term “antibody” includes monoclonal antibodies, multispecific (e.g., bispecific) antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bonded Fv (sdFv), intracellular antibodies, minibodies, diabodies, and anti-idiotype (anti-Id) antibodies (e.g., anti-ID antibodies against antigen-specific TCRs), and any of the epitope-binding fragments described above. The terms “antibody” and “antibodies” also refer to covalent diabodies, such as those disclosed in U.S. Patent Application Publication 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication 2009 / 0060910. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0058] The antigen-binding domain of an antibody, for example, the part of the antibody that recognizes and binds to an antigen's epitope, is also called a "paratope." It is a small region (5-10 amino acids) of the antibody's Fv region, i.e., part of the antigen-binding fragment (Fab region), and may contain portions of the antibody's heavy and / or light chain. A paratope binds specifically to an epitope when it binds to the epitope with high affinity. The term "high affinity" antibody means that it binds to its target epitope with approximately 10% affinity. -9 M or less (for example, approximately 1 x 10) -9 M, 1x10 -10 M, 1x10 -11 M, or approximately 1x10 -12 M)'s K D This refers to an antibody having K D This is measured by surface plasmon resonance, for example, BIACORE®, and in another embodiment, K DIt is measured by ELISA.

[0059] 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 by, for example, germline sequences or reconstituted or unreconstituted sequences, for example, by naive B cells or mature B cells, or T cells. CDRs can be somatically mutated (unlike sequences encoded in, for example, animal germline sequences), humanized, and / or modified by amino acid substitutions, additions, or deletions. Under certain circumstances (e.g., with respect to CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) which are not contiguous (e.g., in unreconstituted nucleic acid sequences) but are contiguous in B cell nucleic acid sequences as a result of, for example, sequence splicing or concatenation (e.g., VDJ rearrangement to form heavy chain CDR3).

[0060] An "epitope" is a portion of a macromolecule recognized by the immune system, particularly by antibodies, B cells, or cytotoxic T cells. Epitopes are usually thought to originate from non-self proteins, but recognized host-derived sequences are also classified as epitopes. Epitopes have a length of at least 4 amino acids, preferably 4 to 30 amino acids, more preferably 5 to 20 amino acids, and particularly 5 to 15 amino acids. Epitopes can be linear or three-dimensional, typically formed by amino acids that are far apart in the primary protein structure but become closer in the secondary and / or tertiary structure. Epitopes specifically recognized by B cells are called B-cell epitopes.

[0061] The term "inverted end repeat" 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 function as the origin of replication for viral DNA synthesis and are essential cis-components for generating the AAV integration vector.

[0062] 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 and λ light chains, as well as VpreB. Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, full-length light chains include variable domains consisting of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, arranged 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 and generally derive from a repertoire of V and J segments present in germline cells. L Segments and J L This includes segments. The sequences, locations, and nomenclature of V and J light chain segments from 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 a first or second epitope selectively bound by an epitope-binding protein present on the light chain. Light chains also include those that bind to and recognize, or assist in binding to, a heavy chain or another light chain, by binding to and recognizing one or more epitopes selectively bound by an epitope-binding protein present on the light chain. Common 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. Light chains can be constructed that include 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).

[0063] The terms “about” or “approximately” include being within a statistically significant range of values. 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 still more preferably within 5%. The acceptable variation encompassed by the terms “about” or “approximately” depends on the particular system under study and can be readily understood by those skilled in the art.

[0064] The term "affinity tag" includes, for example, polypeptide sequences that are members of a specific binding pair that specifically bind to another polypeptide sequence, such as a high-affinity antibody paratope. Exemplary and non-restrictive affinity tags include hexahistidine tags, FLAG tags, StrepII tags, streptavidin-binding peptide (SBP) tags, cartathione-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tags, HA tags, and c-Myc tags. (Validated in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8, and incorporated herein by reference).

[0065] The term “capsid protein” includes proteins that are part of the viral capsid. In adeno-associated viruses, the capsid proteins are generally referred to as VP1, VP2, and / or VP3, each encoded by a single cap gene. In AAV, the three AAV capsid proteins are generated in a redundant manner from the cap open reading frame (ORF) via alternative mRNA splicing and / or the use of alternative translation start codons, although all three proteins use a common stop codon. Warrington et al. (2004) J. Virol. 78:6595, the entire report 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, with VP2 production using a weaker ACG start codon (amino acid T138) and VP3, the most abundant capsid methane protein, using read-through translation to the next available ATG codon (amino acid M203). See Warrington, above; Rutledge et al. (1998) J. Virol. 72:309-19, the entire text of which 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, specifically in dependoparvovirus. See Rutledge et al., above. For example, Rutledge et al. (1998) provides the amino acid sequence alignments of the VP1, VP2, and VP3 capsid proteins of AAV2, AAV3, AAV4, and AAV6 in Figure 4B, where the respective start sites of the VP1, VP2, and VP3 capsid proteins are indicated by arrows, and variable domains are enclosed in boxes.Therefore, the amino acid positions provided herein may be provided in relation to the VP1 capsid protein of AAV, and furthermore, amino acid positions provided herein that are not specified refer to the AAV2 sequence of the major coat 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. In addition, 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".

[0066] Domain exchange between two AAV capsid protein constructs for the generation of a “chimeric AAV capsid protein” is described; see, for example, Shen et al. (2007) Mol. Therapy 15(11):1955-1962 (the entire work is incorporated herein by reference). A “chimeric AAV capsid protein” includes an AAV capsid protein that contains amino acid sequences, e.g., domains derived from two or more different AAV serotypes, and is capable of and / or forms an AAV-like viral capsid / viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a plurality of nucleotides comprising, e.g., at least two nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a distinct AAV serotype, and together they encode a functional chimeric AAV capsid protein. References regarding chimeric capsid proteins associated with specific AAV serotypes indicate 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 a capsid protein that includes one or more domains from the AAV2 VP1, VP2, and / or VP3 capsid proteins, and one or more domains from the VP1, VP2, and / or VP3 capsid proteins of a different AAV.

[0067] 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.

[0068] 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 inserting a nucleic acid sequence encoding a heterologous epitope, and further comprises VP1, VP2, and / or VP3 proteins encoded by a standard cap gene, e.g., a wild-type standard cap gene encoding wild-type VP1, VP2, and / or VP3 proteins of the same AAV serotype as recombinant VP1, VP2, and / or VP3 proteins, a standard cap gene encoding VP1, VP2, and / or VP3 proteins that are identical to recombinant VP1, VP2, and / or VP3 proteins but lack a heterologous epitope, and a mutant wild-type standard cap gene encoding wild-type VP1, VP2, and / or VP3 proteins of the AAV serotype that are substantially identical to recombinant VP1, VP2, and / or VP3 proteins except for mutations (e.g., insertions, substitutions, deletions) (the mutations preferably reduce the directivity of wild-type VP1, VP2, and / or VP3 proteins). In some embodiments, the standard capsid protein is a chimeric standard protein containing at least one domain of the VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins. In some embodiments, the standard cap gene encodes the chimeric VP1, VP2, and / or VP3 proteins.

[0069] The terms "heavy chain" or "immunoglobulin heavy chain" refer to an immunoglobulin heavy chain sequence that includes an immunoglobulin heavy chain constant region sequence derived from any organism. Unless otherwise specified, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. A heavy chain fragment includes CDRs, CDRs and FRs, and 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 epitopes (e.g., K in the micromolar, nanomolar, or picomolar range).D It is capable of recognizing epitopes having (a specific characteristic), is expressible and secretible from cells, and contains a fragment comprising at least one CDR. The heavy chain variable domain is encoded by a variable region nucleotide sequence and is generally present in germline cells. H , D H , and J H V derived from the segment repertoire H , D H , and J H This includes segments. The sequences, locations, and nomenclature of V, D, and J heavy chain segments of various organisms can be found in the IMGT database, which is accessible globally via the internet at the URL "imgt.org" (www).

[0070] 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 containing a variable domain that is manipulably linked to the 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 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) comprising two immunoglobulin-like chains, each functionally C H This includes both homodimeric single-domain antigen-binding proteins, which contain 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 functionally C1. HIt contains the same variable domain that is operably linked to the same heavy chain constant region lacking one domain. In addition, each immunoglobulin-like chain of a single-domain antigen-binding protein contains a variable domain, which is a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segment (e.g., V L , J L ), or combinations thereof, may originate from the heavy chain constant region (and optionally the hinge region) of a gene encoding, for example, IgG, IgA, IgE, IgD, or combinations thereof. H Heavy chain constant region containing deletion or inactivation mutation in 1 (C H ) Can be linked to gene sequences. Single-domain antigen-binding proteins containing variable domains derived from heavy chain gene segments are "V H These may be referred to as "single-domain antibodies" or "VH single-domain antibody-binding proteins," see, for example, U.S. Patent No. 8,754,287, U.S. Patent Publication Nos. 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 referred to as "VH single-domain antibodies." L These may be referred to as "single-domain antigen-binding proteins," see, for example, U.S. Publication No. 2015 / 0289489 (the entire publication is incorporated by reference).

[0071] 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 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 consisting of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus, and the light chain constant region amino acid sequence. The light chain variable domain is encoded by the light chain variable region nucleotide sequence and generally derives from a repertoire of light chain V and J gene segments present in germline cells, light chain V L and light chain J L This includes segments. The sequences, locations, and nomenclature of light chain V gene segments and light chain J gene segments from various organisms can be found in the IMGT database, which is accessible via the internet on the global web (www) at the URL "imgt.org". Light chains include, for example, light chains that do not selectively bind to either a first or second epitope selectively bound by an epitope-binding protein present on the light chain. Light chains also include those that bind to and recognize or assist in binding to a heavy chain by the binding and recognition of one or more epitopes selectively bound by an epitope-binding protein present on the light chain. Light chains also include those that bind to and recognize or assist in binding to a heavy chain by the binding and recognition of one or more epitopes selectively bound by an epitope-binding protein present on the light 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).

[0072] When 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 are positioned to be involved in a biological event, and that this juxtaposition achieves or enables such interaction and / or positioning. For example, a regulatory sequence in a nucleic acid (e.g., an expression regulatory sequence) is said to be “operably linked” to the coding sequence if it is located in such a position that its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, “operable linkage” includes covalent bonding of related components or elements. However, those skilled in the art will readily understand that in some embodiments, covalent bonding is not necessary to achieve effective operable linkage. For example, in some embodiments, a nucleic acid regulatory sequence that is operably linked to the coding sequence it controls is contiguous with the nucleotide of interest. Alternatively or in addition, in some embodiments, one or more such regulatory sequences function trans or at a distance to control the coding sequence of interest. In some embodiments, the term “expression regulatory sequence,” as used herein, refers to polynucleotide sequences necessary and / or sufficient to influence the expression and processing of the coding sequence to which they are ligated. In some embodiments, expression regulatory sequences may be, or 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 improve translation efficiency (e.g., Kozak consensus sequences), sequences that improve protein stability, and / or, in some embodiments, sequences that improve protein secretion. In some embodiments, one or more regulatory sequences are preferentially or exclusively 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 from this context that in many embodiments, the term “regulatory sequence” refers to a component whose presence is essential for expression and processing, and in some embodiments, includes components whose presence is advantageous for expression (e.g., including leader sequences, targeting sequences, and / or fusion partner sequences).

[0073] The term “recombinant capsid protein” includes a capsid protein having at least one mutation compared to the corresponding capsid protein of a wild-type virus, which may be a standard and / or control virus for comparative studies. Recombinant capsid proteins include capsid proteins containing heterologous epitopes that may be inserted into and / or presented by the capsid protein. “Heterologous” in this context means heterologous to the virus from which the capsid protein originates. The inserted amino acid may simply be inserted between two given amino acids of the capsid protein. Amino acid insertion may also involve the deletion of a given amino acid of the capsid protein at the insertion site (for example, one or more capsid protein amino acids may be replaced by five or more heterologous amino acids).

[0074] Terms such as “multispecific binding molecule” and “bispecific binding molecule” generally refer to binding molecules containing at least two and only two non-identical binding components, respectively, where each binding component specifically binds to either a different epitope—two different molecules (e.g., different epitopes on two different immunogens) or the same molecule (e.g., different epitopes on the same immunogen). Generally, in this specification, one of the binding components of a bispecific binding molecule specifically binds to a heterologous epitope presented by a viral capsid protein, and the second binding component is specific to a target cell, for example, a protein primarily and / or preferentially expressed by a T cell marker (e.g., CD3, CD28, etc.), such as a cell surface marker. Bispecific binding molecules can be constructed, for example, by combining binding components that recognize different epitopes on the same immunogen. For example, nucleic acid sequences encoding binding components that recognize different epitopes (e.g., light chain or heavy chain variable sequences) may be fused to nucleic acid sequences encoding the same or different heavy chain constant regions (multiple), the same or different light chain constant regions (multiple), or one heavy chain constant region and one light chain constant region, and such sequences may be expressed intracellularly as multispecific antigen-binding proteins in a format similar to Fab structures, scFab structures, diabody structures, scFv structures, scFv-Fc structures, scFv-zipper structures, tetramer structures similar to those of typical antibodies containing a general-purpose light chain of the same genus, tetramer structures similar to those of typical bivalent antibodies containing a general-purpose light chain of the same genus, and / or additional binding components (e.g., scFV, scFV-zipper structure, scFab, etc.) attached to one or both of the heavy chains (e.g., N-terminus and / or C-terminus) or one or both of the light chains (e.g., N-terminus and / or C-terminus). Various formats of multispecific, specifically bispecific, binding molecules are well known; see, for example, Brinkmann and Konterman (2017) Mabs 9:182-212 (the entire work is incorporated herein by reference).

[0075] An example of a multispecific molecule is a heavy chain CDR followed by (from the N-terminus to the C-terminus) C H 1 domain, hinge, C H 2 domains, and C HIt comprises two heavy chains, each having three domains, and either an immunoglobulin light chain (e.g., a general light chain) that does not confer epitope binding specificity but can associate with each heavy chain, or an immunoglobulin light chain that can associate with each heavy chain and can bind to one or more epitopes bound by the heavy chain epitope binding region, or an immunoglobulin light chain that can associate with each heavy chain and can bind one or both of the heavy chains to one or both of the epitopes. In some embodiments, the multispecific binding molecule comprises (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region containing a first CH3 amino acid sequence of human IgG selected from IgG1, IgG2, IgG4, and combinations thereof, and (2) an immunoglobulin heavy chain variable domain, the second immunoglobulin heavy chain variable domain operably linked to a second heavy chain constant region containing a second CH3 amino acid sequence of human IgG selected from IgG1, IgG2, IgG4, and combinations thereof, wherein the first or second heavy chain variable domain (with or without a congener light chain) is specified herein The modifications include binding to a heterologous epitope described herein, other heavy chain variable domains (with or without a congeneral light chain) binding to a receptor on the target cell, and the first heavy chain constant region associating with the second constant chain region in a manner that provides easy isolation of the multispecific binding protein, for example, the first and second heavy chain constant regions forming a knobs-into-hole (KIH) format, or the second CH3 amino acid sequence reducing or eliminating the binding of the second CH3 amino acid sequence to protein A (see, for example, U.S. Patent No. 8,586,713, which is incorporated herein by reference in its entirety).In some embodiments, the multispecificity binding molecule comprises (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region containing a first CH3 amino acid sequence of human IgG selected from IgG1, IgG2, IgG4, and combinations thereof, and (2) an immunoglobulin heavy chain variable domain, the second immunoglobulin heavy chain variable domain operably linked to a second heavy chain constant region containing a second CH3 amino acid sequence of human IgG selected from IgG1, IgG2, IgG4, and combinations thereof, wherein the first and second heavy chain variable domains (with or without congeneral light chains) are the same or different. The protein binds to an antigen, and the first or second heavy chain constant region is modified to further include an additional binding domain (e.g., an scFV or Fv that binds to a heterologous epitope as described herein, e.g., an additional binding domain is added to the C-terminus or N-terminus of one or both heavy chains), and the first heavy chain constant region associates with the second constant chain region in a manner that provides easy isolation of the multispecific binding protein, and the first and second heavy chain constant regions form a knob-into-hole (KIH) format, or the second CH3 amino acid sequence is modified to reduce or eliminate the binding of the second CH3 amino acid sequence to protein A. In some embodiments, the second CH3 amino acid sequence includes the H95R modification (according to IMGT exon numbering, H435R in EU numbering). In one embodiment, the second CH3 amino acid sequence further includes the Y96F modification (according to IMGT exon numbering, H436F in EU numbering). In another embodiment, the second CH3 amino acid sequence includes both the H95R modification (IMGT exon numbering, H435R in EU numbering) and the Y96F modification (IMGT exon numbering, H436F in EU numbering). In some embodiments, the second CH3 amino acid sequence is derived from modified human IgG1 and further includes mutations selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT, D356E, L38M, N384S, K392N, V397M, and V422I in EU numbering).In some embodiments, the second CH3 amino acid sequence is derived from modified human IgG2 and further includes mutations selected from the group consisting of N44S, K52N, and V82I (according to IMGT, N384S, K392N, and V422I in the EU). In some embodiments, the second CH3 amino acid sequence is derived from modified human IgG4 and further includes mutations selected from the group consisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in the EU). In some embodiments, the heavy chain constant region amino acid sequence is a non-human constant region amino acid sequence and includes one or more of the types of modifications described above.

[0076] In various embodiments, the Fc domain is modified to have altered Fc receptor binding, thereby affecting effector function. In some embodiments, the constructed heavy chain constant region (CH) containing the Fc domain is chimeric. Thus, the chimeric CH region combines CH domains derived from two or more immunoglobulin isotypes. For example, the chimeric CH region includes some or all of the CH2 domains derived from human IgG1, human IgG2, or human IgG4 molecules, combined with some or all of the CH3 domains derived from human IgG1, human IgG2, or human IgG4 molecules. In some embodiments, the chimeric CH region contains a chimeric hinge region. For example, a chimeric hinge may include a combination of an "upper hinge" amino acid sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 216-227 according to EU numbering, and amino acid residues at positions 226-240 according to Kabat numbering) and a "lower hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues at positions 228-236 according to EU numbering, and amino acid positions 241-249 according to Kabat numbering). In some embodiments, the chimeric hinge region includes amino acid residues derived from the human IgG1 or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge.

[0077] In some embodiments, the Fc domain may be constructed to activate all, some, or none of the normal Fc effector functions without affecting the desired pharmacokinetic properties of the Fc-containing protein (e.g., an antibody). For an example of a protein containing a chimeric CH region and having modified effector functions, see WO2014 / 022540, which is incorporated in its entirety herein.

[0078] The term “target cell” includes any cell on which the expression of the target nucleotide is desirable. Preferably, target cells exhibit on their surface proteins, such as receptors, that enable targeting using retargeting ligands, as described below. The targeted protein, e.g., receptor, is preferably specific to the target cell, e.g., “cell-specific marker,” “cell-specific antigen,” etc. Terms such as “cell-specific marker,” “cell-specific antigen,” “organ-specific marker,” and “tissue-specific marker” include those proteins whose expression is enriched by cells, tissues, and / or organs, and which are specific markers for cells, tissues, and / or organs. In the context of protein expression, “enriched” refers to and includes the expression or overexpression of cell / tissue / organ-specific proteins, primarily, preferentially, or simply by cells / tissues / organs whose protein is a specific marker, but such markers may also be expressed at minimal levels by other cells / tissues / or organs. The Human Protein Atlas can be used to determine whether a protein is a cell / tissue / organ-specific marker and also provides a repository of cell / tissue / organ-specific proteins. (See reference) www.proteinatlas.org See also Uhlen et al. (2010) Nat. Biotech. 28:1248-50 (the entire work is incorporated herein by reference).

[0079] Terms such as "transduction" or "infection" refer to the introduction of nucleic acids into target cells by a viral vector. The term "transduction efficiency," which relates to the efficiency of transduction, refers to the fraction (e.g., percentage) of cells that express the target nucleotide after culturing, relative to the number of viral vector sets containing the target nucleotide. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting of transduced cells using a fluorescent reporter gene, and PCR for the expression of the target nucleotide.

[0080] Where used herein, the term “wild-type” includes real-world entities that possess the structure and / or activity found in nature in a “normal” state or circumstances (against mutants, diseases, alterations, etc.). Those skilled in the art will understand that wild-type viral vectors, e.g., wild-type capsid proteins, can be used as standard viral vectors in comparative studies. Generally, a standard viral capsid protein / capsid / vector is identical to a test viral capsid protein / capsid / vector except for the alteration whose effect is being tested. For example, to determine the effect of inserting a heterologous epitope into a test viral vector, e.g., transduction efficiency, the transduction efficiency of the test viral vector (in the absence or presence of appropriate multispecificity binding molecules) can be compared (in the absence or presence of appropriate multispecificity binding molecules, if necessary) to the transduction efficiency of a standard viral vector, which is identical to the test viral vector in all cases (e.g., additional mutations, target nucleotides, number of viral vectors and target cells, etc.), except for the presence of the heterologous epitope.

[0081] Recombinant viral capsid proteins and viral vectors, as well as nucleic acids 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 contains a heterologous epitope in the fiber protein domain, for example, in the fiber protein, fiber knob, and / or at the carboxyl terminus of the HI loop of the fiber knob.

[0082] In some embodiments, the recombinant viral capsid proteins described herein are derived from adeno-associated virus (AAV) capsid genes and are, for example, genetically modified capsid proteins 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, the AAV6 capsid gene, the AAV8 capsid gene, or the AAV9 capsid gene. In some embodiments, the recombinant viral capsid proteins are derived from the AAV2 capsid gene and are, for example, genetically modified AAV2 VP1 capsid proteins, the wild-type amino acid sequences of which are represented as Sequence ID No. 1, respectively. In some embodiments, the recombinant viral capsid protein is derived from the AAV8 capsid gene, for example, the genetically modified AAV8 VP1 capsid protein, whose wild-type amino acid sequence is represented as SEQ ID NO: 21. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, for example, the genetically modified AAV9 VP1 capsid protein, whose wild-type amino acid sequence is represented as SEQ ID NO: 5. In some embodiments, the recombinant viral capsid protein is derived from the AAV6 capsid gene, for example, the genetically modified VP1 capsid protein of AAV6. In some embodiments, a heterologous epitope is inserted at I-453 of the AAV9 capsid protein.

[0083] In general, the recombinant viral capsid proteins described herein include heterologous epitopes inserted into and / or presented by the capsid protein such that the heterologous epitope reduces and / or disables the innate targeting of the capsid protein or the capsid containing it. In some embodiments, the heterologous epitope is inserted into a region of the capsid protein that is involved in the innate targeting of the wild-type standard capsid protein, for example, a region of the capsid protein involved in cell receptors. In some embodiments, the heterologous epitope is inserted into and / or presented by the knob domain of the Adfiber protein. In some embodiments, the heterologous epitope is inserted into and / or presented by the HI loop of the Adfiber protein. In some embodiments, a heterologous epitope 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, Q585 of AAV6 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the heterologous epitope is inserted between amino acids N587 and R588 of the AAV2 VP1 capsid and / or presented therein. In some embodiments, a recombinant viral capsid, a viral vector containing a recombinant viral capsid, and / or a composition containing a recombinant viral capsid includes an amino acid sequence represented as SEQ ID NO: 2. In some embodiments, a recombinant viral capsid, a viral vector containing a recombinant viral capsid, and / or a composition containing a recombinant viral capsid includes an amino acid sequence represented as SEQ ID NO: 4. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence encoded by a nucleic acid sequence represented as Sequence 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 encoded by a nucleic acid sequence represented as SEQ ID NO: 26. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence encoded by a nucleic acid sequence represented as SEQ ID NO: 27. Additional 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 containing specific epitopes inserted at positions 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. Additional 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 proteins described herein may be AAV2 capsid proteins containing heterologous epitopes inserted at positions selected from the group consisting of I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), I-585(AAV5), and combinations thereof.

[0084] As used herein, the nomenclature I-### refers to an insertion site that names the number of amino acids in the AAV capsid protein relative to the VP1 protein as ###. Such insertions may be located directly at the N-terminus or C-terminus, preferably at the C-terminus of one of the five amino acids in the sequence of a given amino acid, preferably at three, more preferably two, and especially at the N-terminus or C-terminus of one(s) amino acids. In addition, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and their mutations) can be easily identified in the VP2 and VP3 capsid proteins encoded by the capsid gene by performing sequence alignment of the VP1, VP2, and VP3 proteins encoded by the standard AAV capsid gene.

[0085] Therefore, when the capsid protein is encoded, by superimposing reading frames of the same gene with shifted start codons, insertions into the corresponding positions of the nucleic acid encoding one of these sites in the cap gene lead to insertions into VP1, VP2, and / or VP3. Thus, for example, in AAV2, according to this nomenclature, insertions of amino acids 1-138 are inserted only into VP1, insertions of 138-203 are inserted into both VP1 and VP2, and insertions of 203-C-terminus are inserted into VP1, VP2, and VP3, and this naturally also applies to insertion site I-587. Therefore, the present invention encompasses structural genes of AAV having corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0086] In addition, due to the high degree of conservation of at least a wide range of closely related family members, corresponding insertion sites to AAVs other than those listed can be identified by performing amino acid alignment or by comparing capsid structures. For example, see Rutledge et al. (1998) J. Virol. 72:309~19, each incorporated herein in its entirety by reference, and U.S. Patent No. 9,624,274 for exemplary alignments of different AAV capsid proteins.

[0087] In some compositions disclosed herein, comprising recombinant viral capsids (for example, in the absence of a multispecific binding molecule), the recombinant viral capsid protein is the AAV2 capsid protein VP1 with a heterologous epitope inserted at the I587 site, and the heterologous epitope does not contain the Arg-Gly-Asp(RGD) motif, the NGR motif, or c-myc. In some compositions disclosed herein, comprising recombinant viral capsids (for example, in the absence of a multispecific binding molecule), the recombinant viral capsid protein is the VP1 capsid protein with a heterologous epitope inserted between T448 and N449, and the heterologous epitope does not contain c-myc. In some compositions disclosed herein, comprising a recombinant viral capsid (for example, in the absence of a multispecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein in which a heterologous epitope is inserted at the I-447 site, and the heterologous epitope does not contain L14 or HA.

[0088] In some compositions comprising a recombinant viral capsid (for example, further comprising a multispecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at the I587 site, the heterologous epitope comprising an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions disclosed herein comprising a recombinant viral capsid (for example, further comprising a multispecific binding molecule), the viral capsid is a VP1 capsid, the heterologous epitope comprises c-myc, and the heterologous epitope is inserted between T448 and N449, or between N587 and R588. In some compositions disclosed herein comprising a recombinant viral capsid (for example, further comprising a multispecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at the I-447 site, the heterologous epitope comprising L14 or HA. In some compositions disclosed herein, including recombinant viral capsids (for example, in the presence of a multispecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein in which a heterologous epitope is inserted between T448 and N449, and the heterologous epitope contains c-myc. U.S. Patent No. 9,624,274 describes I-453 of the AAV capsid protein as a preferred insertion site for a heterologous epitope.

[0089] In some embodiments, insertion (presentation) of a heterologous epitope neutralizes the innate targeting of the viral vector, for example, transduction of cells that are naturally tolerant to infection by a wild-type standard viral vector and / or target cells is undetectable in the absence of appropriate multispecificity binding molecules. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector compared to transduction of cells that are naturally tolerant to infection by a wild-type standard viral vector. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 5%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 5%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 10%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 20%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 30%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 40%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 50%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 60%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 70%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 80%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 90%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 95%. In some embodiments, insertion (presentation) of a heterologous epitope reduces the innate targeting of the viral vector by at least 90%.In these embodiments where the insertion (presentation) of a heterologous epitope does not invalidate the innate targeting of the recombinant viral capsid, the innate targeting of such recombinant viral capsid may be invalidated by a second different mutation. For example, in one embodiment, the recombinant viral capsid protein described herein may be derived from the AAV9 capsid gene, contain a heterologous epitope, and further contain a mutation, such as the W503A mutation.

[0090] In particular, when considering systemic versus topical or localized administration of viral vectors, it is important to detarget the virus from its native host cells, as the uptake of the viral vector by its 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, especially hepatocytes. For AAV2, HSPG binding activity is dependent on a group of five basic amino acids: R484, R487, R585, R588, and K532 (Kern et al., (2003) J Virol. 77(20):11072-81). More recently, the amino acid substitution K531E from lysine to glutamate has been reported to lead to an inhibition of AAV6's ability to bind to heparin or HSPG ((Wu et al., 2006) J. of Virology 80(22):11393-11397). Therefore, a preferred point mutation is one that reduces the transduction activity of the viral vector to a given target cell mediated by the innate receptor, and, when HSPG is the primary receptor, the binding of the viral vector to HSPG, by at least 50%, preferably at least 80%, and particularly at least 95%.

[0091] As a result, preferred further mutations in HSPG-binding viral vectors are those that eliminate or replace a basic amino acid, such as R, K, or H, preferably R or K, which is involved in HSPG binding of each virus, with a non-basic amino acid, such as A, D, G, Q, S, and T, preferably A, or a different but highly conserved amino acid present at the corresponding position of the AAV serotype lacking such basic amino acid at this position. Consequently, preferred amino acid substitutions are R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A, or R588T, particularly R585A and / or R588A in AAV2, and K531A or K531E in AAV6. A particularly preferred embodiment of the present invention is a capsid protein mutation of such AAV2 that additionally contains two point mutations R585A and R588A, which are sufficient to significantly reduce HSPG binding activity. These point mutations allow for efficient detargeting from HSPG-expressing cells for targeting purposes and increase the specificity of the respective mutant viruses to their new target cells.

[0092] One embodiment of the present invention is a multimer structure comprising recombinant viral capsid proteins of the present invention. The multimer structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 recombinant viral capsid proteins, each comprising heterologous epitopes as described herein. They can form ordinary viral capsids (empty viral particles) or viral vectors (capsids that encapsulate target nucleotides). The formation of viral vectors capable of packaging a viral genome is a highly desirable feature for the use of recombinant viral capsids described herein as viral vectors.

[0093] One embodiment of the present invention is a nucleic acid encoding the capsid protein described above. Preferably, the nucleic acid is a vector comprising the nucleic acid sequence of the claims. The nucleic acid, and in particular the vector, is required for recombinant expression of the capsid protein of the present invention.

[0094] Further embodiments of the present invention include the use of at least one recombinant viral capsid protein and / or nucleic acid encoding it, preferably at least one multimeric structure (e.g., a viral vector) for production and use as a transfer vector.

[0095] heterogeneous epitopes Generally, viral vectors comprising recombinant viral capsid proteins and / or recombinant viral capsids include heterologous epitopes that enable retargeting of the viral vector, for example, via multispecific binding molecules. In some embodiments, the heterologous epitope is a B-cell epitope, for example, about 1 to 35 amino acids long, and forms a binding pair with an antibody paratope, for example, an immunoglobulin variable domain. In some embodiments, the heterologous epitope includes an affinity tag.

[0096] Numerous tags are known in the art. (See, for example, Nilsson et al. (1997) “Affinity fusion strategies for detection, purification, and immobilization of recombinant protein” Protein Expression and Purification 11:1-16, Terpe et al. (2003) “Overview of tag protein fusions: From molecular and biochemical fundamentals to Commercial Microbiology and Biotechnology 60:523-533, and references). Affinity tags include, but are not limited to, immobilized divalent cations (e.g., Ni2+ Examples of tags include polyhistidine tags that bind to (e.g., His-6, His-8, or His-10 tags), biotin moieties that bind to immobilized avidin (e.g., on biotinylated polypeptide sequences in vivo), GST (glutathione S-transferase) sequences that bind to immobilized glutathione, S tags that bind to immobilized S proteins, antigens that bind to immobilized antibodies or domains or fragments thereof (e.g., including T7, myc, FLAG, and B tags that bind to corresponding antibodies), FLASH tags (high-affinity tags that link to specific arsenic moieties), receptors or receptor domains that bind to immobilized ligands (or vice versa), protein A or derivatives of protein A or its derivatives that bind to immobilized IgG (e.g., Z), maltose-binding proteins (MBPs) that bind to immobilized amylose, albumin-binding proteins that bind to immobilized albumin, chitin-binding domains that bind to immobilized chitin, calmodulin-binding peptides that bind to immobilized calmodulin, and cellulose-binding domains that bind to immobilized cellulose. Another exemplary tag is the SNAP-tag, commercially available from Covalys. www.covalys.com In some embodiments, the heterologous epitopes disclosed herein include affinity tags that are recognized only by antibody paratopes. In some embodiments, the heterologous epitopes disclosed herein include affinity tags that are recognized by antibody paratopes and other specific binding pairs.

[0097] In some embodiments, the heterologous epitope and / or affinity tag does not form a binding pair with the immunoglobulin constant domain. In some embodiments, the heterologous epitope and / or affinity tag is a metal ion, for example, Ni 2+ Co 2+ Cu 2+ Zn 2+ Fe 3+ It does not form binding pairs with such molecules. In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of streptavidin, StrepII, HA, L14, 4C-RGD, LH, and protein A.

[0098] In some embodiments, the affinity tag is selected from the group consisting of FLAG (SEQ ID NO: 7), HA (SEQ ID NO: 8), and c-myc (EQKLISEEDL; SEQ ID NO: 6). In some embodiments, the heterologous epitope is c-myc.

[0099] In some embodiments, the recombinant viral capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV VP1 capsid protein. In some embodiments, the recombinant essential capsid described herein includes an amino acid sequence EQKLISEEDL (represented as SEQ ID NO: 6) adjacent to and / or operably linked to at least five consecutive amino acids of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid described herein includes EQKLISEEDL (represented as SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid protein described herein includes an amino acid sequence represented as SEQ ID NO: 2. In some embodiments, the recombinant viral capsid protein described herein includes an amino acid sequence represented as SEQ ID NO: 4. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence encoded by a nucleic 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 encoded by a nucleic acid sequence represented as SEQ ID NO: 26. In some embodiments, recombinant viral capsids, viral vectors containing recombinant viral capsids, and / or compositions containing recombinant viral capsids include an amino acid sequence encoded by a nucleic acid sequence represented as SEQ ID NO: 27.

[0100] In some embodiments, the heterologous epitope includes an affinity tag and one or more linkers. In some embodiments, the heterologous epitope includes an affinity tag adjacent to the linker, for example, the heterologous epitope includes a first linker, an affinity tag, and a second linker, from the N-terminus to the C-terminus. 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.

[0101] Generally, the heterologous epitopes described herein, for example, affinity tags themselves or affinity tags combined with one or more linkers, are about 5 to 35 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is at least 5 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 6 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 7 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 8 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 9 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 10 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 11 amino acid lengths. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 12 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 13 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 14 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 15 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 16 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 17 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 18 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 19 amino acid long.In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 20 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 21 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 22 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 23 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 24 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 25 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 26 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 27 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 28 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 29 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 30 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 31 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 32 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 33 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 34 amino acid long. In some embodiments, the heterologous epitope (by itself or in combination with one or more linkers) is 35 amino acid long.

[0102] retargeting part The viral vectors described herein have reduced or incapacitated transduction ability in the absence of a multispecific binding molecule, specifically comprising (i) an antibody paratope that specifically binds to an epitope, and (ii) a retargeting ligand that specifically binds to a receptor that can be conjugated on the surface of beads (e.g., for purification) or expressed by target cells. Thus, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to an epitope, and (ii) a retargeting ligand that specifically binds to a receptor, retargets the viral vector. Such “retargeting” or “reorientation” may include scenarios in which a wild-type viral vector targets several cells within tissues and / or several organs within an organism, and by insertion of a heterologous epitope, the broad targeting of tissues or organs is reduced or incapacitated, and retargeting to more specific cells in tissues or more specific organs within an organism is achieved using a multispecific binding molecule. Such retargeting or reorientation may also include scenarios in which a wild-type viral vector targets a tissue, and the insertion of a heterologous epitope reduces or neutralizes the targeting of that tissue, thereby enabling retargeting to a completely different tissue, which can be achieved using a multispecificity conjugate molecule. The antibody paratopes described herein generally contain, at a minimum, a complementarity-determining region (CDR) that specifically recognizes the heterologous epitope, e.g., a CDR3 region of the heavy and / or light chain variable domains. In some embodiments, the multispecificity conjugate molecule comprises an antibody (or a portion thereof) containing an antibody paratope that specifically binds to the heterologous epitope. For example, the multispecificity conjugate molecule may contain a single-domain heavy chain variable region or a single-domain light chain variable region, and the single-domain heavy chain variable region or single-domain light chain variable region contains an antibody paratope that specifically binds to the heterologous epitope. In some embodiments, the multispecificity conjugate molecule may contain an Fv region, e.g., an scFv containing an antibody paratope that specifically binds to the heterologous epitope. In some embodiments, the multispecificity binding molecules described herein include antibody paratopes that specifically bind to c-myc.

[0103] In some embodiments, the multispecific binding molecules described herein include antibody paratopes that specifically bind to c-myc, the paratopes including single-chain Fvs (scFvs), the heavy and light chain variable domains of scFvs, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences encoded by a nucleic acid sequence represented as SEQ ID NO: 28, such as an scFV including an amino acid sequence represented as SEQ ID NO: 37. In some embodiments, the multispecific binding molecules described herein include an Fv or sCfv encoded by a nucleic acid sequence represented as SEQ ID NO: 28.

[0104] Thus, the present invention includes, as an antibody that specifically binds to c-myc, an antigen-binding fragment of an antibody, and a multispecific binding protein, wherein the antibody, the fragment of the antibody, and the multispecific binding protein include a paratope including a heavy chain variable region (HCVR) including SEQ ID NO: 29 and a light chain variable region (LCVR) including SEQ ID NO: 30. The present invention also includes an antibody, an antigen-binding fragment of an antibody, and / or a multispecific binding protein including a paratope that specifically binds to c-Myc, the paratope including a heavy chain complementarity-determining region 1 (HCDR1) including SEQ ID NO: 31, an HCDR2 including SEQ ID NO: 32, an HCDR3 including SEQ ID NO: 33, a light chain complementarity-determining region 1 (LCDR1) including SEQ ID NO: 34, an LCDR2 including SEQ ID NO: 35, and an LCDR3 including SEQ ID NO: 36.

[0105] The present invention provides an antibody, an antigen-binding fragment thereof, and / or a multispecific binding protein including a paratope including a HCVR including the amino acid sequence represented as SEQ ID NO: 29 or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0106] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising an LCVR containing an amino acid sequence represented as SEQ ID NO: 30, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity therewith.

[0107] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope containing an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair, which includes an HCVR amino acid sequence represented as SEQ ID NO: 29 paired with an LCVR amino acid sequence represented as SEQ ID NO: 30. In some embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 29 / 30.

[0108] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence represented as SEQ ID NO: 31, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0109] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising a heavy chain CDR2 (HCDR2) having an amino acid sequence represented as SEQ ID NO: 32, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0110] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising a heavy chain CDR3 (HCDR3) having an amino acid sequence represented as SEQ ID NO: 33, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0111] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising a light chain CDR1 (LCDR1) containing the amino acid sequence represented as SEQ ID NO: 34, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0112] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising a light chain CDR2 (LCDR2) containing the amino acid sequence represented as SEQ ID NO: 35, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0113] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecificity-binding proteins comprising a paratope comprising a light chain CDR3 (LCDR3) having an amino acid sequence represented as SEQ ID NO: 36, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0114] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope containing an HCDR3 and LCDR3 (HCDR3 / LCDR3) amino acid sequence pair, which includes an LCDR3 amino acid sequence represented as SEQ ID NO: 36 and an HCDR3 amino acid paired with an HCDR3 amino acid represented as SEQ ID NO: 33. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is represented as SEQ ID NO: 33 / 36.

[0115] The present invention provides an antibody, its antigen-binding fragment, and / or a multispecific binding protein comprising a paratope comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) encoded by a nucleotide sequence represented as SEQ ID NO: 28. In certain embodiments, the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequence set is represented as SEQ ID NOs: 31-32-33-34-35-36.

[0116] In related embodiments, the present invention provides an antibody, its antigen-binding fragment, and / or multispecific binding protein comprising a paratope containing a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within an HCVR / LCVR amino acid sequence pair represented as SEQ ID NOs. 29 / 30. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (1991), Al-Lazikani et al., J.Mol.Biol.273:927-948 (1997), and Martin et al., Proc.Natl.Acad.Sci.USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0117] The present invention also provides nucleic acid molecules encoding an anti-myc antibody or a portion thereof.

[0118] The multispecific binding molecules described herein further comprise a retargeting ligand in addition to a paratope (e.g., an antibody or a portion thereof) that specifically binds to a heterologous epitope inserted into / presented on a recombinant viral capsid protein. In some embodiments, the retargeting ligand binds to a protein expressed on the surface of a cell, e.g., a cell surface protein on a (human) eukaryotic cell (e.g., a target cell). Numerous cell surface proteins, e.g., suitable cell surface receptors, can be targeted by the retargeting ligand, and retargeting ligands for them, e.g., antibodies or portions 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 growth hormone, 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 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, vascular endothelial growth factor, lipoprotein This includes, but is not limited to, proteins (including further or other type 1 transmembrane receptors such as PRLR, G protein-binding receptors such as GCGR, and ion channels such as Nav1.7, ASIC1, or ASIC2), cell adhesion molecules, transport molecules of metabolites such as amino acids, light source receptors of 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 a multispecific binding molecule containing a retargeting ligand that binds to a differentiated cell surface antigen as a target for the viral vector complex.

[0119] In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) hepatocytes (e.g., alone), i.e., a liver-specific marker. In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) brain cells (e.g., alone), i.e., a brain cell-specific marker. In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) hematopoietic cells (e.g., alone), i.e., a hematopoietic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) T cells (e.g., alone), i.e., a T cell-specific marker. In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) B cells (e.g., alone), i.e., a B cell-specific marker. In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) dendritic cells (e.g., alone), i.e., a dendritic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein primarily expressed by (human) macrophages (e.g., alone), i.e., a macrophage-specific marker. In some embodiments, the retargeting ligand binds to proteins primarily expressed by (human) NK cells (e.g., alone), i.e., NK cell-specific markers. In some embodiments, the retargeting ligand binds to proteins primarily expressed by (human) renal hepatocytes (e.g., alone), i.e., kidney-specific markers. In some embodiments, the retargeting ligand binds to proteins primarily expressed by (human) pancreatic hepatocytes (e.g., alone), i.e., pancreas-specific markers. In some embodiments, the retargeting ligand binds to proteins primarily expressed by (human) intestinal cells (e.g., alone), i.e., intestinal-specific markers. In some embodiments, the retargeting ligand binds to proteins primarily expressed by (human) cancer cells, i.e., tumor-associated antigens (e.g., alone). In some embodiments, the retargeting ligand binds to proteins primarily expressed by (human) cells (e.g., alone) infected with heterologous pathogens.Proteins are well known that (1) 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 retargeting ligands as described herein. www.proteinatlas.org See also Uhlen et al. (2010) Nat. Biotech. 28:1248-50 (the whole is incorporated herein by reference). Table 1 below provides exemplary and non-limiting organ-specific markers available for antigen-binding proteins that may be useful as retargeting ligands, and cells / tissues / organs that express such markers. [Table 1-1] [Table 1-2]

[0120] In some embodiments, the retargeting ligand binds to receptors expressed by (human) hepatocytes, such as the asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the retargeting ligand binds to receptors expressed by (human) brain cells. In some embodiments, the retargeting ligand binds to receptors expressed by (human) T cells, such as CD3, e.g., CD3ε. In some embodiments, the retargeting ligand binds to receptors expressed by (human) kidney cells. In some embodiments, the retargeting ligand binds to receptors expressed by (human) muscle cells, such as integrins. In some embodiments, the retargeting ligand binds to receptors expressed by (human) cancer cells, such as tumor-associated antigens, e.g., E6 and E7. In some embodiments, the retargeting ligand binds to the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds to human ENTPD3.

[0121] In some embodiments, the retargeting ligand binds to tumor-associated antigens expressed by tumor cells. Non-limiting examples of specific tumor-associated antigens include, for example, adipophyllin, AIM-2, ALDH1A1, alpha-actin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, C PSF, CSNK1A1, CTAG1, CTAG2, Cyclin D1, Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH(hMena), Ep-CAM, EpCAM, EphA3, Epithelial carcinoma 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, GP NMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal Carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 (also known as CCDC110), LAGE-1, LDLR-Fucosyltransferase AS Fusion Protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10 MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malate 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, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secretin 1, SIRT2, SNRPD1, SOX10, Sp17 SPA17, SSX-2, SSX-4, STEAP1, Survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-beta-RII, 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], gp100, tyrosinase, TRP-1 / gp75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); mucin [MUC-1] (in breast, pancreatic, colon, and prostate cancer); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colorectal, breast, and gastrointestinal cancers), as well as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1, 2, 8, CAGE-3 TO 7. It binds to such covalent tumor-specific antigens as LAGE-1, NY-ESO-1 / LAGE-2, NA-88, GnTV, and TRP2-INT2. In some embodiments, the tumor-associated antigen is ErBb2 / Her2. In some embodiments, the tumor-associated antigen is E6 and / or E7.

[0122] In some embodiments, the retargeting ligand binds to CD markers associated with an immune response, such as CD3, CD4, CD8, CD19, CD20, etc. In some embodiments, the CD marker is CD3.

[0123] In certain exemplary embodiments, the multispecific binding molecule is a bispecific antibody. Each antigen-binding domain of the bispecific antibody comprises a heavy-chain variable domain (HCVR) and a light-chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising first and second antigen-binding domains, the CDRs of the first antigen-binding domain may be denoted with the prefix "A1", and the CDRs of the second antigen-binding domain may be denoted with the prefix "A2". Thus, the CDRs of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3, and the CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0124] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly linked to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen-binding domain may each be linked to a separate multimerizing domain. The association of one multimerizing domain with another facilitates the association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid having the ability to associate with a second multimerizing domain of the same or similar structure or configuration. For example, the multimerizing domain may be a polypeptide containing an immunoglobulin CH3 domain. Non-limiting examples of multimerizing components include not only any allotype within each isotype group, but also the Fc portion of immunoglobulins (containing CH2-CH3 domains), e.g., the Fc domain of IgG selected from isotypes IgG1, IgG2, IgG3, and IgG4.

[0125] The bispecific antigen-binding molecule of the present invention will typically contain two multimerizing domains, for example, two Fc domains, each being an individual part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype, for example, IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes, for example, IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4.

[0126] In certain embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerizing domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerizing domains include peptides or polypeptides containing or comprising a leucine zipper, a helix-loop motif, or a coiled-coil motif.

[0127] Any bispecific antibody format or technique may be used to produce the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment having a first antigen-binding specificity can be functionally linked (e.g., by chemical coupling, gene fusion, or non-covalent association, or vice versa) to one or more other molecular entities, such as another antibody or antibody fragment having a second binding specificity, to produce a bispecific antigen-binding molecule. Specific exemplary bispecificity formats that may be used in the context of the present invention include, but are not limited to, scFv-system or diabody bispecificity formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadromas, knob-into-hole, general light chains (e.g., general light chains with knob-into-hole), CrossMab, CrossFab, (SEED) antibodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab2 bispecificity formats (for the consideration of the aforementioned formats, see, for example, Klein et al. 2012, mAbs 4:6, 1-11 and the references cited therein, and also see Brinkmann and Konterman (2017) mAbs 9:182-212 (each of which is incorporated by reference in its entirety)).

[0128] The present invention also comprises a bispecific antigen-binding molecule comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and the difference by at least one amino acid reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds to protein A, and the second Ig CH3 domain contains a mutation that reduces or disables protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). The second CH3 may further include the Y96F modification (according to IMGT, Y436F in EU). Further modifications that may be found in the second CH3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in the EU); and for IgG2 antibodies, N44S, K52N, and V82I (according to IMGT). For IgG4 antibodies, see N384S, K392N, and V422I in the EU, and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in the EU), see, for example, WO2010 / 151792.

[0129] In certain embodiments, the Fc domain may be a chimera combining Fc sequences derived from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may include some or all of the CH2 sequences derived from the human IgG1, human IgG2, or human IgG4 CH2 region, and some or all of the CH3 sequences derived from human IgG1, human IgG2, or human IgG4. The chimeric Fc domain may also contain a chimeric hinge region. For example, the chimeric hinge may include an "upper hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region combined with a "lower hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules represented herein includes [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3] from the N-terminus to the C-terminus. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules represented herein includes [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3] from the N-terminus to the C-terminus. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the present invention are described in PCT application WO2014 / 022540, which is incorporated in its entirety by reference. Chimeric Fc domains and their variants having these common structural arrangements may have altered Fc receptor binding, thereby affecting Fc effector function.

[0130] 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 transmission plasmid that generally contains 5' and 3' inverted terminal repeat (ITR) sequences adjacent to the reporter gene(s) or therapeutic gene(s) (which may be under the control of a viral or non-viral promoter when contained within an AAV vector). In one embodiment, the target nucleotide is a transmission plasmid containing, from 5' to 3', a 5' ITR, a promoter, a gene (e.g., a reporter and / or therapeutic gene), and a 3' ITR.

[0131] 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 phosphoglycerate kinase (PGK) promoter. Internal enhancers may also be present in the viral construct to increase the expression of the target gene. For example, the CMV enhancer can be used (Karasuyama et al. 1989. J. Exp. Med. 169:13 (the entire document is incorporated herein by reference)). In some embodiments, the CMV enhancer can be used in combination with the chicken 13-actin promoter.

[0132] A variety of reporter genes (or detectable regions) can be encapsulated in a multimeric structure containing the recombinant viral capsid protein described herein. Examples of reporter genes include, for example, β-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), high-sensitivity blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), high-sensitivity yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or combinations thereof. The methods described herein demonstrate the construction of a targeted vector using a reporter gene encoding a green fluorescent protein; however, those skilled in the art will understand that the non-human animals described herein can be generated in the absence of a reporter gene or using any reporter gene known in the art.

[0133] Diverse therapeutic genes can be encapsulated, for example, as part of a transmission vector, in a multimeric structure containing the recombinant viral capsid protein described herein. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or parts thereof, antisense RNA, siRNA, shRNA, and the like.

[0134] Further embodiments of the present invention are processes for the preparation of recombinant capsid proteins, the methods being: a) A step of expressing nucleic acid encoding a recombinant capsid protein under suitable conditions, b) The process includes isolating the capsid protein expressed in step a).

[0135] Further embodiments of the present invention are methods for altering the directivity of a virus, comprising: (a) inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding a viral capsid protein to form a nucleic acid sequence encoding a genetically modified capsid protein containing the heterologous epitope; and / or (b) culturing packaging cells under conditions sufficient for the production of a viral vector, wherein the packaging cells contain the nucleic acid sequence. Further embodiments of the present invention are methods for presenting a heterologous epitope on the surface of a capsid protein, comprising: a) expressing a nucleic acid according to the present invention under suitable conditions; and b) isolating the capsid protein expressed in step a).

[0136] In some embodiments, the packaging cells further comprise a transfer plasmid containing a helper plasmid and / or a 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) treating the supernatant containing the viral vector with DNase I and MgCl2, (c) enriching the viral vector, (d) purifying the viral vector, and (e) any combination of (a) to (d).

[0137] Examples of packaging cells useful for generating the viral vectors 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 the viral vectors described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., the American Cell Culture and Cell Lineage Preservation Center [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.

[0138] 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).

[0139] For further information on packaging cells and systems, 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.

[0140] Further embodiments include methods for reorienting a virus and / or delivering a reporter or therapeutic gene to target cells, the methods including a method for transducing cells in vitro or in vivo, the method including contacting target cells with a combination of a viral vector comprising a capsid containing a recombinant viral capsid that presents a heterologous epitope and a multispecificity binding molecule, the multispecificity binding molecule comprising i) an antibody paratope that specifically binds to the epitope, and (ii) a retargeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the recombinant viral vector and the multispecificity binding molecule are included in the compositions described herein, or the methods described herein, in a molecule:molecule ratio that restores the transduction efficiency of the viral vector to that of a wild-type control viral vector. In some embodiments, the recombinant viral vector to multispecificity binding molecule ratio (molecule:molecule) is in the range of 1:0.5 to 1:100. In some embodiments, the recombinant viral vector to multispecificity binding molecule ratio (molecule:molecule) is in the range of 1:4 to 1:20. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is in the range of 1:8 to 1:15. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is 1:4. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is 1:8. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is 1:20. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is less than 1:100. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is less than 1:50. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is less than 1:20. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is less than 1:15.In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) is less than 1:10.

[0141] In some embodiments, the target cells are located outside the body. In other embodiments, the target cells are located inside a subject, such as a human body.

[0142] target cell Using the recombinant viral vectors disclosed herein, a wide variety of cells can be targeted to deliver the target nucleotide. The target cells will generally be selected based on the target nucleotide and the desired effect.

[0143] In some embodiments, target nucleotides can be delivered to enable target cells to produce proteins that compensate for deficiencies in the organism, such as enzyme deficiencies or immune deficiencies like severe combined immunodeficiency linked to X-linking. Therefore, in some embodiments, cells that would normally produce the protein in an animal are targeted. In other embodiments, cells within a region where the protein is most beneficial are targeted.

[0144] In other embodiments, a target nucleotide, such as a gene encoding siRNA, can inhibit the expression of specific genes in target cells. For example, the target nucleotide may inhibit the expression of genes involved in the pathogen life cycle. Therefore, cells that are susceptible to or infected with a pathogen can be targeted. In other embodiments, the target nucleotide may inhibit the expression of genes responsible for toxin production in target cells.

[0145] In other embodiments, the target nucleotide may encode a toxic protein that kills cells expressing the toxic protein. In this case, tumor cells or other unwanted cells may be targeted.

[0146] In other embodiments, target nucleotides encoding collected proteins, such as therapeutic proteins, may be used to target cells capable of producing and secreting proteins.

[0147] Once a specific population of target cells is identified in which the expression of a target nucleotide is desirable, a target receptor specifically expressed on that population of target cells is selected. The target receptor may be expressed only in that cell population, or to a higher degree in that cell population than in other cell populations. The more specific the expression, the more specifically the delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and therefore, gene delivery) can vary. For example, high specificity is most preferable to introduce a toxic gene, in order to avoid killing non-target cells. Lower marker specificity may be required for the expression of a protein for harvesting, or for the expression of a secreted product where a comprehensive effect is desired.

[0148] As described above, the target receptor can be any receptor from which a retargeting ligand can be identified or constructed. Preferably, the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target receptor can be a carbohydrate or other molecule that can be recognized by a binding partner. If the binding partner of the target receptor, e.g., the ligand, is known, it may 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. In that case, the antibody can be used as a retargeting ligand as part of a multispecific binding molecule.

[0149] Therefore, target cells can be selected based on a variety of factors, including, for example, (1) specific uses (e.g., therapy, expression of proteins to be collected, and conferral of disease resistance), and (2) expression of markers with desired specificity.

[0150] The target cells are not limited in any way and include germline cells and cell lines, as well as both somatic cells and cell lines. The target cells may be stem cells derived from any of these origins. When the target cells are germline cells, they are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).

[0151] Pharmaceutical compositions, dosage forms, and administration Further embodiments provide a pharmaceutical product comprising at least one recombinant viral capsid protein and a suitable multispecificity binding molecule according to the present invention, and / or a nucleic acid according to the present invention, preferably at least one multimeric structure according to the present invention. Such a pharmaceutical product is preferably a useful gene transfer vector.

[0152] Furthermore, pharmaceutical compositions comprising the viral vector described herein, as well as pharmaceutically acceptable carriers and / or excipients, are disclosed herein. In addition, pharmaceutical dosage forms comprising the viral vector described herein are disclosed herein.

[0153] As discussed herein, the viral vectors described herein can be used for a variety of therapeutic applications (in vivo and ex vivo) and as research tools.

[0154] The viral vector-based pharmaceutical compositions disclosed herein may be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. Viral vectors may be prescribed for administration, for example, by injection, inhalation (through either the mouth or nose), or isolation, or by oral, intraoral, parenteral, or rectal administration, or by direct administration to a tumor.

[0155] Pharmaceutical compositions can be formulated for a variety of administration modes, including systemic, topical, or localized administration. Techniques 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.

[0156] 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, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate), 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 using a composition containing water or other suitable medium 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, flavoring agents, coloring agents, and sweeteners.

[0157] Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may optionally contain preservatives and be presented in unit dosage forms, for example, in ampoules or multi-dose containers. Pharmaceutical compositions can be further formulated as suspensions, solutions, or emulsions in oily or aqueous media and may contain other agents, including suspending agents, stabilizers, and / or dispersants.

[0158] In addition, pharmaceutical compositions can also be formulated as depot preparations. These long-acting preparations 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. Another suitable delivery system is microspheres, which offer the possibility of local, non-invasive delivery of drugs over a long period. 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.

[0159] Systemic administration is also possible by transmucosal or transdermal means. For transmucosal or transdermal administration, a suitable penetrating agent for the penetration barrier is used in the formulation. Such penetrating agents are generally known in the art, and examples for transmucosal administration include bile salts and fusidic acid derivatives. In addition, detergents may be used to promote penetration. Transmucosal administration may be performed using intranasal sprays or suppositories. For topical administration, the viral vectors 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.

[0160] Suitable pharmaceutical forms for injectable use 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 certain storage parameters (e.g., cooling and freezing) and protected from microbial contamination such as bacteria and fungi.

[0161] 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.

[0162] 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 a coating such as lecithin, by maintaining the required size of the viral vector in the case of a dispersion, and by the use of a surfactant. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents known in the art. In many cases, it would be preferable to include an isotonic agent, such as sugar or sodium chloride. Long-term absorption of the injectable composition can be achieved by the use of an absorption-delaying agent composition, such as aluminum monostearate and gelatin.

[0163] 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.

[0164] When prescribed, the solution may be administered in a form compatible with the administered formulation and in a therapeutically effective amount. The formulation can be readily administered in a variety of dosage forms, such as the injectable solution types described above, but sustained-release capsules or microparticles and microspheres may also be used.

[0165] 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 infusion site.

[0166] 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 vector 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).

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] Further embodiments disclosed herein may relate to kits for use in conjunction with methods and compositions. Kits may also include suitable containers, such as vials, tubes, mini-tubes or microtubes, test tubes, flasks, bottles, syringes, or other containers. When additional components or agents are provided, the kit may contain one or more additional containers in which these agents or components may be placed. Kits herein also typically include means for containing viral vectors and any other commercially available tightly sealed reagent containers. Such containers may include syringes or blow-molded plastic containers that hold the desired vials. Optionally, the compositions described may require one or more additional activators, such as anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antitumor agents.

[0172] The dose range and frequency of administration may vary depending on the properties of the viral vector, the medical condition and parameters of the particular patient, and the route of administration used. In some embodiments, the viral vector composition can be administered to a subject in doses ranging from about 1 × 10⁵ plaque-forming units (pfu) to about 1 × 10¹⁵ pfu, depending on the mode of administration, route of administration, and the nature and condition of the disease being treated. In some cases, the viral vector composition can be administered in doses ranging from about 1 × 10⁸ pfu to about 1 × 10¹⁵ pfu, or about 1 × 10¹⁰ pfu to about 1 × 10¹⁵ pfu, or about 1 × 10⁸ pfu to about 1 × 10¹² pfu. More precise doses may also depend on the subject being administered. For example, a lower dose may be required if the subject is young, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, the more precise dose may depend on the weight of the subject. In a particular embodiment, for example, a young human subject may receive a dose of approximately 1 × 10⁸ pfu to approximately 1 × 10¹⁰ pfu, while an adult human subject may receive a dose of approximately 1 × 10¹⁰ pfu to approximately 1 × 10¹² pfu.

[0173] 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, intrafocally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreously, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucosa, intrapericardially, subumbilically, intraocularly, orally, locally, by inhalation, injection, drip infusion, continuous drip infusion, local perfusion, via catheter, lavage, in a cream, or in a lipid composition.

[0174] Any method known to those skilled in the art may be used for the large-scale production of the viral vectors, packaging cells, and vector constructs described herein. For example, master seed and working seed stocks may be prepared under GMP conditions in a primary CEF that meets the requirements, or by other means. Packaging cells may be plated onto a large surface area flask and grown to near-dense density to purify the viral vector. The cells may be harvested and the viral vector released into culture medium may be isolated and purified, or the intracellular viral vector may be released by mechanical disintegration (cell debris can be removed by large-pore depth filtration and by digesting the host cell DNA with an endonuclease). The viral vector may then be purified and concentrated by tangential flow filtration, followed by dialysfiltration. The resulting concentrated bulk can be formulated by diluting it with a buffer containing a stabilizer, filling it into vials, and lyophilizing it. The compositions and formulations may be stored for later use. For use, the lyophilized viral vector may be reconstituted by adding a diluent.

[0175] Certain additional drugs used in combination therapy can be formulated and administered by any means known in the art.

[0176] The compositions disclosed herein may also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, media, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants can stimulate Th1 or Th2 immunization. The compositions and methods disclosed herein may also include adjuvant therapy. [Examples]

[0177] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0178] Example 1: Generation of an adeno-associated virus vector containing a heterologous epitope AAV capsid proteins are modified using PCR to contain one of several heterologous epitopes, such as FLAG, c-myc, or hexahistidine, to generate plasmids encoding recombinant capsid proteins. Briefly, after the codons encoding N587 for AAV2 capsid protein, Q585 for AAV6 VP1 capsid protein, N590 for AAV8 VP1 capsid protein, A589 for AAV9 VP1 capsid protein, and G453 for AAV9 VP1 capsid protein are used, a sequence encoding FLAG, c-myc, or hexahistidine is inserted into the frame.

[0179] Adeno-associated virus generation is performed using a triple transfection method with HEK293 cells (see, for example, Erik Arden and Joseph M. Metzger, J Biol Methods. 2016;3(2)). The cells are then given an appropriate vector: Helper plasmid, pHelper(Agilent, Cat#240074); For example, plasmids encoding wild-type or modified AAV rep / cap genes (such as pAAV RC2 (Cell Biolabs, Cat#VPK-422)), pAAV RC8, pAAV RC2-N587myc, pAAV RC2 / 6-Q585myc, pAAVRC8-N590myc, pAAV RC9-A589myc; and A plasmid encoding an AAV ITR sequence, such as pscAAV-CMV-eGFP, pAAV-CMVGFP (Agilent Cat#240074), pAAV-EF1a-eGFP, or pAAV-CAGG-eGFP, is used to combine the target nucleotide with the plasmid. The product is plated one day before PEFpro (Polyplus transfection, New York, NY) mediated transfection.

[0180] 72 hours after transfection, collect the culture medium and lyse the cells in buffer [50 Mm Tris-HCl, 150 Mm NaCl, and 0.5% sodium deoxycholate (Sigma, Cat#D6750-100G)]. Next, add benzonase (Sigma, St. Louis, MO) until both the medium and cell lysate reach a final concentration of 0.5 U / μl, then incubate at 37°C for 60 minutes. Centrifuge the cell lysate at 4000 rpm for 30 minutes. Combine the cell lysate and medium and precipitate with PEG8000 (Teknova Cat#P4340) to a final concentration of 8%. Resuspend the pellet in 400 Mm NaCl and centrifuge at 10000 g for 10 minutes. Pellet the virus in the supernatant by ultracentrifugation at 149,000 g for 3 hours and titrate by qPCR.

[0181] For qPCR to titrate the AAV genome, AAV samples are treated with DNase I (Thermofisher Scientific, Cat#EN0525) at 37°C for 1 hour and lysed with DNA extract All Reagents (Thermofisher Scientific, Cat#4403319). The primers are applied directly to the AAV2 ITR, and the capsid-forming viral genome is quantified using the QuantStudio 3 Real-Time PCR System (Thermofisher Scientific). The sequences of the AAV2 ITR primers are 5'-GGAACCCCTAGTGATGGAGTT-3' (forward ITR, SEQ ID NO: 9) and 5'-CGGCCTCAGTGAGCGA-3' (reverse ITR, SEQ ID NO: 10) (Aurnhammer et al., 2012), derived from the left internal inverted repeat (ITR) sequence and the right internal inverted repeat (ITR) sequence of AAV, respectively. The sequence of the AAV2 ITR probe is 5'-6-FAM-CACTCCCTCTCTGCGCGCTCG-TAMRA-3' (SEQ ID NO: 11) (Aurnhammer C., Haase M., Muether N., et al., 2012, Hum. Gene Ther. Methods 23, 18-28). After an activation step of 10 minutes at 95°C, 40 cycles of two PCR cycles were performed: 15 seconds at 95°C and 30 seconds at 60°C. TaqMan Universal PCR Master Mix (Thermofisher Scientific, Cat#4304437) was used for qPCR. A DNA plasmid (Agilent, Cat#240074) was used as a reference for determining absolute titer.

[0182] An adeno-associated virus vector containing a capsid in which the c-myc epitope is generated. In this example, the c-myc epitope (EQKLISEEDL, SEQ ID NO: 6) was inserted between amino acids N587 and R588 of the AAV2 VP1 capsid protein, or between amino acids A589 and Q590 of the AAV9 VP1 capsid protein. In other words, the nucleotide sequence encoding the c-myc epitope (GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG, SEQ ID NO: 12) was inserted into plasmid pAAV RC2 (Cell Biolabs, Inc., San Diego, CA) or plasmid pAAV RC2 / 9, and each of the modified pAAV RC2-N587Myc and pAAV RC9-A589Myc plasmids were used to encode modified capsid proteins of AAV virus vectors with reduced or inactivated directivity.

[0183] To produce pAAV RC2-N587Myc, a first polymerase chain reaction (PCR) product containing the BsiW1 restriction site (from 5' to 3'), the nucleotide sequence between positions 3050 and 3773 of pAAV RC2, and the c-myc epitope overhang nucleotide sequence, and a second PCR product containing the c-myc epitope overhang nucleotide sequence (from 5' to 3'), the nucleotide sequence between positions 3774-4370 of pAAV RC2, and the Pme1 restriction site were prepared using the primers shown in Table 2. By digesting pAAV RC2 with BsiW1 (New England Biolabs, R0553L) and Pme1 (New England Biolabs, R0560L), we constructed the pAAV RC2-N587Myc plasmid (i.e., a pAAV RC2 plasmid modified to encode a c-myc epitope between amino acids N587 and R588 of the VP1 capsid protein), and inserted two PCR products via ligation-independent cloning as described in (2012)Methods Mol. Biol. 52:51-9.

[0184] To construct pAAV RC2 / 6-Q585Myc, gblock DNA fragments containing positions 3700 and 3940 of pAAV RC2 / 6, which have a c-myc epitope sequence inserted between 3757 and 3758, were ordered from Integrated DNA Technologies (Coralville, Iowa). The pAAV RC2 / 6-Q585Myc plasmid was constructed by inserting the gblock fragments into pAAV RC2 / 6 digested with MscI (New England Biolabs, Cat#R0534L) and AflII (New England Biolabs, Cat#R0520L) via ligation-independent cloning, as described in (2012)Methods Mol. Biol. 52:51-9.

[0185] To produce pAAV RC9-A589Myc, a first polymerase chain reaction (PCR) product containing the BsiW1 restriction site (from 5' to 3'), the nucleotide sequence between positions 3052 and 3779 of pAAV RC9, and the c-myc epitope overhang nucleotide sequence, and a second PCR product containing the c-myc epitope overhang nucleotide sequence (from 5' to 3'), the nucleotide sequence between positions 3779-4404 of pAAV RC9, and the Pme1 restriction site were prepared using the primers shown in Table 2.

[0186] By digesting pAAV RC9 with BsiW1 (New England Biolabs, R0553L) and Pme1 (New England Biolabs, R0560L), we constructed the pAAV RC9-A589Myc plasmid (i.e., a pAAV RC2 / 9 plasmid modified to encode a c-myc epitope between amino acids A589 and Q590 of the VP1 capsid protein), and inserted two PCR products via ligation-independent cloning as described in (2012)Methods Mol. Biol. 52:51-9. [Table 2]

[0187] pscAAV-CMV-eGFP was generated by introducing a GFP fragment into a pscAAV MCS vector (Cell Biolabs, Cat#VPK-430) using the BamHI and NotI restriction sites. pAAV-EF1a-eGFP plasmid and pAAV-CAGG-eGFP were constructed by de novo synthesis from Thermofisher Scientific (Waltham, MA).

[0188] Example 2: In vitro antibody-mediated retargeting of AAV virus vectors HepG2 is a human hepatocellular carcinoma cell line that expresses asialoglycoprotein receptor 1 (ASGR1), a liver-specific marker. To test whether the scAAV2-N587Myc-CMV-hrGFP viral vector can infect HepG2, mixtures containing scAAV2-N587Myc-CMV-hrGFP and bispecific anti-myc-ASGR1 antibody were cultured at room temperature for 30 minutes at a number of ratios of viral genomes (5e9 viral genomes) versus antibody molecules, for example, via a bispecific antibody that recognizes both c-myc and ASGR1 (anti-myc-ASGR1 antibody). As a control, HepG2 cells were also cultured with either the wild-type scAAV2-CMV-eGFP viral vector alone, the scAAV2-N587Myc-CMV-eGFP viral vector alone, or the scAAV2-N587Myc-CMV-eGFP viral vector containing a monospecific anti-myc antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) in a 1:8 ratio. Three days after infection, GFP expression in infected HepG2 cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 1). GFP expression was also detected at a comparable percentage to that of HepG2 cells when cultured with wild-type scAAV2-CMV-eGFP and when cultured with the 1:8 ratio scAAV2-N587Myc-CMV-eGFP viral vector and bispecific anti-myc-ASGR1 antibody (44.6% and 44.1%, respectively, Figure 1A and 1G, upper and lower panels). Lower GFP expression was detected by FACS in HepG2 cells cultured with a mixture of the scAAV2-N587Myc-CMV-eGFP viral vector and a higher or lower bispecific anti-myc-ASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) (Figure 1C, bottom panel).In addition, GFP was not detected in HepG2 cells infected with scAAV-N587Myc-CMV-eGFP in the absence of anti-myc-ASGR1 antibody, or in HepG2 cells cultured with scAAV2-N587Myc-CMV-eGFP and a monospecific anti-myc antibody.

[0189] Similarly, 293T cells genetically modified to express human (h)ASGR1 on their cell surface, known as -293T-hASGR1 cells, were cultured with a mixture containing scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody in different ratios (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, or 1:100) of viral genome versus antibody molecules. Wild-type 293T cells (that do not express hASGR1) were cultured with a mixture of scAAV2-N587Myc-CMV-eGFP and a bispecific anti-myc-ASGR1 antibody in a 1:8 ratio. 293T-hASGR1 cells were cultured with (a) wild-type scAAV virus vector alone, (b) scAAV2-N587Myc-CMV-eGFP virus vector alone, or (c) an unrelated bispecific anti-myc-GCGR antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) in a 1:8 ratio, which functions as a control and recognizes the glucagon receptor (GCGR) not expressed by c-myc and 293T cells. Three days after infection, 293T-hASGR1 or 293T cells were stained with human anti-hASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY), followed by APC-conjugated goat anti-human antibody (Jackson ImmunoResearch laboratories Inc., Cat#109-136-098, West Grove, PA), and GFP expression was analyzed by FACS. hASGR1 expression was detected on the surface of 293T-hASGR1 cells (Figure 2Ai~2Ax and 2Axii), but not in 293T cells (Figure 2Axi). After culturing with a mixture of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody in ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, and 1:50, GFP-positive 293T-hASGR1 cells were detected (56.9%~68.3%) at levels comparable to those of 293T-hASGR1 cells cultured with wild-type scAAV (56.7%) (Figures 2Ai and 2Aiii~2Aix).A 1:100 ratio of scAAV2-N587Myc-CMV-eGFP to bispecific anti-myc-ASGR1 antibody resulted in reduced infectivity (Figure 2Ax). Culture with scAAV2-N587Myc alone, or with scAAV-N587Myc containing unrelated bispecific anti-myc-GCGR antibody, did not result in GFP expression by 293T-hASGR1 cells (Figures 2Aii and 2Axii).

[0190] In similar experiments, 293T-hASGR1 cells were cultured with cells cultured with either the unmodified AAV9-CAGG-GFP viral vector alone, the AAV9-A589Myc-CAGG-eGFP viral vector alone, or the AAV9-A589Myc-CAGG-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody in different proportions. Three days after infection, GFP expression was analyzed by FACS. After culturing cells with a mixture of AAV9-A589Myc-CAGG-eGFP and bispecific anti-myc-ASGR1 antibody in ratios of 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, and 1:100, GFP-positive 293T-hASGR1 cells were detected at levels comparable to those of 293T-hASGR1 cells cultured with wild-type AAV9-CAGG-eGFP (9.72%) (41.1%~91%) (Figure 2B(i) and Figures 2B(iii)~(ix). Culturing with AAV9-A589Myc-CAGG-eGFP alone did not result in GFP expression by 293T-hASGR1 cells (Figure 2B(ii)).

[0191] The ability of a bivalent anti-hASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) to inhibit infection of 293T-hASGR1 cells was tested. 293T-hASGR1 cells were cultured at room temperature for 1 hour with different concentrations of the bivalent anti-hASGR1 antibody, and then cultured with a mixture containing scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody in a 1:8 ratio. Three days after infection, cells were fixed and analyzed by FACS as described above. Figure 3 provides data demonstrating that entry of scAAV2-N587Myc-CMV-eGFP can be inhibited in a dose-dependent manner by the bivalent anti-hASGR1 antibody.

[0192] To determine whether sequential administration of bispecific antibodies and modified AAV can lead to antibody-mediated retargeting of modified AAV, a number of different bispecific anti-myc-ASGR1 antibody molecules (1x10) were used. 9 ~1x10 12 (Numerator in the range) 2x10 5 Add to 293T-hASGR1 cells for 1 hour, then 1 x 10 9 The scAAV-N587Myc viral vector was added. As controls, (1) 293T-hASGR1 cells cultured with wild-type scAAV alone, i.e., in the absence of antibodies, and (2) 1x10 11 Unrelated bispecific anti-myc-GCGR antibody molecule and 1x10 9 293T-hASGR1 cells cultured sequentially with the scAAV2-N587Myc-CMV-eGFP viral vector, and (3) 1x10 11 The bispecific anti-myc-ASGR1 antibody molecule and 1x10 9One example was 293T cells that do not express hASGR1 and were cultured sequentially with the scAAV-N587Myc viral vector. Two days after infection, GFP expression by infected 293T-hASGR1 cells was visualized by microscopy (Figure 4). GFP expression by 293T-hASGR1 cells was observed after culture with wild-type scAAV alone, and after sequential culture with all concentrations of anti-ASGR1 antibody molecules and scAAV2-N587Myc-CMV-eGFP (Figure 4A, 4C-4I). GFP was not detected in 29T3-hASGR1 cells cultured with scAAV2-N587Myc-CMV-eGFP alone (Figure 4B), in 293T cells that do not express ASGR1 after being cultured sequentially with anti-myc-hASGR1 antibody and the scAAV2-N587Myc-CMV-eGFP viral vector (Figure 4J), or in 29T3-hASGR1 cells that were cultured sequentially with an unrelated bispecific anti-myc-GCGR antibody and the scAAV2-N587Myc-CMV-eGFP viral vector (Figure 4K).

[0193] As described herein, the directivity of self-complementary AAVs (scAAVs) can be (1) inactivated, for example, by modification of the capsid protein, e.g., insertion of a c-myc epitope, and optionally (2) reoriented using a bispecific antibody, e.g., a bispecific antibody that recognizes a c-myc epitope and a ligand expressed by the target cell. To determine whether the directivity of single-stranded AAV (ssAAV) can similarly be (1) reduced or inactivated, and (2) selectively reoriented, 293T-hASGR1 cells were cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector produced as described in Example 1, either in the absence of bispecific anti-myc-hASGR1 antibody or in the presence of bispecific anti-myc-hASGR1 antibody at different viral vector:antibody ratios (1:0, 1:1, 1:2, 1:4, 1:8, 1:20, 1:100, or 1:1000). As controls, 293T-hASGR1 cells cultured with wild-type ssAAV, 293T-hASGR1 cells cultured with ssAAV2-N587Myc-CMV-hrGFP viral vector in a 1:8 viral vector:antibody ratio and unrelated bispecific anti-myc-GCGR antibody, and 293T cells (not expressing hASGR1) cultured with ssAAV2-N587Myc-CMV-hrGFP viral vector in a 1:8 viral vector:antibody ratio and bispecific anti-myc-hASGR1 antibody. Three days after infection, cells were fixed and GFP expression was detected by FACS (Figure 5). GFP expression was detected in 293T-hASGR1 cells cultured with wild-type ssAAV, and in 293T-hASGR1 cells cultured with a mixture of ssAAV2-N587Myc-CMV-hrGFP viral vectors in all proportions and bispecific anti-myc-ASGR1 antibodies (Figure 5A, 5C-5I).GFP expression was not observed in 29T3 cells cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector and a bispecific anti-myc-ASGR1 antibody (Figure 5J), nor in 293T-hASGR1 cells cultured with only the ssAAV2-N587Myc-CMV-hrGFP viral vector, or with the ssAAV-N587Myc viral vector and an unrelated bispecific anti-myc-GCGR bispecific antibody (Figures 5B, 5K).

[0194] The human (h) glucagon receptor (GCGR) is not normally expressed by 293T cells. However, 293T-hGCGR is a stable 293T cell line genetically modified to express hGCGR on its cell surface. To test whether retargeting of the scAAV2-N587Myc-CMV-eGFP viral vector can be mediated by a bispecific anti-myc-GCGR antibody via the hGCGR receptor, 293T-hGCGR cells were cultured with different mixtures containing different ratios of scAAV2-N587Myc-CMV-eGFP:bispecific anti-myc-GCGR antibody. The scAAV2-N587Myc-CMV-eGFP viral vector was mixed with bispecific anti-myc-GCGR antibodies at room temperature in ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, or 1:100 30 minutes before adding it to 293T-hGCGR cells. Three days after infection, GFP expression in infected 293T-hGCGR cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 6). Significant percentages of GFP-positive cells were detected after culturing with wild-type scAAV (Figure 6A) or with scAAV2-N587Myc-CMV-eGFP / bispecific anti-myc-GCGR antibodies in ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, and 1:100 (Figures 6C-6K). GFP was not detected in 293T-hGCGR cells cultured with the scAAV2-N587myc-CMV-eGFP viral vector alone or with the scAAV2-N587Myc-CMV-eGFP viral vector containing a monospecific anti-myc antibody (Figures 6B and 6L, respectively).

[0195] Jurkat is a human acute T-cell leukemia cell line that expresses human (h)CD3. To test whether retargeting of the AAV6-Q585Myc-EF1a-eGFP viral vector can be mediated by a bispecific anti-myc-CD3 antibody via the hCD3 receptor, Jurkat cells were cultured with different mixtures containing different ratios of AAV6-Q585Myc-EF1a-eGFP:bispecific anti-myc-CD3 antibody. The AAV6-Q585Myc-EF1a-eGFP viral vector was mixed with the bispecific anti-myc-CD3 antibody at room temperature in ratios of 1:1, 1:5, 1:10, 1:100, and 1:1000 30 minutes before adding it to Jurkat cells. Three days after infection, GFP expression by infected Jurkat cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 7). Significant percentages of GFP-positive cells were detected after culturing with wild-type AAV6-EF1a-eGFP (Figure 7B), or with AAV6-Q585Myc-EF1a-eGFP / bispecific anti-myc-CD3 antibodies in 1:1, 1:5, 1:10, and 1:100 ratios (Figures 7D-7G). GFP was not detected in Jurkat cells cultured with the AAV6-Q585Myc-EF1a-eGFP viral vector (Figure 7C).

[0196] Described in this embodiment is the innate targeting of several self-complementary (sc) or single-stranded (ss) AAV serotypes, demonstrated by the inactivation of scAAV2, or genetically modified ssAAV2 (scAAV-N587myc or ssAAV-N587myc) in which a c-myc epitope is inserted between amino acids N587 and R588 of the VP1 capsid protein, AAV6 in which a c-myc epitope is inserted between Q585 and S586, and AAV9, including infected cells normally infected with wild-type AAV (Figures 1A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B, and 7). In addition, this embodiment demonstrates the ability of a bispecific antibody that recognizes the c-myc epitope and a second ligand expressed by target cells (e.g., hASGR1, hGCGR, or hCD3) to retarget the modified AAV, and to mediate infection of target cells with pseudotyped AAV in a ligand-specific manner (Figures 1-7). Furthermore, this embodiment demonstrates that co-administration of genetically modified sc- or ss-AAV and the bispecific antibody is not necessary for infection; that is, continuous administration is sufficient for in vitro retargeting of genetically modified sc- or ss-AAV (Figure 4).

[0197] Example 3: Antibody-mediated reorientation of a modified viral vector in vivo Retargeting of viral vectors to the liver using different multispecific binding molecule formats To determine whether a bispecific anti-myc-ASGR1 antibody can retarget the scAAV2-N587myc-CMV-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 1 x 10⁶ mice genetically modified to background expression of hASGR1 in hepatocytes in C57BL / 6 mice, and control wild-type C57BL / 6 mice were subjected to a study. 11The wild-type scAAV2-CMV-eGFP alone (titrated by qPCR) or the scAAV2-N587myc-CMV-eGFP viral vector combined with bispecific anti-myc-ASGR1 in a 1:8 ratio of viral genome to antibody molecule was intravascularly injected. Controls included mice injected with physiological saline [250 mm NaCl] or the scAAV2-N587myc-CMV-eGFP viral vector alone. Ten days after injection, the mice were sacrificed and perfused transcardiacally with 4% PFA. The liver, kidneys, and heart were collected and dehydrated in 15% sucrose followed by 30% sucrose. Next, organs were cryosectioned on slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488 conjugate anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) (Figures 8A-8C). GFP-positive cells were detected in livers from genetically transformed animals modified to express ASGR1 in the liver and injected with wild-type scAAV2-CMV-eGFP or scAAV2-N587myc-CMV-eGFP combined with bispecific anti-myc-ASGR1 antibody (Figures 8A(i) and 8A(iv)), as well as in livers from wild-type C57BL / 6 mice injected with wild-type scAAV2-CMV-eGFP (Figure 8A(v)). GFP was not detected in any spleen or kidney samples injected with physiological saline or the scAAV2-N587myc-CMV-eGFP viral vector alone (Figures 8B and 8C), or in any liver, spleen, or kidney samples from any animal (Figure 8A(ii, iii, vi, vii)), or in any liver sample taken from wild-type C57BL / 6 animals injected with scAAV2-N587myc-CMV-eGFP combined with bispecific anti-myc-ASGR1 antibody (Figure 8A(viii)).In summary, the combination of the scAAV2-N587myc-CMV-eGFP viral vector and the bispecific anti-myc-ASGR1 antibody infected only (hepatic) cells expressing hASGR1, which strongly suggests that the scAAV2-CMV-eGFP viral vector was inactivated by modification of its capsid protein, for example, that the innate targeting of the scAAV viral vector could be reduced or neutralized using, for example, the c-myc epitope, and that such a viral vector could be specifically reactivated, for example, in vivo, specifically in hepatocytes, by, for example, the bispecific anti-myc-ASGR1 antibody, or specifically retargeted.

[0198] Similarly, to determine whether the bispecific anti-myc-ASGR1 antibody can retarget the ssAAV2-N587myc-CAGG-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, we used 2.18x10⁻¹⁰ C57BL / 6 mice genetically modified to background-express hASGR1 in hepatocytes and control wild-type C57BL / 6 mice. 11The ssAAV2-N587myc-CAGG-eGFP viral vector was intravascularly injected either wild-type ssAAV2-CAGG-eGFP alone (titrated by qPCR) or in combination with bispecific anti-myc-ASGR1 in a 1:4 ratio of viral genome to antibody molecule. Controls included mice injected with PBS or the ssAAV2-N587myc-CAGG-eGFP viral vector alone. Four weeks after injection, the mice were sacrificed and perfused transcardiacally with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose, followed by 30% sucrose. Next, organs were cryo-sectioned on slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488 conjugate anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). GFP-positive cells were detected in livers from genetically transformed animals modified to express ASGR1 in the liver and injected with wild-type ssAAV2-CAGG-eGFP or ssAAV2-N587myc-CAGG-eGFP combined with bispecific anti-myc-ASGR1 antibody (Figures 9E-9F, 9P-9R), and in livers from wild-type C57BL / 6 mice injected with wild-type ssAAV2-CAGG-eGFP (Figures 9B-9C). Surprisingly, the infection efficiency of ssAAV2-N587myc-CAGG-eGFP combined with bispecific anti-myc-ASGR1 antibody was significantly higher than that of WT ssAAV2-CAGG-GFP (Figures 9E-9F, 9P-9R). Liver samples from all animals injected with physiological saline or the ssAAV2-N587myc-CAGG-eGFP viral vector alone (Figures 9A, 9D, 9G-9L), or liver samples taken from wild-type C57BL / 6 animals injected with ssAAV2-N587myc-CAGG-eGFP combined with bispecific anti-myc-ASGR1 antibody (Figures 9M-9O), were either undetectable or barely detectable.In summary, the combination of the ssAAV2-N587myc-CAGG-eGFP viral vector and the bispecific anti-myc-ASGR1 antibody infected only (hepatic) cells expressing hASGR1, which strongly suggests that the ssAAV2-N587myc-CAGG-eGFP viral vector was inactivated by modification of its capsid protein, for example, that the innate targeting of the scAAV viral vector could be reduced or neutralized using, for example, the c-myc epitope, and that such a viral vector could be specifically reactivated, for example, in vivo, specifically in hepatocytes, by, for example, the bispecific anti-myc-ASGR1 antibody, or specifically retargeted.

[0199] Further experiments using pseudotype AAV9 virus vectors were conducted in mice genetically modified at C57BL / 6 to express hASGR1 in hepatocytes. These mice were given 1x10⁻¹⁰ 11Mice were injected with wild-type AAV9-CAGG-eGFP (titrated by qPCR) or AAV9-A589myc-CAGG-eGFP virus particles combined with a bispecific anti-myc-ASGR1 antibody in a 1:100 ratio of viral genome to antibody molecule. Controls included mice injected with PBS or AAV9-N587myc-CAGG-eGFP virus particles combined with an unrelated bispecific anti-myc-hCD3 antibody. Four weeks after injection, the mice were sacrificed. The livers were fixed in 10% formalin and sent to HistoWiz Inc (New York, NY) for GFP staining. GFP-positive cells were detected in the livers of animals injected with wild-type AAV9-CAGG-eGFP or AAV9-N587myc-CAGG-eGFP virus particles combined with a bispecific anti-myc-ASGR1 antibody (Figures 10A and 10D). In all liver samples from animals injected with physiological saline or AAV9-N587myc-CAGG-eGFP virus particles combined with a bispecific anti-myc-hCD3 antibody, GFP was either not detected or barely detected (Figures 10B and 10C). In summary, similar to AAV2, the innate targeting of AAV9 can be reduced or neutralized by modification of the capsid protein, for example, by insertion of the c-myc epitope, and such modified viral vectors, e.g., AAV9-A589myc, can be retargeted to specific cell types using corresponding bispecific anti-epitope cell-specific marker-binding proteins, e.g., a bispecific anti-myc-ASGR1 antibody against hepatocytes.

[0200] Anti-hASGR1 IgG4 knob-in-hole Fc format with added anti-myc scFc To determine whether various multispecific binding molecular formats can mediate AAV infection, anti-myc scFv was fused to the C-terminus of one strand of a knob-into-hole anti-hASGR1 IgG4 antibody (see Figure 11A). Specifically, gblocks encoding anti-myc scFv (SEQ ID NO: 28) adjacent to the same arms as 1459-1478 and 1479-1498 of pRG7078 encoding the anti-hASGR1 stealth knob IgG4 heavy chain were obtained from Integrated DNA Technologies (Skokie, Illinois). The nucleotide sequences (NA) encoding the amino acid sequences (AA) of scFv (SEQ ID NO: 37) encoded by SEQ ID NO: 28 are provided in Table 3. [Table 3]

[0201] gblock was cloned into pRG7078 encoding the anti-hASGR1 stealth knob IgG4 heavy chain to create pRG7078 encoding the anti-hASGR1 stealth knob IgG4 anti-myc scFv fusion peptide, "anti-hASGR1-IgG4-Fc / anti-myc bispecificity". Modified pRG7078 plasmid using c-myc, unmodified pRG7078 anti-hASGR1 IgG4 stealth Hallstar, and plasmids containing the antibody light chain were transfected into 293T cells and cultured with OPTI-MEM (Cat#31985070, Thermo Fisher). Four days after transfection, the culture medium was collected and the anti-hASGR1-IgG4-Fc / anti-myc bispecificity binding molecule was purified using a Protein A column (cat#89948, Thermo Fisher).

[0202] To construct a pseudotyped AAV8 viral vector, we ordered a gblock containing pAAV RC8 N590myc and a c-myc epitope (SEQ ID NO: 22) inserted between N590 and T591 of the AAV8 VP1 capsid protein from Integrated DNA Technologies (Skokie, Illinois). The pAAV RC8 plasmid (SEQ ID NO: 23) was digested with Mlu1 and Sbf1 enzymes, and the gblock was cloned into a vector using SLIC to produce pAAV RC8 N590myc (SEQ ID NO: 24).

[0203] -293T-hASGR1 cells, which are 293T cells genetically modified to express human (h)ASGR1 on their cell surface, were cultured with either a mixture containing either AAV8-N590Myc-CAGG-eGFP versus -hASGR1-IgG4-Fc / anti-myc bispecific binding molecule or the AAV8-CAGG-eGFP viral genome in different ratios (1:0, 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50, or 1:100). Three days after infection, GFP expression was analyzed by FACS. After culturing cells with a mixture of AAV8-N590Myc-CAGG-eGFP and an anti-hASGR1-IgG4-Fc / anti-myc bispecific molecule in ratios of 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50, or 1:100, GFP-positive 293T-hASGR1 cells were detected (2.12%~22.2%, Figures 12D~12K) at a rate comparable to that of 293T-hASGR1 cells cultured with wild-type AAV8-CAGG-eGFP (46.1%) (Figure 12A). Neither culture with pseudo-infected cells (Figure 12B) nor with AAV8-N590Myc-CAGG-eGFP alone resulted in GFP expression by 293T-hASGR1 cells (Figure 12C).

[0204] To determine whether the anti-hASGR1-IgG4-Fc / anti-myc bispecific binding protein can retarget AAV8 N590myc-CAGG-eGFP virus particles to hepatocytes expressing hASGR1 in vivo, 1x10⁶ mice genetically modified to background-express hASGR1 in hepatocytes at C57BL / 6 were subjected to a study. 11Wild-type AAV8-CAGG-eGFP (titrated by qPCR) or AAV8 N590myc-CAGG-eGFP viral particles combined with an anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecule in a 1:12 ratio were injected intravascularly. Control animals were injected with AAV8 N590myc-CAGG-eGFP viral particles combined with an unrelated anti-hCD3x anti-myc bispecific binding molecule having a similar format to the anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecule. Three weeks after injection, mice were sacrificed and perfused transcardiacally with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Next, organs were cryo-sectioned on slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa488 conjugate anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). GFP-positive cells were detected in livers from animals injected with wild-type AAV8-CAGG-eGFP (Figures 13A-13C) or AAV8 N590myc-CAGG-eGFP virus particles combined with an anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecule (Figures 13G-13I). GFP was either not detected or barely detected in liver samples from animals injected with AAV8 N590myc-CAGG-eGFP virus particles combined with an unrelated anti-hCD3 / myc binding protein (Figures 13D-13F). In summary, epitope-labeled AAV8, such as AAV8-N590myc, can be retargeted to specific cell types using a bispecific binding molecule that specifically binds to the epitope and a cell-specific marker expressed by the target cell type.

[0205] Retargeting of viral vectors to the intestines and / or pancreas 293T cells ("293T-ENTPD3") genetically modified to express human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3) on their cell surface were cultured with a mixture containing the AAV2-N587Myc-CAGG-eGFP or AAV2-CAGG-GFP viral genome and a bispecific binding molecule formed by fusing anti-myc scFv to the C-terminus of one strand of a knob-into-hole anti-hENTPD3 IgG4 antibody (anti-hENTPD3-IgG4-Fc / anti-myc) in different ratios (1:0, 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, 1:100, or 1:200). Three days after infection, GFP expression was analyzed by FACS. After culturing with a mixture of AAV2-N587Myc-CAGG-eGFP and anti-hENTPD3-IgG4-Fc / anti-myc, GFP-positive 293T-ENTPD3 cells were detected at levels comparable to those of 293T-ENTPD3 cells cultured with wild-type AAV2-CAGG-eGFP (66%) (Figure 14A) (1.48%~16%, Figures 14C~14J). Culturing with AAV2-N587Myc-CAGG-eGFP alone resulted in very low levels of GFP expression in 293T-ENTPD3 cells (Figure 14B).

[0206] ENTPD3 was determined to be expressed in the intestinal mucosa (data not shown). To determine whether the anti-hENTPD3-IgG4-Fc / anti-myc binding protein can retarget AAV2-N587myc-CAGG-eGFP virus particles to intestinal cells expressing ENTPD3 in vivo, 5x10⁶ C57BL / 6 mice were subjected to a test. 11AAV2-N587myc-CAGG-eGFP viral particles were intravascularly injected with anti-hENTPD3-IgG4-Fc / anti-myc binding proteins in a 1:20 ratio of viral genome-to-binding protein molecules (titation by qPCR). Control mice were injected with AAV2-N587myc-CAGG-eGFP viral particles combined with PBS, wild-type AAV9, or unrelated binding protein molecules. Three weeks after injection, the mice were sacrificed. The liver, intestines, and pancreas were fixed in 10% formalin and sent to HistoWiz Inc (New York, NY) for GFP staining. GFP-positive cells were detected in mice injected with wild-type AAV9 (Figure 15B(ii)), but not in the livers of mice injected with PBS, or AAV2-N587myc-CAGG-eGFP virus particles combined with unrelated binding molecules, or anti-hENTPD3-IgG4-Fc / anti-myc binding protein (Figures 15A(iii) to 15A(iv) respectively), indicating that the AAV2-N587myc virus vector does not infect ENTPD3-negative hepatocytes, even when co-injected with hENTPD3-IgG4-Fc / anti-myc binding protein. GFP was detected only in the intestines of mice injected with wild-type AAV9 or AAV2-N587myc-CAGG-eGFP virus particles combined with hENTPD3-IgG4-Fc / anti-myc binding protein (Figures 15B(i) to 15B(iv)). GFP was detected in the pancreatic islets of mice injected with AAV2-N587myc-CAGG-eGFP virus particles combined with an anti-hENTPD3-IgG4-Fc / anti-myc binding protein (Figure 15C(iv)). WT AAV9 infected both islet and non-islet cells of the pancreas (Figure 15C(ii)). GFP was not detected in pancreatic samples from mice injected with saline or AAV2-N587myc-CAGG-eGFP virus particles combined with unrelated binding proteins (Figures 7C(i) and 7C(iii)).In summary, the innate targeting of AAVs can be reduced or neutralized by inserting heterologous epitopes, and they can be retargeted to the same or different cells using multispecific binding proteins that bind to epitope tags and markers expressed by the cells or tissues being retargeted.

[0207] This embodiment demonstrates that several different serotypes of adeno-associated virus vectors can be genetically modified using heterologous epitopes described herein (e.g., c-myc) to inactivate their infectivity, and that the viral vectors can be retargeted and delivered to target nucleotides using bispecific antibodies that recognize both the heterologous epitopes (e.g., c-myc) and markers expressed by target cells (regardless of the bispecific format).

[0208] Example 4: Use of a genetically modified AAV-N587Myc virus vector to deliver therapeutic cargo to specific cells This embodiment demonstrates the ability of the AAV-N587Myc viral vector to specifically deliver therapeutic cargo, such as one or more suicide genes, biological therapeutic agents (e.g., antibodies), CRISPR / Cas gene editing systems, or shRNAs, to target cell types. Specifically, this embodiment describes the delivery of suicide genes, antibody coding sequences, or CRISPR / Cas gene editing systems to cells expressing target ligands.

[0209] Delivery of suicide genes to cells expressing targeted ligands To test the ability of the scAAV2-N587Myc viral vector to deliver suicide genes to specific cells, HER2 + We will use a xenograft nude mouse model of breast cancer (described in Wang et al. ((2010) Cancer Gene Therapy 17:559-570)).

[0210] Viral vectors: The scAAV2-N587Myc or ssAAV2-N587Myc viral vector (scAAV2-N587Myc-EGFP or ssAAV2-N587MycEGFP) carrying the reporter EGFP gene is purified as described in Example 1. The scAAV2-N587Myc or ssAAV2-N587Myc viral vector carrying the suicide gene (SG) is produced in the same manner. In short, as described above, 293T17 cells are transfected under the control of a promoter, e.g., CMV, using a pAAV vector carrying (1) a pAd helper, and (2) pAAV RC2 (encoding a wild-type capsid) or a pAAV RC2-N587Myc vector (encoding a capsid modified with a c-myc epitope), and (3) a suicide gene, e.g., a cytosine deaminase gene or a herpes simplex virus thymidine kinase gene. The ssAAV-N587MycSG or scAAV-N587SG viral vector is isolated and titrated as described in Example 1.

[0211] Cell lines: The BT474 breast cancer, SK-BR-3 breast cancer, and Calu-3 lung cancer cell lines 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 are obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in media recommended by ATCC.

[0212] Mice: Obtain female nude mice, 6-8 weeks old, and raise them under conditions free of specific pathogens. On day 0, simultaneously administer (1)10 to the mice. 7(1) Subcutaneously inject BT474, SK-BR-3, Calu-3, A-673, or HeLa tumor cells into the right flank, and (2) intravenously treat with a bispecific anti-myc-HER2 antibody and an ss- or scAAV-N587Myc viral vector carrying a reporter (e.g., EGFP) or suicide gene. Untreated animals (animals injected with tumor cells only), animals injected with a wild-type ss- or scAAV viral vector carrying a reporter or suicide gene, animals injected with bispecific anti-myc-HER2 antibody only, or animals injected with an ss- or scAAV-N587Myc viral vector carrying only a reporter or suicide gene serve as controls. All animals are treated with appropriate prodrugs one day after injection and treatment. The size of each tumor is measured twice weekly using calipers, and the tumor volume is length × width 2 Calculate using a multiplier of 0.52. (Pathological condition, tumor ulceration, tumor diameter 15 mm, or tumor volume 1000 mm) 3 At this stage, 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 wild-type ss- or scAAV, or ss- or scAAV-N587Myc virus vectors carrying the reporter gene, are fixed, and reporter gene expression is visualized.

[0213] While targeted delivery of suicide-inducing genes has been described (Zarogoulidis P., et al. (2013) J. Genet. Syndr. Gene Ther. 4:16849), this example describes the delivery of a suicide gene to cells expressing a targeted HER2 ligand using a viral vector described herein containing a heterologous epitope, e.g., c-myc, as well as a bispecific antibody that specifically binds to a targeted ligand and the heterologous epitope. In additional experiments, a suicide gene is delivered to another cell type expressing one or more other target ligands using a viral vector containing a heterologous epitope described herein, as well as a bispecific antibody that specifically binds to the heterologous epitope (e.g., c-myc) and the target receptor. Exemplary and non-limiting examples of receptors suitable for targeting include receptors that mediate the endocytosis of viral vectors, such as carcinoembryonic antigen (CEA) (Qiu Y, et al. (2012) Cancer Lett. 316:31-38) and vascular endothelial growth factor receptor (VEGFR) (Leng A, et al. (2013) Tumour Biol. 32:1103-1111, Liu T, et al. (2011) Exp Mol Pathol. 91:745-752).Additional receptors that can be targeted include epidermal growth factor receptor (EGFR) (Heimberger AB, et al. (2009) Expert Opin Biol Ther. 9:1087-1098), leukocyte differentiation antigen 44 (CD44) (Heider KH, et al. (2004) Cancer Immunol Immunother. 53:567-579), leukocyte differentiation antigen 133 (CD133, also known as 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 leukocyte differentiation antigen 71 (CD71) (Habashy HO, et al., Breast Cancer Res). Examples include Treat.119(2):283-93), mucin (Torres MP, et al., (2012) Curr Pharm Des.2012;18(17):2472-81), stage-specific embryonic antigen 4 (SSEA-4) (Malecki M., et al., (2012) J Stem Cell Res Ther.2(5)), and tumor resistance antigen 1-60 (TRA-1-60) (Malecki M., et al., (2013) J Stem Cell Res Ther.3:134).

Claims

1. A recombinant adeno-associated virus (AAV) capsid protein modified to include a heterologous epitope containing the amino acid sequence EQKLISEEDL (shown as SEQ ID NO: 6), The aforementioned heterogeneous epitope, (a) AAV2 I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-4 71, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713 or I-716; (b) AAV6 I-585; (c) AAV8 I-590; (d) AAV9 I-453 or I-589; (e) I-587 or I-589 of AAV1; (f) AAV3 I-585; (g) I-585 of AAV4; and (h) AAV5 I-585 It is inserted immediately after an amino acid position selected from the group consisting of, or replaces the said amino acid position. The aforementioned heterologous epitope specifically binds to the antibody paratope, Recombinant AAV capsid protein wherein the recombinant AAV capsid protein forms a recombinant AAV capsid with reduced or inactivated intrinsic targeting.

2. The recombinant AAV capsid protein according to claim 1, further comprising substitutions at amino acid positions involved in the innate directivity of the AAV capsid, such that the recombinant AAV capsid protein forms a recombinant AAV capsid having reduced or inactivated innate directivity, wherein the substitutions are (a) AAV2 R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R588A or R588T; (b) K531A or K531E of AAV6; or (c) AAV9 W503A Recombinant AAV capsid proteins selected from the group consisting of the following.

3. The recombinant AAV capsid protein according to claim 1, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope, and the AAV is AAV2.

4. The recombinant AAV capsid protein according to claim 1, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope, and the AAV is AAV6.

5. The recombinant AAV capsid protein according to claim 1, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope, and the AAV is AAV8.

6. The recombinant AAV capsid protein according to claim 1, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope, and the AAV is AAV9.

7. (i) The AAV capsid protein is a genetically modified AAV2 VP1 capsid protein, and the heterologous epitope is inserted immediately after the amino acid at position I-453 or I-587, or replaces that amino acid. (ii) The AAV capsid protein is a genetically modified AAV6 VP1 capsid protein, and the heterologous epitope is inserted immediately after the amino acid at position I-585, or replaces that amino acid. (iii) The AAV capsid protein is a genetically modified AAV8 VP1 capsid protein, and the heterologous epitope is inserted immediately after the amino acid at position I-590, or replaces that amino acid, or (iv) The recombinant AAV capsid protein according to any one of claims 1 or 3 to 6, wherein the AAV capsid protein is a genetically modified AAV9 VP1 capsid protein, and the heterologous epitope is inserted immediately after or replaces the amino acid at position I-453 or I-589.

8. The recombinant AAV capsid protein according to any one of claims 1 to 3 or 7, wherein the recombinant AAV capsid protein is encoded by an AAV2 capsid gene modified to express the heterologous epitope between amino acids N587 and R588 of the AAV2 VP1 capsid protein.

9. The recombinant AAV capsid protein according to any one of claims 1, 2, 4, or 7, wherein the recombinant AAV capsid protein is encoded by an AAV6 capsid gene modified to express the heterologous epitope immediately after amino acid Q585 of the AAV6 VP1 capsid protein.

10. The recombinant AAV capsid protein according to any one of claims 1, 5, or 7, wherein the recombinant AAV capsid protein is encoded by an AAV8 capsid gene modified to express the heterologous epitope immediately after amino acid N590 of the AAV8 VP1 capsid protein.

11. The recombinant AAV capsid protein according to any one of claims 1, 2, 6, or 7, wherein the recombinant AAV capsid protein is encoded by an AAV9 capsid gene modified to express the heterologous epitope immediately after or by replacing the amino acid G453 or A589 of the AAV9 VP1 capsid protein, and the recombinant AAV capsid protein further comprises a mutation in addition to the heterologous epitope, the mutation further reduces or neutralizes the directivity of the recombinant AAV capsid compared to a standard AAV capsid lacking the mutation.

12. The recombinant AAV capsid protein according to claim 11, wherein the recombinant AAV capsid protein further comprises a W503A substitution.

13. The recombinant AAV capsid protein according to any one of claims 1 to 12, wherein the heterologous epitope is adjacent to and / or operably linked to at least five consecutive amino acids of the AAV capsid protein.

14. The recombinant AAV capsid protein according to any one of claims 1 to 13, wherein the recombinant AAV capsid protein comprises the amino acid sequence represented as SEQ ID NO: 2, the amino acid sequence represented as SEQ ID NO: 4, the amino acid sequence represented as SEQ ID NO: 25, the amino acid sequence represented as SEQ ID NO: 26, or the amino acid sequence represented as SEQ ID NO:

27.

15. The recombinant AAV capsid protein, or the recombinant AAV capsid containing the recombinant AAV capsid protein, is unable to infect target cells in the absence of the multispecific binding molecule containing the antibody paratope, and selectively, in the absence of the multispecific binding molecule containing the antibody paratope, the transduction efficiency of the recombinant AAV capsid protein, or the recombinant AAV capsid containing the recombinant AAV capsid protein, is (i) Reduced by at least 10%, (ii) It will be reduced by at least 20%, (iii) It will be reduced by at least 30%, (iv) Reduced by at least 40%, (v) Reduced by at least 50%, (vi) Reduced by at least 60%, (vii) Reduced by at least 70%, (viiii) It will be reduced by at least 80%, (ix) Reduced by at least 90%, or (x) will be invalidated, Recombinant AAV capsid protein according to any one of claims 1 to 14.

16. A recombinant AAV capsid comprising the recombinant AAV capsid protein described in any one of claims 1 to 15, wherein the recombinant AAV capsid is optionally a mosaic AAV capsid, and optionally the mosaic AAV capsid is in a specific recombinant AAV capsid protein:standard AAV capsid protein ratio. Recombinant AAV capsid protein according to any one of claims 1 to 15, The standard AAV capsid protein of the same serotype that does not contain the aforementioned heterologous epitope and Recombinant AAV capsids, including [specifically, a type of AAV capsid].

17. A recombinant AAV vector comprising a target nucleotide encapsulated by the recombinant AAV capsid described in claim 16.

18. The recombinant AAV vector according to claim 17, 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.

19. The recombinant AAV vector according to claim 18, wherein the target nucleotide is under the control of a non-human promoter.

20. The recombinant AAV vector according to claim 19, wherein the target nucleotide is adjacent to the AAV ITR sequence.

21. The recombinant AAV vector according to any one of claims 17 to 20, wherein the target nucleotide is a reporter gene.

22. The recombinant AAV vector according to claim 21, wherein the reporter gene encodes a green fluorescent protein.

23. The recombinant AAV vector according to any one of claims 17 to 20, wherein 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 a CRISPR / Cas system or a part(s) of it, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, and combinations thereof.

24. (a) A recombinant AAV vector according to any one of claims 17 to 23, (b) A pharmaceutically acceptable carrier and A pharmaceutical composition containing the above.

25. An in vitro method for delivering target nucleotides to target cells expressing cell surface proteins or cell surface markers, (a) The target nucleotide, (i) an AAV capsid comprising the recombinant AAV capsid protein described in any one of claims 1 to 15, or (ii) Recombinant AAV capsid according to claim 16 To enclose in; (b) The AAV capsid containing the target nucleotides from (a) (i) the antibody paratope that specifically binds to the heterologous epitope; and (ii) Retargeting ligands that specifically bind to the cell surface protein or cell surface marker. To complex with multispecific binding molecules that include; (c) bringing the target cells into contact with the complex of (b) Methods that include...

26. An in vitro method for delivering target nucleotides to target cells expressing cell surface proteins or cell surface markers, (a) A recombinant AAV vector according to any one of claims 17 to 23, or a recombinant AAV vector of a pharmaceutical composition according to claim 24, (i) the antibody paratope that specifically binds to the heterologous epitope; and (ii) Retargeting ligands that specifically bind to the cell surface protein or cell surface marker. To complex with multispecific binding molecules that include; (b) Contacting the target cells with the complex of (a) or the pharmaceutical composition thereof. Methods that include...

27. (a) The target cell is a human cell, or (b) The target cell is a human nerve cell, or (c) The target cell is a human muscle cell, or (d) The target cells are selected from the group consisting of hepatocytes, brain cells, T cells, kidney cells, intestinal cells, pancreatic cells, cancer cells, and cells infected with a different pathogen. The extracorporeal method according to claim 25 or claim 26.

28. (a) The target cells are human hepatocytes, and the retargeting ligand optionally binds to human asialoglycoprotein receptor 1 (hASGR1) expressed by the human hepatocytes, or (b) The retargeting ligand binds to the GABA receptor expressed by the human nerve cell, or (c) The retargeting ligand binds to a transferrin receptor expressed by the human nerve cell, or (d) The target cell is a human T cell, and the retargeting ligand optionally binds to CD3 expressed by the human T cell, or (e) The target cell is a human T cell, and the retargeting ligand optionally binds to CD3ε expressed by the human T cell, or (f) The target cells are human hematopoietic cells, and the retargeting ligand optionally binds to CD34, or (g) The target cells are human cancer cells, and the retargeting ligand optionally binds to tumor-associated antigens, or (h) The target cells are human cancer cells, and the retargeting ligand optionally binds to tumor-associated antigens selected from the group consisting of E6, E7, and Her2, or (i) The cell surface protein is a human glucagon receptor (hGCGR), or (j) The target cell is an intestinal cell or a pancreatic cell, and the cell surface protein or cell surface marker is ENTPD3, The in vitro method according to claim 27.

29. (i) The retargeting ligand binds to CD20, or (ii) The retargeting ligand binds to the human glucagon receptor, or (iii) The retargeting ligand specifically binds to CD63 or human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3), The in vitro method according to any one of claims 25 to 27.

30. An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant AAV capsid protein according to any one of claims 1 to 15.

31. A method for preparing recombinant AAV capsid protein, (a) Expressing the isolated nucleic acid described in claim 30 in cells under conditions suitable for the production of the recombinant AAV capsid protein, (b) Isolating the recombinant AAV capsid protein expressed in step (a) from the cells. Methods that include...

32. (a) A first isolated nucleic acid encoding the recombinant AAV capsid protein according to any one of claims 1 to 15, (b) A composition comprising a second isolated nucleic acid encoding a standard AAV capsid protein of the same serotype that does not contain the heterologous epitope.

33. The composition according to claim 32, wherein the first isolated nucleic acid and the second isolated nucleic acid are present in a ratio in the range of 1:1 to 1:

15.

34. A method for preparing recombinant mosaic AAV capsid protein, (a) Administering the composition according to claim 32 or claim 33 to cells, (b) Maintaining the cells under conditions suitable for the expression of the recombinant mosaic AAV capsid protein, (c) Isolating the recombinant mosaic AAV capsid protein expressed in step (b) from the cells. Methods that include...

35. A method for generating AAV vectors, Culture packaging cells under conditions sufficient for AAV vector production. The packaging cells include, (a) A plasmid encoding the recombinant AAV capsid protein according to any one of claims 1 to 15 or the recombinant AAV capsid according to claim 16. (b) A plasmid comprising the isolated nucleic acid described in claim 30, or (c) The composition according to claim 32 or claim 33 Methods that include...

36. The method according to claim 35, further comprising isolating a self-complementary AAV vector from the culture supernatant.

37. The method according to claim 35 or claim 36, further comprising lysing the packaging cells and isolating the single-stranded AAV vector from the cell lysate.

38. (a) Removing cell fragments, (b) Processing the supernatant containing the AAV vector with DNase I and MgCl2, (c) Enriching the AAV vector, (d) Purifying the AAV vector, (e) Any combination of (a) to (d) and The method according to any one of claims 35 to 37, further comprising:

39. Packaging cells for generating AAV vectors, (a) A plasmid encoding the recombinant AAV capsid protein according to any one of claims 1 to 15 or the recombinant AAV capsid according to claim 16. (b) A plasmid comprising the isolated nucleic acid described in claim 30, or (c) The composition according to claim 32 or claim 33 Packaging cells, including those mentioned above.

40. The packaging cell according to claim 39, wherein the packaging cell further comprises a transfer plasmid containing a helper plasmid and / or a target nucleotide.

41. The packaging cell according to claim 40, wherein the transfer plasmid comprises 5' and 3' AAV inverted terminal repeat (ITR) nucleotide sequences adjacent to the target nucleotide.

42. The packaging cell according to claim 40 or claim 41, wherein the target nucleotide comprises a nucleic acid sequence encoding a reporter or therapeutic molecule.

43. The packaging cell according to claim 42, wherein the reporter is selected from the group consisting of β-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), high-sensitivity blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), high-sensitivity yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulea, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

44. The packaging cell according to claim 42, wherein the therapeutic molecule is selected from the group consisting of a toxin (e.g., encoded by a suicide gene), a therapeutic antibody or a fragment thereof, a CRISPR / Cas system or a part(s) thereof, antisense RNA, siRNA, shRNA, or a combination thereof.

45. The packaging cell according to any one of claims 40 to 44, wherein the transfer plasmid further comprises a viral promoter or a non-viral promoter.

46. The packaging cell according to claim 45, wherein the target nucleotide is operably linked to the viral promoter or non-viral promoter and is under the control of the viral promoter or non-viral promoter.

47. The packaging cell according to any one of claims 40 to 46, wherein the transfer plasmid comprises, from 5' to 3', a 5' AAV ITR, a viral promoter or a non-viral promoter, a target nucleotide, and a 3' AAV ITR.

48. A method for reducing or neutralizing the natural directivity of an AAV capsid, (a) Inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding an AAV capsid protein to form a nucleotide sequence encoding a genetically modified recombinant AAV capsid protein containing the heterologous epitope, wherein the heterologous epitope includes the amino acid sequence EQKLISEEDL (shown as Sequence ID No. 6), The aforementioned heterogeneous epitope, (a) AAV2 I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-4 71, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713 or I-716; (b) AAV6 I-585; (c) AAV8 I-590; (d) AAV9 I-453 or I-589; (e) I-587 or I-589 of AAV1; (f) AAV3 I-585; (g) I-585 of AAV4; and (h) AAV5 I-585 It is inserted immediately after an amino acid position selected from the group consisting of, or replaces the said amino acid position. The recombinant AAV capsid protein forms a recombinant AAV capsid with reduced or inactivated natural targeting, (b) Culture packaging cells under conditions sufficient for the generation of AAV capsids. A method comprising the following: wherein the packaging cell contains the nucleotide sequence.

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