Targeted modified recombinant viral vectors for targeted transfer of genetic material into human cells and their uses

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] Sequence Listing Reference Submitted as a text file via EFS Web The sequence listing set forth in 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 tropic modified recombinant viral vectors and compositions comprising same that are useful for the targeted transfer of genetic material into cells and / or tissues. [Background technology]

[0003] The 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 a variety of chronic and genetic diseases. Currently, the lack of an ideal gene delivery vehicle has limited progress in the clinical application of gene therapy. To achieve therapeutic success, a gene delivery vehicle must be capable of transducing target cells while avoiding transduction of non-target cells. Specifically, when the natural tropism of a virus does not meet urgent therapeutic needs, recombinant viral vectors are needed in which the natural tropism has been deleted or attenuated, and the desired tropism has been successfully engineered. (Buchholz et al.,)

[0004] In recent years, most progress in vector development has been achieved using not only enveloped viruses (e.g., viruses whose capsids are surrounded by a lipid bilayer) such as retroviruses, lentiviruses, and herpes simplex viruses, but also naked viruses (e.g., viruses that contain a capsid formed by viral capsid proteins without an envelope (e.g., a lipid bilayer)) such as adeno-associated viruses (AAV) and adenoviruses (Ad). AAV is a non-enveloped virus that is only mildly immunogenic but capable of transducing a wide range of species and tissues in vivo without overt toxicity, and therefore AAV-based vectors have been the focus of much research.

[0005] AAV is a small, non-enveloped, single-stranded DNA virus. The AAV genome is 4.7 kb and features two inverted terminal repeats (ITRs) and two open reading frames encoding the Rep and Cap proteins, respectively. The two ITRs are the only cis-receptors essential for AAV replication, 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 AAV integration into host cell 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 relative abundances of approximately 1:1:10. In vitro, the three proteins spontaneously assemble into virion-like structures, such as viral capsids. Thus, viral encapsidation in infected cells appears to proceed independently of viral DNA synthesis (as verified by Kotin et al. (1994) Hum. Gene Ther. 5:793).

[0006] Among all known AAV serotypes, AAV2 is perhaps the best characterized, since its infectious clone was first produced. (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) In general, all AAVs share more than 80% identity in nucleotide sequence.

[0007] AAV is a promising vector for human gene therapy because, unlike other viral vectors, AAV has not been shown to be associated with any known human disease and is generally not considered pathogenic (Muzyczka et al. (1992) Current Topics in Microbiology and Immunology 158:97-129). Furthermore, AAV can safely transduce postmitotic tissues with relatively low immunogenicity and can integrate into host chromosomes in a site-specific manner and into tissue culture cells on chromosome 19 when Rep proteins are 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 multiple 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 through a virus / ligand:cell / receptor interaction that ultimately leads to endocytosis of the virus by the infected cell. This ligand:receptor interaction is the focus of much of viral vector research and can be manipulated to redirect the natural tropism of the virus away from cells that are naturally permissive for infection of target cells by, for example, wild-type virus, via receptors expressed by the target cells.

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

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

[0011] Such techniques have been used with 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 al. al. (2005) Int. J. Oncol. 29:1359-69), and coronaviruses (Haijema et al. (2003) J. Virol. 77:4528-438, Wurdinger et al. (2005) Gene Therapy 12:1394-1404).

[0012] The more common approach is the recombinant genetic modification of viral capsid proteins, and thus the surface of viral capsids. In the indirect recombinant approach, viral capsids are modified with heterologous "scaffolds" that are then linked to adaptors. The adaptors bind to the scaffolds 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). Scaffolds such as (1) Fc-binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of antibody adapters, (2) (strept)avidin that binds to biotin-labeled adapters, (3) biotin that binds to adapters fused to (strept)avidin, and (4) protein:protein binding pairs that form isometric peptide bonds, such as SpyCatcher that binds to Spy-labeled adapters, have been incorporated into Ad (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. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988), and so on have been incorporated into AAV (Gigout et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988). (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, targeting ligands are directly inserted into or linked to viral capsids, i.e., the protein viral capsid is modified to express heterologous targeting ligands. The ligands then redirect, for example, bind to receptors or markers that are preferentially or only expressed on target cells. (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), paramyxoviruses (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 herpesviruses (Shah and Breakefield (2006) Current Gene Therapy 6:361-370, Campadelli-Fiume et al. (2011) Reviews in Medical. It is used in Virology 21:213-226). Each of these three approaches has its advantages and disadvantages. The main advantage of the direct recombinant approach is that the specificity of the viral vector is inherent in the viral genome and is maintained during replication. However, with this and indirect recombinant approaches, the ability to genetically modify the virus requires maintaining the capsid structure and placing the targeting ligand or scaffold in a position that will tolerate and properly present the targeting ligand or scaffold, thus limiting the repertoire of suitable ligands or scaffolds that can be used. Thus, recombinant retargeting methods are limited by the naturally occurring molecules that are useful as targeting ligands, leading to the incorporation of other binding ligands, such as antibodies or portions thereof. Both non-recombinant and recombinant adapter platforms offer the advantage of flexibility in the adapters used. However, achieving optimal transduction efficiency is difficult with these two-component systems. Provided herein is a viral retargeting strategy that overcomes the problems inherent in conventional recombinant retargeting strategies by inserting a heterologous epitope into the viral capsid, which exhibits reduced or abolished native targeting, which is restored and redirected, when combined with a multispecific, optionally bispecific, binding molecule comprising an antibody paratope, such as an Fv, that specifically binds to a heterologous epitope and a retargeting ligand that specifically binds to a target cell, particularly within a certain ratio of viral vector:multispecific binding molecule. [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 Summary of the Invention [Means for solving the problem]

[0016] Disclosed herein is a recombinant viral capsid protein, wherein the viral capsid comprises a recombinant viral capsid protein and a viral vector comprising a nucleotide of interest encapsulated by the recombinant viral capsid, wherein the capsid protein, capsid, and viral vector are genetically engineered to comprise (present) a heterologous epitope, wherein the heterologous epitope (a portion thereof or in combination with the viral capsid protein) forms a binding pair with an antibody paratope, and wherein the recombinant viral capsid protein or the viral capsid comprising the recombinant viral capsid protein exhibits a low The recombinant viral capsid protein / capsid / vector may further comprise mutations, insertions, or deletions, e.g., at amino acid positions involved in receptor binding, that correspond to the (natural) tropism of the viral capsid protein / capsid / vector, such that the (natural) tropism of the viral capsid protein / capsid / vector is reduced or abolished (e.g., a recombinant viral capsid protein that has a transduction efficiency in the absence of a multispecific, optionally bispecific, binding molecule that is lower than the transduction efficiency of a standard viral capsid protein / capsid / vector lacking a heterologous epitope, or an undetectable transduction efficiency in the absence of a multispecific, optionally bispecific binding molecule). The reduced or abolished (natural) tropism of such a recombinant viral capsid protein / capsid / vector can be improved or restored in the presence of an appropriate multispecific, optionally bispecific binding moiety. Accordingly, compositions comprising (1) a recombinant viral vector having a capsid comprising a recombinant capsid protein described herein, and (2) a multispecific, optionally bispecific, binding molecule comprising an antibody paratope and a targeting ligand, including compositions comprising a certain ratio of viral vector:multispecific binding molecule, are also described herein, as are their uses for directing and / or introducing genetic material into target cells.Also described are methods for retargeting recombinant viral vectors, e.g., to target and deliver a nucleotide of interest to a target cell, comprising contacting the recombinant viral vector with a multispecific, optionally bispecific, binding molecule, as well as methods for producing recombinant viral vectors.

[0017] Described herein are recombinant viral capsid proteins comprising an epitope, wherein the epitope is heterologous to the capsid protein, and wherein the epitope, or a portion thereof, specifically binds to an antibody paratope, and wherein the recombinant viral capsid protein, or a viral capsid comprising the recombinant viral capsid, has a native tropism that is reduced or abolished, e.g., in the absence of the multispecific, optionally bispecific, binding moiety.

[0018] In some embodiments, the insertion and / or display of the heterologous epitope reduces or abolishes the (natural) tropism of the viral capsid compared to a standard viral capsid lacking the heterologous epitope, e.g., the heterologous epitope is inserted (displayed) into a recombinant viral capsid protein such that the viral capsid comprises a mutation comprising an insertion of the epitope at an amino acid position and / or a substitution of an amino acid at an amino acid position with the epitope, and the mutation reduces or abolishes the (natural) tropism of the capsid protein, e.g., in the absence of a multispecific, optionally bispecific, binding moiety. In some embodiments, a heterologous epitope is inserted (displayed) into a viral capsid protein such that the insertion and / or display partially reduces the (native) tropism of the recombinant viral capsid, e.g., in the absence of the multispecific, optionally bispecific binding moiety, compared to a standard viral capsid lacking the heterologous epitope, and the viral capsid further comprises additional mutations (e.g., substitutions, deletions, insertions other than the insertion of the heterologous epitope) that further reduce and / or abolish the (native) tropism of the recombinant viral capsid or a recombinant viral vector comprising it, in the absence of the multispecific, optionally bispecific binding moiety, e.g., compared to a standard viral capsid lacking the mutation.

[0019] Generally, the recombinant viral capsid proteins described herein can be derived from a capsid gene, e.g., encoded by a capsid gene modified to express an epitope, and / or are genetically modified non-enveloped viruses that generally infect human cells, such as adenoviruses, 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 derived from an AAV capsid gene, e.g., encoded by a capsid gene modified to express an epitope, and / or are AAV serotypes of genetically modified adeno-associated virus (AAV) capsid proteins that infect primates, optionally wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant viral capsid protein is derived from an AAV2, AAV6, AAV8, or AAV9 capsid gene, such as 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 an AAV2 capsid gene, such as 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 by SEQ ID NO: 1, respectively. In some embodiments, the recombinant viral capsid proteins are derived from the AAV6 capsid gene, e.g., are genetically modified AAV6 VP1, VP2 and / or VP3 capsid proteins encoded by an AAV6 capsid gene modified to express an epitope, and / or the amino acid sequence of wild-type AAV6 VP1 is represented as SEQ ID NO: 3.In some embodiments, the recombinant viral capsid proteins are derived from the AAV8 capsid gene, e.g., genetically modified AAV8 VP1, VP2, and / or VP3 capsid proteins, encoded by an AAV8 capsid gene modified to express an epitope and / or the wild-type AAV VP1 protein amino acid sequence is each represented as SEQ ID NO: 21. In some embodiments, the recombinant viral capsid proteins are derived from the AAV9 capsid gene, e.g., genetically modified AAV9, encoded by an AAV9 capsid gene modified to express an epitope and / or the wild-type AAV9 VP1 amino acid sequence is each represented as SEQ ID NO: 5. The capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from an 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 an 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 an 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 an AAV9 capsid gene, e.g., is encoded by an AAV9 capsid gene that has been 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, wherein the chimeric AAV capsid gene comprises multiple nucleic acid sequences, each of which encodes a portion of a capsid protein of a different AAV serotype, and the multiple nucleic acid sequences together encode the chimeric AAV capsid protein. In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV2 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV6 capsid gene. In some embodiments, the viral capsid protein is derived from a chimeric AAV8 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from a chimeric AAV9 capsid gene.

[0021] Generally, the recombinant viral capsid proteins described herein comprise a heterologous epitope inserted into and / or presented by the recombinant capsid protein such that the heterologous epitope itself reduces and / or abolishes the native tropism of the recombinant capsid protein or capsids comprising it, compared to a standard capsid lacking the heterologous epitope or a standard capsid lacking the heterologous epitope, respectively. In some embodiments, the heterologous epitope is inserted into (presented by) a region of the capsid protein responsible for the native tropism of the wild-type standard capsid protein, e.g., a region of the capsid protein responsible for cellular 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 an AAV2 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position 1453 or 1587 of the AAV2 VP1 capsid protein and / or the amino acid at the corresponding position in the AAV2 VP2 and / or VP3 capsid protein;(ii) the viral capsid protein is derived from an AAV6 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position 1585 of the AAV6 VP1 capsid protein and / or the amino acid at the corresponding position of the AAV6 VP2 and / or VP3 capsid protein; (iii) the viral capsid is derived from an AAV8 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position 1590 of the AAV8 VP1 capsid protein and / or the amino acid at the corresponding position of the AAV8 VP2 and / or VP3 capsid protein; or (iv) the viral capsid protein is derived from an AAV9 capsid gene, and the epitope is inserted after and / or replaces the amino acid at position 1453 or 1589 of the AAV9 VP1 capsid protein and / or the amino acid at the corresponding position of the AAV9 In some embodiments, the heterologous epitope is inserted after and / or replaces an amino acid at a corresponding position in the VP2 and / or VP3 capsid protein. In some embodiments, the heterologous epitope is inserted after and / or replaces an amino acid at a corresponding position in the VP2 and / or VP3 capsid protein. In some embodiments, the heterologous epitope is inserted after and / or replaces an amino acid at a corresponding position in the VP2 and / or VP3 capsid protein of an AAV2 capsid protein, such as G453 of the AAV2 capsid protein VP1 (or a corresponding amino acid at a corresponding position in the VP2 and / or VP3 capsid protein encoded by the same capsid gene, or a corresponding amino acid at a corresponding position in the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). or a corresponding amino acid in the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9), Q585 of the AAV6 capsid protein VP1 (or a corresponding amino acid in the VP2 and / or VP3 capsid protein encoded by the same capsid gene), or a corresponding amino acid in the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9,and / or the corresponding amino acid in the VP3 capsid protein), N590 of the AAV8 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid protein encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV9), G453 of the AAV9 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid protein encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV9). , AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8), or A589 of AAV9 capsid protein VP1 (or the corresponding positions of VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids of VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused C-terminally to) G453 of the AAV2 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused C-terminally to) N587 of the AAV2 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) Q585 of the AAV6 capsid protein VP1 (or the corresponding amino acid in the VP2 and / or VP3 capsid protein encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid protein of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C-terminus of) N590 of the AAV8 capsid protein VP1 (or at the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or at the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused C-terminally to) G453 of the AAV9 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused C-terminally to) A589 of the AAV9 capsid protein VP1 (or the corresponding position in the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acid in the VP1, VP2, and / or VP3 capsid proteins of a different AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8).and / or the corresponding amino acids of the VP2 and / or VP3 capsid protein encoded by the same capsid gene). 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 in the VP2 and / or VP3 capsid protein encoded by the same capsid gene) and / or is presented by the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO:2. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises the amino acid sequence represented as SEQ ID NO:4. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises the amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO:25. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises an amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 26. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises an amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 27.

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

[0023] Generally, recombinant viral capsid proteins and / or viral vectors comprising recombinant viral capsids comprise a heterologous epitope that is at least one amino acid in length. In some embodiments, the heterologous epitope can be about 5 amino acids to about 35 amino acids in length and forms a binding pair with an antibody paratope (e.g., an immunoglobulin variable domain). In some embodiments, the heterologous epitope comprises at least 10 amino acids in length. In some embodiments, the heterologous epitope comprises an affinity tag. In some embodiments, the heterologous epitope and / or affinity tag does not form a binding pair with an immunoglobulin constant domain. In some embodiments, the heterologous epitope and / or affinity tag binds to a metal ion, e.g., Ni. 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ and the like. 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 comprises c-myc (EQKLISEEDL, SEQ ID NO: 6).

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

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

[0026] In some embodiments, the recombinant viral capsid described herein comprises the 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 comprises 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 comprises EQKLISEEDL (represented as SEQ ID NO:6) inserted between Q585 and S586 of the AAV6 VP1 capsid protein, e.g., comprises the amino acid sequence represented as SEQ ID NO:4.

[0027] In some embodiments, the recombinant viral capsid described herein comprises the 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 comprises EQKLISEEDL (represented as SEQ ID NO:6) inserted at I590 of the AAV8 VP1 capsid protein. In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented as SEQ ID NO:6) inserted between N590 and T591 of the AAV8 VP1 capsid protein, e.g., comprising the amino acid sequence represented as SEQ ID NO:25.

[0028] In some embodiments, the recombinant viral capsid described herein comprises the 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 comprises EQKLISEEDL (represented as SEQ ID NO:6) inserted at I453 of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid comprises the AAV9 In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented as SEQ ID NO: 6) inserted between G453 and S454 of the VP1 capsid protein, e.g., the amino acid sequence represented as SEQ ID NO: 26. In some embodiments, the recombinant viral capsid comprises AAV9 In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented as SEQ ID NO: 6) inserted between A589 and Q590 of the AAV9 VP1 capsid protein, e.g., 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 flanked by linkers, e.g., the heterologous epitope comprises, from N-terminus to C-terminus, a first linker, an affinity tag, and a second linker. In some embodiments, the first and second linkers are each independent polypeptides at least one amino acid long. In some embodiments, a heterologous epitope described herein, e.g., an affinity tag by itself or in combination with one or more linkers, is about 5 amino acids to about 35 amino acids long. In some embodiments, the first and second linkers are the same length and / or comprise the same amino acid sequence.

[0030] Generally, recombinant viral capsids comprising the recombinant viral capsid proteins described herein, in the absence of an appropriate multispecific, optionally bispecific, binding molecule, have reduced or abolished natural tropism, e.g., reduced or incapable of targeting and binding to standard cells that are naturally permissive for transduction, compared to a standard viral capsid, e.g., a capsid comprising a standard viral capsid protein, e.g., a viral capsid protein identical to the recombinant viral capsid protein but lacking a heterologous epitope. In some embodiments, and in the absence of an appropriate multispecific, optionally bispecific binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 10% reduced transduction efficiency compared to the standard viral capsid. In some embodiments, and in the absence of an appropriate multispecific, optionally bispecific binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 20% reduced transduction efficiency compared to the standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 30% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 40% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 50% reduced transduction efficiency compared to a standard viral capsid.In some embodiments, and in the absence of a suitable multispecific, optionally bispecific binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 60% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 70% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 75% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least an 80% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least an 85% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 90% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 95% reduced transduction efficiency compared to a standard viral capsid.In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, recombinant viral capsids comprising the recombinant viral capsid proteins described herein exhibit at least a 99% reduced transduction efficiency compared to a standard viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, transduction of control cells by recombinant viral capsids comprising the recombinant viral capsid proteins described herein is abolished, e.g., undetectable.

[0031] In some embodiments, a viral capsid comprising a recombinant viral capsid protein described herein is a mosaic capsid, e.g., comprising a specific ratio of a recombinant viral capsid protein comprising a heterologous epitope and a standard capsid protein not comprising a heterologous epitope. In some embodiments, the standard capsid protein is a wild-type standard capsid protein in that it comprises the amino acid sequence of a wild-type capsid protein having the same serotype as the recombinant viral capsid protein. In some embodiments, the standard capsid protein is a reference capsid protein in that it comprises the amino acid sequence of a recombinant viral capsid protein, except that the reference capsid protein lacks the heterologous epitope. In some embodiments, the standard capsid protein is a mutant wild-type standard protein in that it comprises substantially the same amino acid sequence as a wild-type capsid protein having the same serotype as the recombinant viral capsid protein, except for mutations (e.g., amino acid sequence insertions, chimerization, etc.) that reduce the tropism of the wild-type capsid protein. In some embodiments, the compositions described herein comprise, or the methods described herein combine, recombinant viral capsid proteins and standard capsid proteins in a ratio ranging from 1:1 to 1:15. In some embodiments, the ratio is 1:2. In some embodiments, the ratio is 1:3. In some embodiments, the ratio is 1:4. In some embodiments, the ratio is 1:5. In some embodiments, the ratio is 1:6. In some embodiments, the ratio is 1:7. In some embodiments, the ratio is 1:8. In some embodiments, the ratio is 1:9. In some embodiments, the ratio is 1:10. In some embodiments, the ratio is 1:11. In some embodiments, the ratio is 1:12. In some embodiments, the ratio is 1:13. In some embodiments, the ratio is 1:14. In some embodiments, the ratio is 1:15.

[0032] Also disclosed herein are nucleic acids encoding the recombinant viral capsid proteins disclosed herein (e.g., that may be used in methods of making recombinant viral capsids), compositions comprising such nucleic acids, and / or recombinant viral capsid proteins (e.g., a composition consisting essentially of recombinant viral capsid proteins, a composition comprising only a viral vector encapsulated by a capsid comprising a viral capsid protein described herein, a composition comprising such a viral vector and a multispecific, optionally bispecific, binding molecule (e.g., at a certain ratio of viral vector to multispecific, optionally bispecific binding molecule (molecule:molecule)), a composition comprising such a viral vector, a retargeting molecule, and a pharmaceutically acceptable carrier, etc.). 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 virus capsid protein.

[0033] In some embodiments, the nucleic acids described herein comprise a nucleotide sequence encoding the 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 comprise 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 comprise 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 described nucleic acids encode an amino acid sequence comprising the amino acid sequence represented as SEQ ID NO:2.

[0034] In some embodiments, the nucleic acids described herein comprise a nucleotide sequence encoding the 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 comprise 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 comprise 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 described nucleic acids encode an amino acid sequence comprising the amino acid sequence represented as SEQ ID NO:4.

[0035] In some embodiments, the nucleic acids described herein comprise a nucleotide sequence encoding the 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 comprise 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 comprise 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 described nucleic acids encode an amino acid sequence comprising the amino acid sequence represented as SEQ ID NO:25.

[0036] In some embodiments, the nucleic acids described herein comprise a nucleotide sequence encoding the 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 comprise 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 comprise 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 described nucleic acids encode an amino acid sequence comprising the amino acid sequence represented as SEQ ID NO:26. In some embodiments, the nucleic acids described herein comprise 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 comprise a nucleotide sequence encoding EQKLISEEDL (represented as SEQ ID NO:6) inserted between A589 and Q590 of the AAV9 VP1 capsid protein, e.g., in some embodiments, the described nucleic acids encode an amino acid sequence comprising the amino acid sequence represented as SEQ ID NO:27.

[0037] Generally, the recombinant viral vectors described herein comprise a viral capsid comprising a recombinant viral capsid protein described herein, wherein the viral capsid encloses a nucleotide of interest. In some embodiments, the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide of interest is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter.

[0038] Generally, the nucleotides of interest may be one or more genes, which may encode a detectable marker, e.g., a reporter, or a therapeutic polypeptide. In some embodiments, the nucleotides of interest are reporter genes. In some embodiments, the nucleotides of interest are reporter genes encoding a detectable marker selected from the group consisting of green fluorescent protein, luciferase, β-galactosidase, and the like. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the nucleotides of interest are selected from the group consisting of suicide genes, nucleotides encoding antibodies or fragments thereof, nucleotides encoding CRISPR / Cas systems or portion(s) thereof, nucleotides encoding antisense RNA, nucleotides encoding siRNA, secreted enzymes, and the like. In one embodiment, the nucleotides of interest encode a multidomain therapeutic protein, e.g., a protein comprising at least two domains that provide two distinct functions.

[0039] The compositions described herein generally include a viral vector comprising a recombinant viral capsid protein described herein, e.g., a capsid comprising a recombinant viral capsid protein, wherein the capsid encapsulates a nucleotide of interest. In some embodiments, the compositions described herein include (1) a viral vector having a capsid comprising a recombinant viral capsid protein genetically modified to include 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 comprise, at a minimum, a complementarity-determining region (CDR) that specifically recognizes a heterologous epitope, e.g., the CDR3 region of a heavy and / or light chain variable domain. In some embodiments, the multispecific, optionally bispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a heterologous epitope. For example, the multispecific, optionally bispecific binding molecule may comprise a single-domain heavy chain variable region or a single-domain light chain variable region, wherein the single-domain heavy chain variable region or the single-domain light chain variable region comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the multispecific, optionally bispecific binding molecule may comprise an Fv region, e.g., the multispecific, optionally bispecific binding molecule may comprise an scFv comprising an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the multispecific, optionally bispecific, binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a heterologous epitope, and the antibody (or portion thereof) further comprises one or more antibody constant domains (e.g., a heavy chain constant domain (e.g., CH1, hinge, CH2, CH3, CH4, etc.), and / or a light chain constant domain (e.g., CL), wherein one or more antibody constant domains do not bind to the heterologous epitope.

[0041] The multispecific, optionally bispecific, binding molecules described herein further comprise a retargeting ligand in addition to a paratope (e.g., an antibody or portion thereof comprising the paratope) that specifically binds to a heterologous epitope inserted / displayed in a recombinant viral capsid protein. In some embodiments, the retargeting ligand specifically binds to a receptor on the surface of a bead (e.g., for the isolation and / or purification of a recombinant viral capsid protein described herein). In some embodiments, the retargeting ligand specifically binds to a cell surface protein, e.g., a receptor, a cell surface marker, etc., expressed on the surface of a mammalian (e.g., human) eukaryotic cell, e.g., a target cell. In some embodiments, the retargeting ligand binds to (human) liver cells, (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 a (human) liver cell-specific marker, a (human) brain cell-specific marker, a (human) T cell-specific marker, a (human) kidney cell-specific marker, a (human) intestinal cell-specific marker, a (human) pancreatic cell-specific marker, a (human) tumor cell-specific marker, and / or a pathogenic epitope.

[0042] In some embodiments, the retargeting ligand binds to a receptor expressed by (human) hepatocytes, e.g., an asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) neuronal cells, e.g., GABA, transferrin, etc. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) T cells, e.g., CD3, e.g., CD3ε. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) hematopoietic stem cells, e.g., CD34. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) kidney cells. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) muscle cells, e.g., integrin, etc. In some embodiments, the retargeting ligand is a receptor expressed by (human) cancer cells, e.g., a tumor-associated antigen, e.g., adipophilin, 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, malic enzyme, mammaglobin A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide p53, PAP, PAX5, PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, 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, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigens (in lymphoma and other solid tumors), ErbB receptors, 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 colon, breast, and gastrointestinal cancers), and 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, and the like. 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 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 portion thereof) and the retargeting ligand are directly fused to each other. In some embodiments, the paratope (e.g., an antibody or portion thereof) that specifically binds to a heterologous epitope and the retargeting ligand are covalently linked 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 / displayed 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 / displayed in a recombinant viral capsid protein, and the second antigen-binding domain specifically binds to a receptor expressed by a target cell, the first antigen-binding domain being operably linked to a first heavy chain region comprising a first CH3 domain, the second antibody-binding domain being operably linked to a second heavy chain region comprising a second CH3 domain, and the first and second Ig C H In one embodiment, the first Ig C domain is a IgG1 domain, and the first Ig C domain is a IgG2 domain. H The 3 domain binds to protein A and the second Ig C H The C3 domain contains mutations that reduce or abolish Protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). H The C3 domain may further comprise a Y96F modification (Y436F in EU according to IMGT). HAdditional modifications that may be found within the three domains include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU by IMGT), N44S, K52N, and V82I for IgG2 antibodies (N384S, K392N, and V422I in IMGT, EU), and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT).

[0045] In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to an affinity tag displayed by a 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 multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by a 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 multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by a recombinant viral capsid protein described herein, and the second antigen-binding domain binds to hASGR1. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody, comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to a CD3 protein, e.g., CD3ε. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody, comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to an integrin.In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to an integrin, e.g., hGCGR. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first and a second antigen-binding domain, wherein the first antigen-binding domain binds to the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by the recombinant viral capsid protein described herein, and the second antigen-binding domain binds to ENTPD3.

[0046] Also described herein are methods for making and using recombinant viral capsid proteins, viral vectors, compositions, and the like, comprising the same. In some embodiments, a method for redirecting a virus, such as an adenovirus or adeno-associated virus, to deliver a diagnostic / therapeutic cargo to a target cell comprises combining a recombinant viral vector comprising a recombinant viral capsid protein described herein, e.g., a viral vector comprising a capsid comprising a recombinant viral capsid displaying a heterologous epitope, with a bispecific binding molecule, wherein the bispecific binding molecule comprises (i) an antibody paratope that specifically binds to the epitope and (ii) a retargeting ligand that specifically binds to a receptor. Such a method can include, as a first step in generating a recombinant viral vector, culturing packaging cells under conditions sufficient for the production of the viral vector, the packaging cells comprising a plasmid encoding the capsid protein comprising the epitope. When delivering diagnostic / therapeutic cargo to a target cell, the methods described herein can include contacting the target cell with a combination of a viral vector comprising a capsid containing a recombinant viral capsid displaying a heterologous epitope and a multispecific binding molecule, where 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 cell is ex vivo. In other embodiments, the target cell is in vivo in a subject, e.g., a human.

[0047] In some embodiments, the compositions described herein comprise, or the methods described herein combine, a recombinant viral vector comprising a nucleotide of interest encapsulated in a capsid comprising a recombinant capsid protein described herein at a molecule:molecule ratio that restores transduction efficiency of the viral vector similar to that of a standard viral vector, and a multispecific binding molecule. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) ranges from 1:0.5 to 1:100. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) ranges from 1:4 to 1:20. In some embodiments, the ratio of recombinant viral vector to bispecific binding molecule (molecule:molecule) ranges from 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.

[0048] Also described herein are methods for inactivating viral capsids and / or producing viral vectors, generally comprising: (a) inserting a nucleic acid encoding a heterologous protein into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the heterologous protein; and / or (b) culturing packaging cells under conditions sufficient to produce a viral vector, wherein the packaging cells comprise the nucleotide sequence. In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising a nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementary adeno-associated viral vector from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral vector from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the supernatant containing the viral vector with DNase I and MgCl2, (c) concentrating the viral vector, (d) purifying the viral vector, and (e) any combination of (a)-(d). Also provided herein are viral vectors produced according to the methods described herein, and packaging cells useful for producing the viral vectors described herein, e.g., packaging cells containing plasmids encoding the recombinant capsid proteins described.

[0049] In particular embodiments, for example, the following items are provided: (Item 1) a recombinant viral capsid protein comprising an epitope, said epitope being heterologous to said capsid protein; the heterologous epitope or a portion thereof specifically binds to an antibody paratope; A recombinant viral capsid protein, wherein said viral capsid protein forms a recombinant viral capsid with a reduced or abolished native tropism. (Item 2) 2. The recombinant viral capsid protein of item 1, wherein the epitope is at least one amino acid in length. (Item 3) 3. The recombinant viral capsid protein of claim 1 or 2, comprising a substitution, insertion, or deletion at an amino acid position involved in the native tropism of the viral capsid, such that the recombinant viral capsid protein forms a reduced or abolished viral capsid. (Item 4) 4. The recombinant viral capsid protein according to items 1 to 3, wherein the viral capsid protein is derived from an adeno-associated virus (AAV) capsid gene, and optionally the heterologous epitope is 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 a primate, and optionally the AAV serotype that infects a primate is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. (Item 5) 5. The recombinant viral capsid protein according to item 4, wherein the adeno-associated virus is AAV2. (Item 6) 5. The recombinant viral capsid protein according to item 4, wherein the adeno-associated virus is AAV6. (Item 7) 5. The recombinant viral capsid protein according to item 4, wherein the adeno-associated virus is AAV8. (Item 8) 5. The recombinant viral capsid protein according to 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 and / or replaces the amino acid at position 1453 or 1587; (ii) the viral capsid protein is a genetically modified AAV6 VP1 capsid protein, and the epitope is inserted after and / or replaces the amino acid at position 1585; (iii) the viral capsid is a genetically modified AAV8 VP1 capsid protein, and the epitope is inserted after and / or replaces the amino acid at position 1590; (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 1453 or 1589. (Item 10) 10. The recombinant viral capsid protein of 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) 10. The recombinant viral capsid protein according to any one of items 1 to 9, wherein the viral capsid protein is derived from an AAV6 capsid gene and the epitope is inserted immediately after amino acid Q585. (Item 12) Item 13. The recombinant viral capsid protein according to any one of Items 1 to 9, wherein the viral capsid protein is derived from an AAV8 capsid gene and the epitope is inserted immediately after amino acid N590 of the AAV8 VP1 capsid protein. 10. The recombinant viral capsid protein of any one of items 1 to 9, wherein the viral capsid protein is derived from an AAV9 capsid gene, the epitope is inserted into the capsid protein after and / or replaces an amino acid at position G453 or A589 of the AAV9 VP1 capsid protein, and the capsid protein further comprises additional mutations. (Item 14) 14. The recombinant viral capsid protein according to item 13, wherein the additional mutation is W503A. (Item 15) 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 wherein 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; 16. The 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) 17. The recombinant viral capsid protein of 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 a viral capsid comprising the recombinant viral capsid protein, is unable to infect a target cell in the absence of a multispecific binding molecule comprising the antibody paratope, and optionally, the transduction efficiency of the viral capsid protein, or a viral capsid comprising the recombinant viral capsid protein, in the absence of a multispecific binding molecule comprising the antibody paratope is (i) reduced by at least 10%; or (ii) reduced by at least 20%; or (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) The recombinant viral capsid protein according to any one of items 1 to 17, which is disabled. (Item 19) 19. An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant viral capsid protein according to any one of items 1 to 18. (Item 20) 20. The isolated nucleic acid of claim 19, wherein the nucleotide sequence encodes a recombinant viral capsid protein comprising 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) 21. A recombinant viral vector comprising a nucleotide of interest encapsulated by the recombinant viral capsid of item 20, optionally wherein the recombinant viral capsid is a mosaic capsid. (Item 22) 22. The recombinant viral vector of item 21, wherein the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. (Item 23) 23. The recombinant viral vector of item 22, wherein the nucleotide of interest is under the control of a non-human promoter. (Item 24) 24. The recombinant viral vector of item 23, wherein the nucleotides of interest are flanked by AAV ITR sequences. (Item 25) 25. The recombinant viral vector according to any one of items 21 to 24, wherein the target nucleotide is a reporter gene. (Item 26) 26. The recombinant viral vector of item 25, wherein the reporter gene encodes green fluorescent protein. (Item 27) 25. The recombinant viral vector of any one of Items 21 to 24, wherein the nucleotides of interest are selected from the group consisting of nucleotides encoding a suicide gene, an antibody or a fragment thereof, nucleotides encoding a CRISPR / Cas system or a portion(s) thereof, nucleotides encoding an antisense RNA, nucleotides encoding an siRNA, and combinations thereof. (Item 28) 28. A composition comprising: (a) the recombinant viral vector of any one of items 21 to 27; and (b) a multispecific binding molecule comprising an antibody paratope that specifically binds to an epitope, optionally wherein the multispecific binding molecule further comprises a retargeting ligand that specifically binds to a receptor expressed on a target cell; and optionally wherein the multispecific binding molecule is a bispecific binding molecule. (Item 29) 29. The composition of item 28, wherein the viral vector and the multispecific binding molecule are present in a ratio of 1:4. (Item 30) 30. The composition of claim 28 or 29, wherein the antibody paratope is an Fv domain. (Item 31) 31. The composition of claim 30, wherein the Fv domain is fused directly to a heavy chain constant domain. (Item 32) 32. The composition of 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; 33. The composition of 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) 34. The composition of claim 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 comprising two identical immunoglobulin heavy chains and two identical light chains; 33. The composition of any one of items 28 to 32, wherein the paratope that binds to a heterologous epitope 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 36) 36. The composition of claim 35, wherein the paratope is an scFv, and optionally the scFv comprises the amino acid sequence represented as SEQ ID NO: 37. (Item 37) 37. The composition of 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) 38. The composition of 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) 39. The composition of claim 38, wherein the cell surface marker is asialoglycoprotein 1 (ASGR1). (Item 40) 39. The composition of item 38, wherein the cell surface marker is CD3. (Item 41) 39. The composition of claim 38, wherein the cell surface marker is ENTPD3. (Item 42) 42. The composition according to any one of items 28 to 41, further comprising a pharmaceutically acceptable carrier. (Item 43) A method for directing the recombinant viral vector according to any one of Items 21 to 27 to a target cell, comprising: contacting the recombinant viral vector with a multispecific binding molecule; The 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) 44. The method of claim 43, wherein the target cell is in vivo. (Item 45) 45. The method of claim 43 or 44, wherein the contacting is carried out ex vivo. (Item 46) 43. A method for delivering a nucleotide of interest to a target cell expressing a cell surface protein, comprising contacting said target cell with the composition of any one of items 28 to 42, wherein said multispecific binding molecule comprises a retargeting ligand that binds to said cell surface protein. (Item 47) 47. The method of claim 46, wherein the target cell is in vitro. (Item 48) 47. The method of claim 46, wherein the target cell is in vivo in a subject. (Item 49) 49. The method of claim 48, wherein the subject is a human. (Item 50) 50. The method according to any one of items 46 to 49, wherein the target cells are human cells. (Item 51) 51. The method according to any one of items 46 to 50, wherein the target cells are selected from the group consisting of liver cells, brain cells, T cells, kidney cells, intestinal cells, pancreatic cells, cancer cells, and cells infected with a heterologous pathogen. (Item 52) 52. The method according to any one of items 46 to 51, wherein the target cells are human hepatocytes. (Item 53) 53. The method of any one of items 46 to 52, wherein the cell surface protein is human asialoglycoprotein receptor 1 (hASGR1). (Item 54) 52. The method according to any one of items 46 to 51, wherein the target cells are human T cells. (Item 55) 55. The method of claim 54, wherein the cell surface protein is CD3. (Item 56) 52. The method according to any one of items 46 to 51, wherein the cell surface protein is human glucagon receptor (hGCGR). (Item 57) 51. The method according to any one of items 46 to 50, wherein the target cells are intestinal cells. (Item 58) 51. The method according to any one of items 46 to 50, wherein the target cells are pancreatic cells. (Item 59) Item 60. The method according to items 57 and 58, wherein the cell surface protein is ENTPD3. 1. A method for inactivating a viral capsid protein, comprising: (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 comprising said heterologous epitope; (b) culturing packaging cells under conditions sufficient for the production of a viral vector, wherein the packaging cells contain the nucleotide sequence. (Item 61) 19. A method for producing a viral vector, comprising culturing packaging cells under conditions sufficient for the production of the viral vector, wherein the packaging cells contain a plasmid encoding the recombinant capsid protein of any one of items 1 to 18. (Item 62) 62. The method of claim 60 or 61, wherein the packaging cells further contain a helper plasmid and / or a transfer plasmid containing the nucleotide of interest. (Item 63) 63. The method of any one of items 60 to 62, further comprising isolating the self-complementary adeno-associated virus vector from the culture supernatant. (Item 64) 64. The method of any one of items 60 to 63, further comprising lysing the packaging cells and isolating the single-stranded adeno-associated virus vector from the cell lysate. (Item 65) a. Removing cellular debris; b. treating the supernatant containing the viral vector with DNase I and MgCl2; c. Concentrating the viral vector; d. purifying the viral vector; 65. The method according to any one of items 60 to 64, further comprising any combination of e.a. to d. (Item 66) A viral vector produced according to the method of any one of Items 60 to 65. (Item 67) 19. A packaging cell for producing a viral vector comprising a plasmid encoding the capsid protein according to any one of items 1 to 18. (Item 68) A binding molecule comprising a paratope that binds to SEQ ID NO: 6, wherein the paratope comprises the scFv of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 sequences encoded by the nucleic acid sequence represented as SEQ ID NO: 28. (Item 69) 69. The binding molecule of item 68, wherein the antibody paratope is an Fv domain. (Item 70) 70. The binding molecule of item 69, wherein the Fv domain is fused directly to a heavy chain constant domain. (Item 71) the binding molecule is a bispecific antibody and further comprises a retargeting ligand; 71. The binding molecule of any one of items 68 to 70, wherein the paratope and the retargeting ligand each comprise separate Fv domains fused to first and second heavy chain constant domains. (Item 72) 72. The binding protein of item 71, wherein the first and second heavy chains bind to Protein A with different binding affinities. (Item 73) the binding molecule is a bispecific antibody and further comprises a retargeting ligand; the retargeting ligand comprises a tetrameric antibody structure comprising two identical immunoglobulin heavy chains and two identical light chains; 70. The binding protein of any one of items 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) 74. The binding protein of item 73, wherein the paratope is an scFv, and optionally the scFv comprises the amino acid sequence represented as SEQ ID NO: 37. (Item 75) 73. The binding protein of any one of items 69 to 72, further comprising a retargeting ligand that is an antibody or a portion thereof. (Item 76) 76. The binding protein of claim 75, wherein the retargeting ligand specifically binds to a cell surface protein that is a cell surface marker. (Item 77) 77. The binding protein of item 76, wherein the cell surface marker is asialoglycoprotein 1 (ASGR1). (Item 78) 77. The binding protein of item 76, wherein the cell surface marker is CD3. (Item 79) 77. The binding protein of item 76, wherein the cell surface marker is GCGR. (Item 80) 77. The binding protein of item 76, wherein the cell surface marker is ENTPD3. (Item 81) A composition comprising: (a) the recombinant viral vector according to any one of Items 21 to 27; and (b) the binding protein according to any one of Items 68 to 81. (Item 82) 82. The composition of item 81, wherein the viral vector and the multispecific binding molecule are present in a ratio of 1:4. (Item 83) 83. The composition of claim 81 or 82, further comprising a pharmaceutically acceptable carrier. This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with the color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0050] [Figure 1-1]Immunofluorescence microscopy images (upper panels) or histograms obtained by fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by HepG2 cells cultured with (A) wild-type scAAV2-CMV-eGFP viral vector alone, (B) scAAV2-N587Myc-CMV-hrGFP viral vector alone, or scAAV2-N587Myc viral vector mixed with bispecific anti-Myc-ASGR1 antibody at 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, or (L) scAAV-N587Myc viral vector mixed with monospecific anti-Myc antibody at a ratio of 1:8. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2A-1] Figure 1 shows dot plots obtained from fluorescence-activated cell sorting (FACS) assessing green fluorescent protein (GFP) expression by 29T3-hASGR1 cells cultured with wild-type scAAV2 viral vector alone (i), scAAV2-N587Myc-CMV-eGFP viral vector alone (ii), scAAV2-N587Myc-CMV-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody at 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 viral vector mixed with irrelevant bispecific anti-Myc-GCGR antibody at a ratio of 1:8 (xii). Also shown is GFP expression by 29T3 cells cultured with scAAV-N587Myc viral vector mixed with bispecific anti-myc-ASGR1 antibody at a 1:8 ratio (xi). Each experiment used 2 x 10 cells and 5 x 10 viral vectors. [Figure 2A-2] Same as above. [Figure 2B-1]Histograms are shown from fluorescence-activated cell sorting (FACS) assays assessing green fluorescent protein (GFP) expression by 29T3-hASGR1 cells cultured with unmodified AAV9-CAGG-GFP viral vector alone (i), AAV9-A589Myc-CAGG-eGFP viral vector alone (ii), or AAV9-A589Myc-CAGG-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody at 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 10 cells and 1 x 10 viral vectors (titered by qPCR). [Figure 2B-2] Same as above. [Figure 3-1] Dot plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells pre-incubated with bivalent anti-ASGR1 antibodies 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 subsequently infected with scAAV2-N587Myc-CMV-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibodies at a 1:8 ratio (L). 29T3-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 2x105 cells and 5x109 viral vectors (titrated by qPCR). [Figure 3-2] Same as above. [Figure 4]Immunofluorescence microscopy images are provided assessing green fluorescent protein (GFP) expression by 293T-hASGR1 cells cultured with wild-type scAAV viral vector alone (A), scAAV2-N587Myc-CMV-eGFP viral vector alone (B), or subsequently cultured sequentially with 1x10 (C), 2x10 (D), 4x10 (E), 8x10 (F), 2x10 (G), 1x10 (H), or 1x10 (I) bispecific anti-Myc-ASGR1 antibody followed by 1x10 (C) scAAV2-N587Myc-CMV-eGFP viral vector. Also shown are immunofluorescence microscopy images of 293T cells (J) serially cultured with 1x10 anti-myc-ASGR1 antibody molecules followed by 1x10 scAAV2-N587Myc-CMV-eGFP viral vector, and 293T-hASGR1 cells (K) serially cultured with 1x10 irrelevant bispecific anti-Myc-GCGR antibody molecules followed by 1x10 scAAV2-N587Myc-CMV-eGFP viral vector. [Figure 5-1] Dot plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells cultured with wild-type ssAAV viral vector alone (A), ssAAV2-N587Myc-CMV-hrGFP viral vector alone (B), ssAAV2-N587Myc-CMV-hrGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody at 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 viral vector mixed with irrelevant bispecific anti-myc-GCGR antibody at a ratio of 1:8 (K). Also shown is GFP expression by 29T3 cells cultured with ssAAV-N587Myc viral vector mixed with bispecific anti-Myc-ASGR1 antibody at a 1:8 ratio (J). Each experiment used 2 x 10 cells and 5 x 10 viral vectors. [Figure 5-2] Same as above. [Figure 6-1]Dot plots obtained from fluorescence-activated cell sorting (FACS) are provided assessing green fluorescent (GFP) expression by 29T3-hGCGR cells cultured with wild-type ssAAVP viral vector alone (A), scAAV2-N587Myc-CMV-eGFP viral vector alone (B), scAAV2-N587Myc-CMV-eGFP viral vector mixed with bispecific anti-Myc-GCGR antibody at 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 viral vector mixed with an irrelevant monospecific anti-Myc antibody (Regeneron Pharmaceuticals, Tarrytown, NY) at a ratio of 1:8 (L). Each experiment used 2x105 cells and 5x109 viral vectors. [Figure 6-2] Same as above. [Figure 7-1] Dot plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by Jurkat cells cultured alone (A), with wild-type scAAV6-EF1-eGFP viral vector alone (B), with AAV6-Q585Myc-EF1a-eGFP viral vector alone (C), or with AAV6-Q585Myc-EF1a-eGFP viral vector mixed with bispecific anti-Myc-CD3 antibody at the following ratios: 1:1 (D), 1:5 (E), 1:10 (F), 1:100 (G), or 1:1000 (H). 2 x 10 cells and 1 x 10 viral vector were used in each experiment. [Figure 7-2] Same as above. [Figure 8A] 1 provides immunofluorescence microscopy images of the liver. [Figure 8B] 1 provides immunofluorescence microscopy images of the spleen. [Figure 8C]Immunofluorescence microscopy images of kidneys are shown. All samples were collected from C57BL / 6 mice genetically engineered to express human ASGR1 in hepatocytes (i-iv) or wild-type C57BL / 6 mice (v-viii) 10 days after intravenous injection of 1 x 10 wild-type scAAV2-CMV-eGFP (i, v), saline (ii, vi), 1 x 10 scAAV2-N587myc-CMV-eGFP viral vector alone (iii, vii), or scAAV2-N587myc-CMV-eGFP viral vector containing a bispecific anti-myc-ASGR1 antibody (iv, viii). [Figure 9-1] Immunofluorescence microscopy images of liver samples collected from C57BL / 6 mice genetically engineered to express human ASGR1 in hepatocytes (D-F, J-L, P-R) or wild-type C57BL / 6 mice (A-C, G-I, M-O) 4 weeks after intravenous injection of 2.18 x 10 wild-type ssAAV2-CAGG-eGFP (B, C, E, F), saline (A, D), 2.18 x 10 ssAAV2-N587myc-CAGG-eGFP viral vector alone (G-I, J-L), or ssAAV2-N587myc-CAGG-eGFP viral vector containing a 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] Immunofluorescence microscopy images of liver samples taken from C57BL / 6 mice genetically engineered to express human ASGR1 by hepatocytes are provided 10 days after intravenous injection of (A) wild-type AAV9, (B) 250 nM NaCl, (C) AAV9-A589myc-CAGG-eGFP viral particles combined with a bispecific anti-myc-hCD3 antibody, or (D) AAV9-A589myc-CAGG-eGFP viral particles combined with a bispecific anti-myc-ASGR1 antibody. [Figure 11]Illustrative, not to scale, non-limiting example multispecific binding molecule formats useful in some embodiments of the invention are provided. [Figure 12-1] Dot plots obtained from fluorescence-activated cell sorting (FACS) are provided to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells cultured with (A) wild-type AAV8 viral vector alone, (C) AA8-N590-myc viral vector alone, or pAAV RC8 N590myc viral vector mixed with the bispecific anti-hASGR1-IgG4-Fc / anti-myc bispecific molecule at 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 by mock-transfected 29T3-hASGR1 cells (B) is also shown. Each experiment used 2 x 10 cells and 1 x 10 viral vector. [Figure 12-2] Same as above. [Figure 13] Immunofluorescence microscopy images are provided of liver samples taken from C57BL / 6 mice genetically engineered to express human ASGR1 by hepatocytes 10 days after intravenous injection of (A)–(C) wild-type AAV8, (D)–(F) AAV8-N590-myc viral vector and control bispecific binding molecule, or (G)–(I) AAV8 N590myc-CAGG-eGFP viral particles combined with anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecule. [Figure 14-1]Dot plots obtained from fluorescence-activated cell sorting (FACS) are provided assessing green fluorescent protein (GFP) expression by 29T3-hENTPD3 cells cultured with (A) wild-type AAV2 viral vector alone, (B) AAV2-N587Myc-CAGG-eGFP viral vector alone, or AAV2-N587Myc-CAGG-eGFP viral vector mixed with the bispecific anti-hENTPD3-IgG4-Fc / anti-myc bispecific molecule at 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 10 cells and 1 x 10 viral vector. [Figure 14-2] Same as above. [Figure 15A] 1 provides immunofluorescence microscopy images of liver samples. [Figure 15B] 1 provides immunofluorescence microscopy images of intestinal samples. [Figure 15C] Fluorescence microscopy images of pancreatic samples are provided. All samples were collected from wild-type C57BL / 6 mice 10 days after intravenous injection of PBS (15A(i), 15B(i), and 15C(i)), 5 x 10 wild-type AAV9 (15A(ii), 15B(ii), and 15C(ii)), 5 x 10 AAV2-N587myc-CAGG-eGFP viral vector containing 1 x 10 irrelevant bispecific IgG4-Fc / anti-myc binding protein (15A(iii), 15B(iii), and 15C(iii)), or 5 x 10 AAV2-N587myc-CAGG-eGFP viral vector containing 1 x 10 bispecific hENTPD3-IgG4-Fc / anti-myc binding protein (15A(iv), 15B(iv), and 15C(iv)). DETAILED DESCRIPTION OF THE INVENTION

[0051] A common problem with adapter approaches that utilize unmodified or scaffold-modified viral capsids is the suboptimal transduction efficiency of the modified capsids. (Grifman et al. (2001) Mol. Ther. 3:964-75). For example, Curiel et al. describe the generation and characterization of recombinant adenovirus vectors containing a fiber with an RGD-4C sequence genetically incorporated into the HI loop of the carboxy-terminal knob domain, demonstrating the utility of the HI loop of the fiber knob as an optimal site for incorporating short peptide ligands. See, for example, U.S. Patent No. 7,297,542; also see Beatty and Curiel (2012) Adv. Cancer Res. 115:39-67. Similarly, insertion of a ligand peptide into the AAV capsid protein allows the ligand to be presented on the capsid surface and mediate transduction through the interaction of the receptor with the ligand, thereby redirecting viral tropism by genetically engineered 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, the insertion of an integrin-binding Arg-Gly-Asp (RGD) motif into the insertion site I-587 of the AAV capsid protein VP1 resulted in the formation of α vIt has been demonstrated that AAV viral vectors can transduce cells via β1 integrins (Girod et al. (1999) supra). In contrast, insertion of the 14-amino acid peptide L14 after amino acid R447 (I-447) resulted in a capsid that was still recognized by the conformation-sensitive antibody A20, but such a recombinant viral vector was unable to transduce cells expressing the L-14 receptor (Girod et al. 1999, see Wu et al. (2000) (who reported insertion of a hemagglutinin (HA) peptide at position I-447 and successful transduction of cells expressing the HA peptide). Insertion of a Myc epitope between T448 and N449 resulted in an inactivated viral vector, although it was recognized by anti-myc antibodies and therefore present on the capsid surface (Grifman et al., 2001). In contrast, successful retargeting by insertion of an NGR motif after N587 was again reported, but insertion of c-myc after N587 did not (Grifman et al. (E. al., 2001). U.S. Patent No. 9,624,274 describes I-453 of the AAV capsid protein as a suitable insertion site for heterologous epitopes. While these studies demonstrate the successful insertion and presentation of heterologous peptides, e.g., epitopes, by AAV capsid proteins, none of these studies offers the promise that multispecific binding molecules, e.g., bispecific binding molecules such as bispecific antibodies, that specifically bind to heterologous peptides and cell surface proteins, can retarget modified viral vectors to cells expressing the cell surface protein and restore their transduction efficiency.

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

[0053] While the present invention has been particularly shown and described with reference to numerous embodiments, those skilled in the art will understand that changes may be made in form and detail to the various embodiments disclosed herein without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to serve as limitations on the scope of the claims.

[0054] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some preferred methods and materials are 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 commonly understood by those skilled in the art to which this invention belongs.

[0055] Definition of Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0056] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0057] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds). Each heavy chain contains a heavy chain variable domain (V- H ) and the heavy chain constant region (C H The heavy chain constant region comprises at least three domains: C H 1. C H 2. C H Each light chain comprises a light chain variable domain (C H ) and the light chain constant region (C L ). Heavy and light chain variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure contains two identical antigen-binding domains, each consisting of a V H and V L formed by the meeting of each C H and C L The domains are combined together to form the antibody Fv region. Single domain antibodies contain a single antigen-binding domain, e.g., 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-linked Fv (sdFv), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the above. The terms "antibody" and "antibodies" also refer to covalent diabodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. 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, e.g., the portion of an antibody that recognizes and binds to an epitope of an antigen, is also referred to as the "paratope." It is a small region (of 5-10 amino acids) of the Fv region of an antibody, i.e., part of the antigen-binding fragment (Fab region), and may contain portions of the heavy and / or light chains of the antibody. A paratope specifically binds to an epitope when the paratope binds to the epitope with high affinity. The term "high affinity" antibody refers to an antibody that specifically binds to its target epitope with approximately 10 -9 M or less (e.g., about 1x10 -9 Medium, 1x10 -10 Medium, 1x10 -11 M, or approximately 1x10 -12 M)'s K D In one embodiment, K D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K Dis measured by ELISA.

[0059] The phrase "complementarity-determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin gene, which amino acid sequence is normally (i.e., in a wild-type animal) found between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). CDRs can be encoded, for example, by germline sequences or rearranged or unrearranged sequences, e.g., by naive or mature B cells, or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the animal germline), humanized, and / or modified with amino acid substitutions, additions, or deletions. Under some circumstances (e.g., with respect to a CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B cell nucleic acid sequence, e.g., as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

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

[0061] The term "inverted terminal repeat" or "ITR" refers to a symmetrical nucleic acid sequence in the genome of an adeno-associated virus that is required for efficient replication. The ITR sequence is located at each end of the AAV DNA genome. The ITR serves as an origin of replication for viral DNA synthesis and is an essential cis-component for generating an AAV integrating vector.

[0062] The term "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa and lambda light chains, as well as surrogate light chains, and VpreB, unless otherwise specified. A 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, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. The light chain variable domain is encoded by a light chain variable region gene sequence and generally includes a V segment derived from a repertoire of V and J segments present in the germline. L Segment and J L The sequences, locations, and nomenclature of V and J light chain segments of various organisms can be found in the IMGT database, www.imgt.org. Light chains include, for example, light chains that do not selectively bind to either the first or second 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, a heavy chain or another light chain by binding and recognizing one or more epitopes selectively bound by an epitope-binding protein present on the light chain. Common or universal light chains include light chains derived from human Vκ1-39Jκ genes or human Vκ3-20Jκ genes, including somatically mutated (e.g., affinity matured) versions thereof. Exemplary human Vκ are: LThe segments include the human Vκ1-39 gene segment, the human Vκ3-20 gene segment, the human Vλ1-40 gene segment, the human Vλ1-44 gene segment, the human Vλ2-8 gene segment, the human Vλ2-14 gene segment, and the human Vλ3-21 gene segment, including somatically mutated (e.g., affinity matured) versions thereof. Light chains can be made that include a variable domain from one organism (e.g., a human or rodent, e.g., a rat or mouse, or an avian, e.g., a chicken) and a constant region from the same or a different organism (e.g., a human or rodent, e.g., a rat or mouse, or an avian, e.g., a chicken).

[0063] The term "about" or "approximately" includes within a statistically significant range of values. Such a range may be within 10-fold, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" will depend on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0064] The term "affinity tag" includes, for example, a polypeptide sequence that is a member of a specific binding pair, specifically binding to another polypeptide sequence, e.g., an antibody paratope, with high affinity. Exemplary and non-limiting affinity tags include hexahistidine tag, FLAG tag, StrepII tag, streptavidin-binding peptide (SBP) tag, calcitonin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S-tag, HA tag, and c-Myc tag. (Zhao et al. (2013) J. Analytical Meth. Chem. 1-8, 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 produced in an overlapping manner from the cap open reading frame (ORF) through alternative mRNA splicing and / or the use of alternative translation initiation codons, but all three proteins share a common stop codon. Warrington et al. (2004) J. Virol. 78:6595, incorporated herein by reference in its entirety. AAV2 VP1 is generally translated from an ATG start codon (amino acid M1) on a 2.4 kb mRNA, whereas AAV2 VP2 and VP3 arise from a smaller 2.3 kb mRNA, using a weaker ACG start codon (amino acid T138) to generate VP2 and read-through translation to the next available ATG codon (amino acid M203) to generate VP3, the most abundant capsid methane protein. Warrington, supra; Rutledge et al. al. (1998) J. Virol. 72:309-19, the entire contents of which are incorporated herein by reference. The amino acid sequences of adeno-associated virus capsid proteins are well known in the art and are generally conserved, particularly among Dependoparvoviruses. See Rutledge et al., supra. For example, Rutledge et al. (1998) supra provides an amino acid sequence alignment of the VP1, VP2, and VP3 capsid proteins of AAV2, AAV3, AAV4, and AAV6 in Figure 4B, where the start sites of each of the VP1, VP2, and VP3 capsid proteins are indicated by arrows and the variable domains are boxed. Thus, while the amino acid positions provided herein may be provided relative to the VP1 capsid protein of AAV, and amino acid positions provided herein not further specified refer to the AAV2 sequence of the major tegument protein VP1, represented as SEQ ID NO: 1, one skilled in the art would readily be able to determine the same amino acid position within the VP2 and / or VP3 capsid proteins of AAV, and the corresponding amino acid positions among different serotypes, respectively. Additionally, one skilled in the art would be able to swap domains between capsid proteins of different AAV serotypes to form "chimeric capsid proteins."

[0066] Domain swapping between two AAV capsid protein constructs to generate "chimeric AAV capsid proteins" has been described; see, e.g., Shen et al. (2007) Mol. Therapy 15(11):1955-1962, incorporated herein by reference in its entirety. A "chimeric AAV capsid protein" includes an AAV capsid protein that contains amino acid sequences, e.g., domains from two or more different AAV serotypes, and is capable of and / or does form an AAV-like viral capsid / viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., nucleotides comprising a plurality, 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 which together encode a functional chimeric AAV capsid protein. References to chimeric capsid proteins relating to a particular AAV serotype indicate that the capsid protein contains one or more domains from a capsid protein of that serotype and one or more domains from a capsid protein of a different serotype. For example, an AAV2 chimeric capsid protein contains a capsid protein that contains one or more domains from an AAV2 VP1, VP2, and / or VP3 capsid protein and one or more domains from a VP1, VP2, and / or VP3 capsid protein of a different AAV.

[0067] A "mosaic capsid" contains at least two sets of VP1, VP2, and / or VP3 proteins, each set encoded by a different cap gene.

[0068] In some embodiments, the mosaic capsids described herein comprise recombinant VP1, VP2, and / or VP3 proteins encoded by cap genes that have been genetically modified by inserting a nucleic acid sequence encoding a heterologous epitope, and further comprise VP1, VP2, and / or VP3 proteins encoded by standard cap genes, e.g., a wild-type standard cap gene that encodes wild-type VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins; a standard cap gene that encodes VP1, VP2, and / or VP3 proteins that are identical to the recombinant VP1, VP2, and / or VP3 proteins but lacking the heterologous epitope; or a mutant wild-type standard cap gene that encodes wild-type VP1, VP2, and / or VP3 proteins of an AAV serotype that is substantially identical to the recombinant VP1, VP2, and / or VP3 proteins except for mutations (e.g., insertions, substitutions, deletions) that preferably reduce the tropism of the wild-type VP1, VP2, and / or VP3 proteins. In some embodiments, the canonical capsid protein is a chimeric canonical protein that includes at least one domain of a VP1, VP2, and / or VP3 protein of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 protein. In some embodiments, the canonical cap gene encodes a chimeric VP1, VP2, and / or VP3 protein.

[0069] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences, including immunoglobulin heavy chain constant region sequences from any organism. A heavy chain variable domain contains three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains contain CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain contains (from the N-terminus to the C-terminus) a CDR, a CDR, and a FR, following the variable domain. H 1 domain, hinge, C H 2 domain, and C H A functional fragment of a heavy chain specifically recognizes an epitope (e.g., a K in the micromolar, nanomolar, or picomolar range).D The heavy chain variable domain is capable of recognizing an epitope having the V (having the V, ... H , D H , and J. H From the segment repertoire, V H , D H , and J. H The sequences, locations, and nomenclature of V, D, and J heavy chain segments from various organisms can be found in the IMGT database, which is accessible via the Internet on the worldwide web (www) at the URL "imgt.org".

[0070] The terms "heavy chain-only antibody," "heavy chain-only antigen binding protein," "single domain antigen binding protein," "single domain binding protein," and the like refer to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable region comprising a variable domain operably linked to a heavy chain constant region, where the heavy chain constant region is typically a functional C H Therefore, the terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," etc., refer to antibodies that lack (i) a functional C H a monomeric single-domain antigen-binding protein comprising one of the immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region lacking one domain, or (ii) two immunoglobulin-like chains, each of which is a functional C H In various embodiments, the homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which is functionally linked to a heavy chain constant region lacking one domain, and a homodimeric single domain antigen binding protein comprising a variable domain operably linked to a heavy chain constant region lacking one domain. HIn addition, each immunoglobulin-like chain of a single domain antigen-binding protein contains a variable domain, which is derived from a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segments (e.g., V L , J L ), or a combination thereof, and the sequence of the heavy chain constant region (and optionally the hinge region) gene, e.g., C encoding IgG, IgA, IgE, IgD, or a combination thereof. H heavy chain constant region (C) containing a deletion or inactivating mutation in H A single domain antigen binding protein comprising a variable domain derived from a heavy chain gene segment may be referred to as a "V" H Single domain antigen binding proteins comprising a variable domain derived from a light chain gene segment may be referred to as a "V single domain antibody" or "VH single domain antibody binding protein," see, e.g., U.S. Patent No. 8,754,287, U.S. Patent Publication Nos. 2014 / 0289876, 2015 / 0197553, 2015 / 0197554, 2015 / 0197555, 2015 / 0196015, 2015 / 0197556, and 2015 / 0197557, each of which is incorporated by reference in its entirety. L These may be referred to as "single domain antigen binding proteins," see, e.g., U.S. Publication No. 2015 / 0289489, which is incorporated by reference in its entirety.

[0071] The term "light chain" includes immunoglobulin light chain sequences from any organism, and includes human kappa (κ) and lambda (λ) light chains, as well as surrogate light chains, and VpreB, unless otherwise specified. A light chain variable domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. The light chain variable domain is encoded by a light chain variable region nucleotide sequence and generally includes a light chain V and J gene segment derived from a repertoire of light chain V and J gene segments present in germline cells. L and light chain J L Light chain V gene segments and light chain J gene segments of various organisms include those sequences, locations, and nomenclature of light chain V gene segments and light chain J gene segments of various organisms can be found in the IMGT database, which is accessible via the Internet at the URL "imgt.org" on the worldwide web (www). Light chains include, for example, light chains that do not selectively bind to either the 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 heavy chains in doing so, by binding and recognizing 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 heavy chains in doing so, by binding and recognizing one or more epitopes selectively bound by an epitope-binding protein present on the light chain. Common or universal light chains include light chains derived from the human Vκ1-39Jκ5 gene or the human Vκ3-20Jκ1 gene, including somatically mutated (e.g., affinity matured) versions thereof.

[0072] The phrase "operably linked," as used herein, includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or are positioned relative to each other so as to participate in a biological event, and this juxtaposition achieves or enables such interaction and / or positioning. For example, a regulatory sequence (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it is positioned relative to the coding sequence such that its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, "operably linked" includes the covalent linkage of the components or elements associated with each other. However, those skilled in the art will readily appreciate that in some embodiments, covalent linkage is not required to achieve effective operably linked. 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 additionally, in some embodiments, one or more such regulatory sequences function in trans or at a distance to control the coding sequence of interest. In some embodiments, the term "expression control sequence," as used herein, refers to polynucleotide sequences necessary and / or sufficient to affect the expression and processing of coding sequences to which they are linked. In some embodiments, expression control sequences may be or include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that 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 control sequences are preferentially or only active in a particular host cell or organism, or type thereof.By way of example, in prokaryotes, control sequences typically include promoters, ribosomal binding sites, and transcription termination sequences, while in eukaryotes, in many embodiments, control sequences typically include promoters, enhancers, and / or transcription termination sequences. Those skilled in the art will understand from their context that in many embodiments the term "control sequences" refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[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 compared to the virus from which the capsid protein is derived. The inserted amino acid may simply be inserted between two given amino acids of the capsid protein. The insertion of an amino acid may also involve the deletion of a given amino acid of the capsid protein at the insertion site (e.g., one or more capsid protein amino acids are replaced by five or more heterologous amino acids).

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

[0075] An exemplary multispecific molecule comprises heavy chain CDRs followed by (N- to C-terminus) C H 1 domain, hinge, C H 2 domain, and C HIt has two heavy chains, each with three domains, and either an immunoglobulin light chain that does not confer epitope binding specificity but can associate with each heavy chain (e.g., a common light chain), or an immunoglobulin light chain that can associate with each heavy chain and bind to one or more of the epitopes bound by the heavy chain epitope-binding region, or an immunoglobulin light chain that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both of the epitopes. In some embodiments, a multispecific binding molecule comprises (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region comprising a first CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and combinations thereof, and (2) an immunoglobulin heavy chain variable domain, wherein a second immunoglobulin heavy chain variable domain is operably linked to a second heavy chain constant region comprising a second CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and combinations thereof, and wherein the first or second heavy chain variable domain (with or without a cognate light chain) is a polypeptide as described herein. It binds to a described heterologous epitope, the other heavy chain variable domain (with or without a cognate light chain) binds to a receptor on a target cell, and the first heavy chain constant region associates with the second constant chain region in a manner that provides for easy isolation of the multispecific binding protein, e.g., the first and second heavy chain constant regions form a knobs-into-hole (KIH) format, or the second CH3 amino acid sequence contains a modification that reduces or eliminates binding of the second CH3 amino acid sequence to Protein A (see, e.g., U.S. Pat. No. 8,586,713, incorporated herein by reference in its entirety).In some embodiments, a multispecific binding molecule comprises: (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region comprising a first CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and combinations thereof; and (2) an immunoglobulin heavy chain variable domain, wherein a second immunoglobulin heavy chain variable domain is operably linked to a second heavy chain constant region comprising a second CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and combinations thereof; and wherein the first and second heavy chain variable domains (with or without cognate light chains) are the same or different. and wherein the first or second heavy chain constant region is modified to further comprise an additional binding domain (e.g., an scFV or Fv that binds a heterologous epitope as described herein, e.g., an additional binding domain is added to the C- 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 for easy isolation of the multispecific binding protein, e.g., the first and second heavy chain constant regions form a knobs-into-holes (KIH) format, or the second CH3 amino acid sequence comprises a modification that reduces or eliminates binding of the second CH3 amino acid sequence to Protein A. In some embodiments, the second CH3 amino acid sequence comprises reduced or eliminated binding to Protein A, the second CH3 amino acid sequence comprises a H95R modification (H435R in EU numbering, according to IMGT exon numbering). In one embodiment, the second CH3 amino acid sequence further comprises a Y96F modification (H436F in EU numbering, according to IMGT exon numbering). In another embodiment, the second CH3 amino acid sequence comprises both an H95R modification (H435R by IMGT exon numbering, EU numbering) and a Y96F modification (H436F by IMGT exon numbering, EU). In some embodiments, the second CH3 amino acid sequence is derived from a modified human IgG1 and further comprises a mutation selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (D356E, L38M, N384S, K392N, V397M, and V422I by IMGT, EU).In some embodiments, the second CH3 amino acid sequence is derived from a modified human IgG2 and further comprises mutations selected from the group consisting of N44S, K52N, and V82I (N384S, K392N, and V422I in EU by IMGT). In some embodiments, the second CH3 amino acid sequence is derived from a modified human IgG4 and further comprises mutations selected from the group consisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU by IMGT). In some embodiments, the heavy chain constant region amino acid sequence is a non-human constant region amino acid sequence, and the heavy chain constant region amino acid sequence comprises any one or more of the types of modifications described above.

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

[0077] In some embodiments, the Fc domain can be engineered 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 examples of proteins containing chimeric CH regions and having altered effector functions, see WO2014 / 022540, which is incorporated herein in its entirety.

[0078] The term "target cell" includes any cell in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit a protein, e.g., a receptor, on their surface that allows the cell to be targeted using a retargeting ligand, as described below. Preferably, the targeted protein, e.g., a receptor, is specific to the target cell, e.g., a "cell-specific marker," "cell-specific antigen," etc. The terms "cell-specific marker," "cell-specific antigen," "organ-specific marker," "tissue-specific marker," etc. include those proteins whose expression is enriched by a cell, tissue, and / or organ that are specific markers for that cell, tissue, and / or organ. "Enriched" in the context of protein expression refers to and includes expression or overexpression of a cell / tissue / organ-specific protein primarily, preferentially, or simply by the cell / tissue / organ for which the protein is a specific marker, although such markers may also be expressed at minimal levels by other cells / tissues / 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 www.proteinatlas.org See also Uhlen et al. (2010) Nat. Biotech. 28:1248-50, which is incorporated herein by reference in its entirety.

[0079] Terms such as "transduction" or "infection" refer to the introduction of nucleic acid into target cells by a viral vector. Efficiency related to transduction, for example, the term "transduction efficiency," refers to the fraction (e.g., percentage) of cells that express a target nucleotide after culturing with a set of viral vectors containing the target nucleotide. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting of transduced cells using a fluorescent reporter gene, PCR for expression of the target nucleotide, etc.

[0080] The term "wild-type," as used herein, includes an entity having a structure and / or activity found in nature under a "normal" state or condition (as opposed to a mutant, diseased, altered, etc.). Those skilled in the art will understand that a wild-type viral vector, e.g., a wild-type capsid protein, can be used as a standard viral vector in comparative studies. Generally, the standard viral capsid protein / capsid / vector is identical to the test viral capsid protein / capsid / vector except for the alteration whose effect is being tested. For example, to determine the effect, e.g., transduction efficiency, of inserting a heterologous epitope into a test viral vector, the transduction efficiency of the test viral vector (in the absence or presence of an appropriate multispecific binding molecule) can be compared to the transduction efficiency of a standard viral vector (in the absence or presence of an appropriate multispecific binding molecule, if necessary) that is identical to the test viral vector in all cases (e.g., additional mutations, nucleotides of interest, number of viral vectors and target cells, etc.) except for the presence of the heterologous epitope.

[0081] Recombinant viral capsid proteins and viral vectors, and nucleic acids In some embodiments, the recombinant viral capsid protein described herein is an Ad capsid protein, e.g., a capsid protein of an Ad serotype selected from the group consisting of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, and Ad7. In some embodiments, the recombinant viral capsid protein is derived from the Ad2 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from the Ad5 capsid gene. In some embodiments, the recombinant Ad viral capsid protein described herein comprises a heterologous epitope in the fiber protein domain, e.g., at the carboxy terminus of the fiber protein, the fiber knob, and / or the HI loop of the fiber knob.

[0082] In some embodiments, the recombinant viral capsid protein described herein is derived from an adeno-associated virus (AAV) capsid gene, such as a genetically modified capsid protein of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, an AAV6 capsid gene, an AAV8 capsid gene, or an AAV9 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, such as a genetically modified AAV2 VP1 capsid protein, the wild-type amino acid sequence of which is represented by SEQ ID NO: 1. In some embodiments, the recombinant viral capsid protein is derived from the AAV8 capsid gene, for example, a genetically modified AAV8 VP1 capsid protein, the wild-type amino acid sequence of which is represented by SEQ ID NO: 21. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein, the wild-type amino acid sequence of which is represented by SEQ ID NO: 5. In some embodiments, the recombinant viral capsid protein is derived from the AAV6 capsid gene, for example, a genetically modified VP1 capsid protein of AAV6. In some embodiments, a heterologous epitope is inserted into I-453 of the AAV9 capsid protein.

[0083] Generally, the recombinant viral capsid proteins described herein comprise a heterologous epitope inserted into and / or presented by the capsid protein such that the heterologous epitope reduces and / or abolishes the natural tropism of the capsid protein or a capsid comprising the capsid protein. In some embodiments, the heterologous epitope is inserted into a region of the capsid protein responsible for the natural tropism of the wild-type, authentic capsid protein, e.g., a region of the capsid protein involved in a cellular receptor. 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 after an amino acid position selected from the group consisting of G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, Q585 of the AAV6 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the heterologous epitope is inserted and / or presented between amino acids N587 and R588 of the AAV2 VP1 capsid. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises the amino acid sequence represented by SEQ ID NO:2. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises the amino acid sequence represented by SEQ ID NO:4. In some embodiments, the recombinant viral capsid, viral vector comprising the recombinant viral capsid, and / or composition comprising the recombinant viral capsid comprises an amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO:25.In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 26. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 27. Additional suitable insertion sites identified using AAV2 are well known in the art (Wu et al. (2000) J. Virol. 74:8635-8647) and include 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, and 1-716. The recombinant viral capsid protein described herein can be an AAV2 capsid protein comprising a specific epitope inserted at a position selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Additional suitable insertion sites identified by using additional AAV serotypes are well known and include I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), and I-585 (AAV5). In some embodiments, the recombinant viral capsid protein described herein can be an AAV2 capsid protein comprising a heterologous epitope inserted at a position selected from the group consisting of I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

[0084] As used herein, the designation I-### refers to an insertion site relative to the VP1 protein of an AAV capsid protein, where ### designates the amino acid number; however, such insertions may be located directly N- or C-terminal, preferably C-terminal to one of the N- or C-terminal amino acids in a sequence of five amino acids of a given amino acid, preferably three, more preferably two, and especially one amino acid(s) N- or C-terminal. In addition, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene; corresponding positions (and mutations thereof) can be readily identified in the VP2 and VP3 capsid proteins encoded by capsid genes by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by standard AAV capsid genes.

[0085] Thus, by overlapping the reading frames of the same gene with shifted start codons when encoding capsid proteins, insertion of a nucleic acid encoding one of these sites in the cap gene at the corresponding position leads to insertion into VP1, VP2, and / or VP3. Thus, for example, in AAV2, according to this nomenclature, an insertion of amino acids 1-138 is inserted only into VP1, an insertion of 138-203 is inserted into VP1 and VP2, and an insertion of 203 to the C-terminus is inserted into VP1, VP2, and VP3, and of course, this also applies to insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0086] Additionally, due to the high degree of conservation of at least most of the closely related family members, corresponding insertion sites in AAVs other than those listed can be identified by performing amino acid alignments or by comparison of capsid structures (see, e.g., Rutledge et al. (1998) J. Virol. 72:309-19, and U.S. Patent No. 9,624,274 for an exemplary alignment of different AAV capsid proteins, each of which is incorporated herein by reference in its entirety).

[0087] In some compositions disclosed herein comprised of recombinant viral capsids (e.g., in the absence of a multispecific binding molecule), the recombinant viral capsid protein is an AAV2 capsid protein VP1 having a heterologous epitope inserted at the I587 site, wherein the heterologous epitope does not comprise an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions disclosed herein comprised of recombinant viral capsids (e.g., in the absence of a multispecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein having a heterologous epitope inserted between T448 and N449, wherein the heterologous epitope does not comprise c-myc. In some compositions disclosed herein that consist of recombinant viral capsids (e.g., in the absence of a multispecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein that has a heterologous epitope inserted at the I-447 site, and the heterologous epitope does not include L14 or HA.

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

[0089] In some embodiments, the insertion (presentation) of a heterologous epitope abolishes the natural tropism of the viral vector, e.g., transduction of cells naturally permissive to infection by the wild-type standard viral vector and / or target cells is undetectable in the absence of the appropriate multispecific binding molecule. In some embodiments, the insertion (presentation) of a heterologous epitope reduces the natural tropism of the viral vector, e.g., compared to transduction of cells naturally permissive to infection by the wild-type standard viral vector. In some embodiments, the insertion (presentation) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (presentation) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (presentation) of a heterologous epitope reduces the natural tropism of the viral vector by at least 10%. In some embodiments, the insertion (presentation) of a heterologous epitope reduces the natural tropism of the viral vector by at least 20%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 30%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 40%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 50%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 60%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 70%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 80%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 90%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 95%. In some embodiments, the insertion (display) of the heterologous epitope reduces the natural tropism of the viral vector by at least 90%.In those embodiments in which the insertion (presentation) of a heterologous epitope does not abolish the native tropism of the recombinant viral capsid, the native tropism of such recombinant viral capsid can be abolished by a second, different mutation. For example, in one embodiment, the recombinant viral capsid protein described herein can be derived from an AAV9 capsid gene, contain a heterologous epitope, and further contain a mutation, e.g., a W503A mutation.

[0090] Detargeting the virus from its native host cells is important, especially when systemic administration of viral vectors is intended versus local or locoregional administration, because uptake of the viral vector by the native host cells limits the effective dose of the viral vector. For AAV2 and AAV6, HSPGs have been reported to be the primary receptors for viral uptake in many 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). Recently, the lysine-to-glutamic acid amino acid substitution K531E was reported to inhibit the ability of AAV6 to bind to heparin or HSPG (Wu et al., 2006) J. of Virology 80(22):11393-11397). Therefore, preferred point mutations are those that reduce the transduction activity of the viral vector into a given target cell mediated by the native receptor, or the binding of the viral vector to HSPG when HSPG is the primary receptor, by at least 50%, preferably at least 80%, and particularly at least 95%.

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

[0092] One embodiment of the present invention is a multimeric structure comprising the recombinant viral capsid proteins of the present invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 recombinant viral capsid proteins containing heterologous epitopes as described herein. They can form either ordinary viral capsids (empty viral particles) or viral vectors (capsids encapsulating nucleotides of interest). The formation of viral vectors capable of packaging viral genomes is a highly desirable feature for the use of the 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 claimed nucleic acid sequence. Nucleic acids, particularly vectors, are required for the recombinant expression of the capsid proteins of the present invention.

[0094] A further embodiment of the present invention is the use of at least one recombinant viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., a viral vector) for production and use as a gene transfer vector.

[0095] Heterologous epitopes Generally, recombinant viral capsid proteins and / or viral vectors comprising recombinant viral capsids comprise a heterologous epitope that allows for retargeting of the viral vector, e.g., via a multispecific binding molecule. In some embodiments, the heterologous epitope is a B-cell epitope, e.g., about 1 amino acid to about 35 amino acids in length, that forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises an affinity tag.

[0096] Numerous tags are known in the art (see, e.g., Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins," 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 therein). Affinity tags include, but are not limited to, immobilized divalent cations (e.g., Ni2+ ), a biotin moiety that binds to immobilized avidin (e.g., on a biotinylated polypeptide sequence in vivo), a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S tag that binds to immobilized S protein, an antigen that binds to an immobilized antibody or domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to the corresponding antibody), a FLASH tag (a high affinity tag linked to a specific arsenic-based moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, maltose-binding protein (MBP) that binds to immobilized amylose, an albumin-binding protein that binds to immobilized albumin, a chitin-binding domain that binds to immobilized chitin, a calmodulin-binding peptide that binds to immobilized calmodulin, and a cellulose-binding domain that binds 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 comprise affinity tags that are recognized only by an antibody paratope. In some embodiments, the heterologous epitopes disclosed herein comprise affinity tags that are recognized by an antibody paratope and another specific binding pair.

[0097] In some embodiments, the heterologous epitope and / or affinity tag does not form a binding pair with an immunoglobulin constant domain. In some embodiments, the heterologous epitope and / or affinity tag binds to a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ 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, a recombinant viral capsid described herein comprises the amino acid sequence EQKLISEEDL (represented as SEQ ID NO:6) adjacent to and / or operably linked to at least five consecutive amino acids of an AAV VP1 capsid protein. In some embodiments, a recombinant essential capsid described herein comprises the amino acid sequence EQKLISEEDL (represented as SEQ ID NO:6) adjacent to and / or operably linked to at least five consecutive amino acids of an AAV2 VP1 capsid protein. In some embodiments, a recombinant viral capsid described herein comprises EQKLISEEDL (represented as SEQ ID NO:6) inserted between N587 and R588 of an AAV2 VP1 capsid protein. In some embodiments, a recombinant viral capsid protein described herein comprises the amino acid sequence represented as SEQ ID NO:2. In some embodiments, a recombinant viral capsid protein described herein comprises the amino acid sequence represented as SEQ ID NO:4. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 25. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 26. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprises the amino acid sequence encoded by the nucleic acid sequence represented as SEQ ID NO: 27.

[0100] 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 flanked by linkers, for example, the heterologous epitope comprises, from N-terminus to C-terminus, a first linker, an affinity tag, and a second linker. In some embodiments, the first and second linkers are each independently at least 1 amino acid long. In some embodiments, the first and second linkers are identical.

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

[0102] retargeting part The viral vectors described herein have reduced or abolished transduction ability in the absence of a multispecific binding molecule, specifically 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 that can be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell. 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" can include scenarios in which a wild-type viral vector targets some cells within a tissue and / or some organ within an organism, and insertion of a heterologous epitope reduces or abolishes broad targeting of tissues or organs, thereby achieving retargeting to more specific cells of a tissue or more specific organ within the organism using the multispecific binding molecule. Such retargeting or redirection can also include scenarios in which a wild-type viral vector targets a tissue, and the insertion of a heterologous epitope reduces or abolishes tissue targeting, allowing retargeting to an entirely different tissue using a multispecific binding molecule. The antibody paratopes described herein generally comprise, at a minimum, a complementarity-determining region (CDR) that specifically recognizes the heterologous epitope, e.g., the CDR3 region of the heavy and / or light chain variable domain. In some embodiments, a multispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a heterologous epitope. For example, a multispecific binding molecule can comprise a single-domain heavy chain variable region or a single-domain light chain variable region, wherein the single-domain heavy chain variable region or the single-domain light chain variable region comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, a multispecific binding molecule can comprise an Fv region, e.g., a multispecific binding molecule can comprise an scFv that comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the multispecific binding molecules described herein comprise an antibody paratope that specifically binds to c-myc.

[0103] In some embodiments, the multispecific binding molecules described herein comprise an antibody paratope that specifically binds to c-myc, wherein the paratope comprises an scFv, the heavy and light chain variable domains of an scFv, and / or an scFV comprising the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences encoded by the nucleic acid sequence set forth as SEQ ID NO: 28, e.g., the amino acid sequence set forth as SEQ ID NO: 37. In some embodiments, the multispecific binding molecules described herein comprise an Fv or sCfv encoded by the nucleic acid sequence set forth as SEQ ID NO: 28.

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

[0105] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising an HCVR comprising the amino acid sequence set forth 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 multispecific binding proteins comprising a paratope comprising an LCVR comprising the amino acid sequence set forth as SEQ ID NO: 30, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0107] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising an HCVR and LCVR (HCVR / LCVR) amino acid sequence pair, comprising the HCVR amino acid sequence represented as SEQ ID NO: 29 paired with the 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 multispecific binding proteins comprising a paratope comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence set forth as SEQ ID NO:31, or a substantially similar sequence thereof 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 multispecific binding proteins comprising a paratope comprising a heavy chain CDR2 (HCDR2) comprising the amino acid sequence set forth as SEQ ID NO: 32, or a substantially similar sequence thereof 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 multispecific binding proteins comprising a paratope comprising a heavy chain CDR3 (HCDR3) comprising the amino acid sequence set forth as SEQ ID NO: 33, or a substantially similar sequence thereof 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 multispecific binding proteins comprising a paratope comprising a light chain CDR1 (LCDR1) comprising the amino acid sequence set forth as SEQ ID NO: 34, or a substantially similar sequence thereof 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 multispecific binding proteins comprising a paratope comprising a light chain CDR2 (LCDR2) comprising the amino acid sequence set forth as SEQ ID NO: 35, or a substantially similar sequence thereof 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 multispecific binding proteins comprising a paratope comprising a light chain CDR3 (LCDR3) comprising the amino acid sequence set forth as SEQ ID NO: 36, or a substantially similar sequence thereof 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 comprising an HCDR3 and LCDR3 (HCDR3 / LCDR3) amino acid sequence pair, which comprises the HCDR3 amino acid sequence set forth as SEQ ID NO: 33 paired with the LCDR3 amino acid sequence set forth as SEQ ID NO: 36. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is set forth as SEQ ID NO: 33 / 36.

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

[0116] In related embodiments, the present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising the set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within the 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. In general terms, 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 approaches. See, e.g., 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 anti-myc antibodies or portions thereof.

[0118] The multispecific binding molecules described herein further comprise a retargeting ligand in addition to a paratope (e.g., an antibody or portion thereof) that specifically binds to a heterologous epitope inserted / displayed in 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). There are many cell surface proteins, e.g., suitable cell surface receptors, that can be targeted by a retargeting ligand, for which retargeting ligands, e.g., antibodies or portions thereof, are already available. Such structures include class I and class II major histocompatibility antigens, receptors for various cytokines (e.g., receptors for IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), cell type-specific growth hormones, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), colony-stimulating growth factors, endothelial growth factors, epidermal growth factors, fibroblast growth factors, glial-derived neurotrophic factors, glial cell growth factors, gro-beta / mip, and the like. 2, hepatocyte growth factor, insulin-like growth factor, interferons (α-IFN, β-IFN, γIFN, consensus IFN), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte growth factor, leukemia inhibitory factor, macrophage / monocyte chemotactic and activating factor, nerve growth factor, neutrophil-activating protein 2, platelet-derived growth factor, stem cell factor, transforming growth factor, tumor necrosis factor, vascular endothelial growth factor, lipoprotein These include, but are not limited to, proteins (including further or other type 1 transmembrane receptors such as PRLR, G-protein coupled receptors such as GCGR, ion channels such as Nav1.7, ASIC1, or ASIC2), cell adhesion molecules, transport molecules for metabolic substances such as amino acids, light receptors of B lymphocytes or T lymphocytes (e.g., B cell receptors and associated proteins (e.g., CD19, CD20, etc.), and T cell receptors and associated proteins (e.g., CD3, CD4, CD8, etc.), tetraspanin proteins (e.g., CD63).The recombinant viral capsids described herein enable specific infection of cell types by using multispecific binding molecules containing retargeting ligands that bind to differentiated cell surface antigens as targets for the viral vector complex.

[0119] In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) liver cells, i.e., a liver-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) brain cells, i.e., a brain cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) hematopoietic cells, i.e., a hematopoietic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) T cells, i.e., a T cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) B cells, i.e., a B cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) dendritic cells, i.e., a dendritic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) macrophages, i.e., a macrophage-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) NK cells, i.e., an NK cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) kidney hepatocytes, i.e., a kidney-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) pancreatic hepatocytes, i.e., a pancreas-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) intestinal cells, i.e., an intestinal-specific marker. In some embodiments, the retargeting ligand binds to a protein that is predominantly expressed by (human) cancer cells, i.e., a tumor-associated antigen (e.g., solely). In some embodiments, the retargeting ligand binds to a protein that is predominantly (e.g., solely) expressed by (human) cells infected with a heterologous pathogen (e.g., solely).Proteins are known that (1) are specifically expressed by or whose expression is enriched 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, which is incorporated herein by reference in its entirety. Table 1 below provides exemplary, non-limiting organ-specific markers that can be used 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 a receptor expressed by (human) liver cells, e.g., an asialoglycoprotein receptor, e.g., hASGR1. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) brain cells. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) T cells, e.g., CD3, e.g., CD3ε. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) kidney cells. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) muscle cells, e.g., an integrin. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) cancer cells, e.g., 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 a tumor-associated antigen expressed by tumor cells. Non-limiting examples of specific tumor-associated antigens include, for example, adipophilin, 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, CCDC110 also known as KMHN1, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10 , MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1,NY-ESO-1 / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RAR alpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, 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, triosephosphate isomerase, TRP-1 / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigens (in lymphomas and other solid cancers), ErbB receptors, 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 colon, breast, and gastrointestinal cancers), and MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1, CAGE-2, CAGE-8, CAGE-3, CAGE-4, CAGE-5, CAGE-6, CAGE-7, CAGE-8, CAGE-9, CAGE-10, CAGE-12, CAGE-1, CAGE-2, CAGE-3, CAGE-4, CAGE-5, CAGE-6 ... 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 a CD marker 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 a 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 a first and a second antigen-binding domain, the CDR of the first antigen-binding domain may be designated with the prefix "A1", and the CDR of the second antigen-binding domain may be designated 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 and second antigen-binding domains can be directly or indirectly connected to each other to form the bispecific antigen-binding molecule of the present invention. Alternatively, the first and second antigen-binding domains can each be connected to a separate multimerizing domain. Association of one multimerizing domain with another multimerizing domain promotes association between the two antigen-binding domains, thereby forming the bispecific antigen-binding molecule. As used herein, a "multimerizing domain" refers to any macromolecule, protein, polypeptide, peptide, or amino acid capable of associating 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 the Fc portion of an immunoglobulin (containing the CH2-CH3 domain), e.g., the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0125] Bispecific antigen-binding molecules of the present invention will typically comprise two multimerization domains, e.g., two Fc domains, each of which is an individual portion of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0126] In certain embodiments, the multimerization 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 multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides that contain or consist of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0127] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity can be operatively linked (e.g., by chemical coupling, genetic 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 generate the bispecific antigen-binding molecule. Certain exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) antibody, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a discussion of the foregoing formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 and references cited therein; see also Brinkmann and Konterman (2017) mAbs 9:182-212, each of which is incorporated by reference in its entirety).

[0128] The present invention also includes bispecific antigen-binding molecules 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 wherein the at least one amino acid difference reduces 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 abolishes Protein A binding, such as an H95R modification (according to IMGT exon numbering, H435R in EU numbering). The second CH3 may further comprise a Y96F modification (according to IMGT, Y436F in EU numbering). Additional modifications that may be found in the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (D356E, L358M, N384S, K392N, V397M, and V422I in EU according to IMGT), and N44S, K52N, and V82I for IgG2 antibodies (IMGT , N384S, K392N, and V422I in EU), and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (by IMGT, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU), see e.g. WO2010 / 151792.

[0129] In certain embodiments, the Fc domain may be a chimera combining Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may comprise part or all of the CH2 sequence from a human IgG1, IgG2, or IgG4 CH2 region and part or all of the CH3 sequence from a human IgG1, IgG2, or IgG4. A chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence from a human IgG1, IgG2, or IgG4 hinge region combined with a "lower hinge" sequence from a human IgG1, IgG2, or IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any of the antigen-binding molecules presented herein comprises, from N- to C-terminus, [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that may be included in any of the antigen-binding molecules depicted herein comprises, from N- to C-terminus, [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG1 CH3]. These and other examples of chimeric Fc domains that may be included in any of the antigen-binding molecules of the invention are described in PCT Application No. WO2014 / 022540, which is incorporated by reference in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, may have altered Fc receptor binding, thereby affecting Fc effector function.

[0130] Use and Preparation A further embodiment of the recombinant viral capsid proteins described herein is their use to deliver a nucleotide of interest, e.g., a reporter gene or a therapeutic gene, to a target cell. Generally, the nucleotide of interest may be a transfer plasmid, which may generally include 5' and 3' inverted terminal repeat (ITR) sequences flanking the reporter gene(s) or therapeutic gene(s) (which, when contained within an AAV vector, may be under the control of a viral or non-viral promoter. In one embodiment, the nucleotide of interest is a transfer plasmid that includes, 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 focus-forming virus (SFFV) promoter, the elongation factor 1 alpha (EF1a) promoter (1.2 kb EF1a promoter, or 0.2 kb EF1a promoter), the chimeric EF1a / IF4 promoter, and the phosphoglycerate kinase (PGK) promoter. Internal enhancers may also be present in the viral construct to increase expression of the gene of interest. For example, the CMV enhancer can be used (Karasuyama et al. 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety). 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 moieties) can be encapsulated in the multimeric structure comprising 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), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or combinations thereof. Although the methods described herein demonstrate the construction of targeting vectors using a reporter gene encoding green fluorescent protein, one of skill in the art upon reading this disclosure will understand that the non-human animals described herein can be generated in the absence of a reporter gene or with any reporter gene known in the art.

[0133] A variety of therapeutic genes can be packaged within the multimeric structures comprising the recombinant viral capsid proteins described herein, e.g., as part of a delivery vector. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.

[0134] A further embodiment of the present invention is a process for the preparation of a recombinant capsid protein, the method comprising: a) expressing nucleic acid encoding the recombinant capsid protein under suitable conditions; b) isolating the expressed capsid protein of step a).

[0135] A further embodiment of the present invention is a method for altering the tropism of a virus, comprising: (a) inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding a viral capsid protein to form a nucleic acid sequence encoding a genetically modified capsid protein comprising the heterologous epitope, and / or (b) culturing packaging cells under conditions sufficient for the production of a viral vector, wherein the packaging cells comprise the nucleic acid sequence. A further embodiment of the present invention is a method for displaying 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 expressed capsid protein of step a).

[0136] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementary adeno-associated viral vector from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral vector from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the supernatant containing the viral vector with DNase I and MgCl2, (c) concentrating the viral vector, (d) purifying the viral vector, or (e) any combination of (a)-(d).

[0137] Packaging cells useful for producing the viral vectors described herein include, for example, animal cells that are permissive for the virus, or cells that have been modified to be permissive for the virus, or packaging cell constructs, for example, through the use of a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral vectors described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), SV40 Large T-antigen-containing HEK-293 cells (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), epithelial glioblastoma-astrocytoma cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH Examples of such cells include 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, and CAP-T cells.

[0138] L929 cells, the FLY virus packaging cell line reviewed in Cosset et al. (1995) J Virol 69, 7430-7436, NS0 (mouse myeloma) cells, human amniotic cells (e.g., CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia pastoris), plant cells (including but not limited to tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (e.g., High5)), or bacterial cells (including but not limited to E. coli).

[0139] For additional packaging cells and systems, packaging techniques and vectors for packaging nucleic acid genomes into pseudotyped viral vectors, see, e.g., Polo, et al., Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express the viral components or transiently transfecting cells with plasmids.

[0140] Further embodiments include methods for redirecting viruses and / or delivering reporter or therapeutic genes to target cells, including methods for transducing cells in vitro or in vivo, comprising contacting target cells with a combination of a viral vector comprising a capsid containing a recombinant viral capsid displaying a heterologous epitope and a multispecific binding molecule, where 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 compositions described herein, or the methods described herein, combine a recombinant viral vector and a multispecific binding molecule at a molecule:molecule ratio that restores transduction efficiency of the viral vector similar to that of a wild-type control viral vector. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) ranges from 1:0.5 to 1:100. In some embodiments, the ratio of recombinant viral vector to multispecific binding molecule (molecule:molecule) ranges from 1:4 to 1:20. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) ranges from 1:8 to 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:4. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:8. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:20. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:100. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:50. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:20. In some embodiments, the ratio of recombinant viral vector to polyspecific 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 ex vivo, hi other embodiments, the target cells are in vivo in a subject, e.g., a human.

[0142] target cell The recombinant viral vectors disclosed herein can be used to target a wide variety of cells for delivery of a nucleotide of interest. Target cells will generally be selected based on the nucleotide of interest and the desired effect.

[0143] In some embodiments, the target nucleotide can be delivered to enable the target cells to produce proteins that compensate for the organism's deficiency, such as an enzyme deficiency or an immune deficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that would normally produce proteins in an animal are targeted. In other embodiments, cells in the region where the protein is most beneficial are targeted.

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

[0145] In other embodiments, the nucleotide of interest may encode a toxic protein that kills cells that express the toxic protein, in which case tumor cells or other unwanted cells may be targeted.

[0146] In still other embodiments, nucleotides of interest that encode a harvested protein, such as a therapeutic protein, may be used to target cells capable of producing and secreting the protein.

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

[0148] As described above, the target receptor can be any receptor for which a retargeting ligand can be identified or generated. 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., a ligand, is known, it can be used as an affinity molecule. However, if the binding molecule is unknown, an antibody against the target receptor can be generated using standard procedures. In this case, the antibody can be used as a retargeting ligand as part of a multispecific binding molecule.

[0149] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the particular use (e.g., therapy, expression of harvested proteins, and conferring disease resistance), and (2) expression of markers with the desired amount of specificity.

[0150] Target cell is not limited in any way, and includes both germline cells and cell lines, and somatic cells and cell lines.Target cell can be stem cells from any source.When target cell is germline cell, target cell is preferably selected from the group consisting of single-cell embryo and embryonic stem cell (ES).

[0151] Pharmaceutical Compositions, Dosage Forms, and Administration A further embodiment provides a pharmaceutical product comprising at least one recombinant viral capsid protein according to the invention and a suitable multispecific binding molecule, and / or a nucleic acid according to the invention, preferably at least one multimeric structure according to the invention. Such a pharmaceutical product is preferably a useful gene transfer vector.

[0152] Also disclosed herein are pharmaceutical compositions comprising the viral vectors described herein and a pharmaceutically acceptable carrier and / or excipient. Additionally, disclosed herein are pharmaceutical dosage forms comprising the viral vectors described 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 pharmaceutical compositions based on the viral vectors disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients.The viral vectors can be formulated for administration, for example, by injection, inhalation (through either the mouth or nose), or isolation, or by oral, buccal, parenteral, or rectal administration, or by direct administration to tumors.

[0155] Pharmaceutical compositions can be formulated for various modes of administration, including systemic administration, topical administration, or localized administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, pharmaceutical compositions can be formulated in a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, pharmaceutical compositions can be formulated in solid form and can be redissolved or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also suitable.

[0156] For oral administration, pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or can be presented as a dry product that is constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., cationized oils, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as appropriate.

[0157] The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of preservatives.The pharmaceutical composition can further be formulated as a suspension, solution, or emulsion in an oily or aqueous medium, and can contain other agents, including suspending agents, stabilizing agents, and / or dispersing agents.

[0158] In addition, pharmaceutical compositions can be formulated as depot preparations. These long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of localized, non-invasive delivery of drugs over long periods of time. This technology can involve microspheres with precapillary sizes that can be injected into any selected part of an organ via a coronary catheter without causing inflammation or ischemia. The administered therapeutic agent is gradually released from the microspheres and absorbed by surrounding cells present in the selected tissue.

[0159] Systemic administration can also be via transmucosal or transdermal routes. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents may be used to enhance penetration. Transmucosal administration may be performed using intranasal sprays or suppositories. For topical administration, the viral vectors described herein can be formulated into ointments, salves, gels, or creams, as generally known in the art. Cleansing solutions can also be used locally to treat wounds or inflammation to accelerate healing.

[0160] Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical forms must be sterile and fluid. They must also be stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing), and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0161] When the formulations disclosed herein are used as therapeutic agents for promoting immune responses in subjects, the therapeutic agents can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0162] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required size of the virus vector in the case of dispersion, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

[0163] Sterile injectable solutions can be prepared by incorporating the active compound or construct in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0164] Upon formulation, solutions can be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although sustained-release capsules or microparticles and microspheres can also be used.

[0165] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration.In this context, those skilled in the art will know which sterile aqueous medium can be used in light of this disclosure.For example, one dose can be dissolved in 1 ml of isotonic NaCl solution, and added to 1000 ml of subcutaneous infusion fluid, or injected into the proposed infusion site.

[0166] The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. For example, a subject can be administered a viral vector described herein daily or weekly for a period of time, or monthly, twice a year, or yearly, depending on need or exposure to pathogenic microorganisms or the subject's condition (e.g., cancer).

[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 form currently in use.

[0168] Nasal or inhalable solutions or sprays, aerosols, or inhalants may also be used. Nasal solutions can be aqueous solutions designed for administration to the nasal passages in drops or sprays. Nasal solutions can be prepared to mimic nasal secretions in many respects. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, if necessary, antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers can be included in the formulation. Various commercially available nasal preparations are known and can contain, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

[0169] Oral formulations may contain excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. In certain defined embodiments, oral pharmaceutical compositions may contain inert diluents or assimilable edible carriers, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be directly incorporated with dietary food. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0170] Tablets, troches, pills, capsules, etc. may also contain the following: binders such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint oil, cornstarch oil, or cherry flavoring. When the dosage unit form is a capsule, in addition to materials of the above type, a liquid carrier may be contained. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain sucrose as a sweetener, methyl and propylparabens as preservatives, dyes, and flavorings such as cherry or orange flavor.

[0171] Further embodiments disclosed herein may relate to kits for use with the methods and compositions. The kits may also include suitable containers, such as vials, tubes, minitubes or microtubes, test tubes, flasks, bottles, syringes, or other containers. When additional components or agents are provided, the kits may contain one or more additional containers into which the agents or components can be placed. The kits herein also typically include a means for containing the viral vector and any other commercially available, tightly sealed reagent containers. Such containers may include syringes or blow-molded plastic containers into which the desired vials are held. Optionally, the compositions described may require one or more additional active agents, such as, for example, 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 nature 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 may be administered to a subject at a dose ranging from about 1 x 10 plaque-forming units (pfu) to about 1 x 10 pfu, depending on the mode of administration, the route of administration, and the nature and condition of the subject's disease. In some cases, the viral vector composition may be administered at a dose ranging from about 1 x 10 pfu to about 1 x 10 pfu, or from about 1 x 10 pfu to about 1 x 10 pfu, or from about 1 x 10 pfu to about 1 x 10 pfu. The more precise dose may also depend on the subject to which it is administered. For example, a younger subject may require a lower dose, while an adult human subject may require a higher dose. In certain embodiments, the more precise dose may depend on the subject's weight. In certain embodiments, for example, a young human subject may receive from about 1×10 pfu to about 1×10 pfu, while an adult human subject may receive a dose of from about 1×10 pfu to about 1×10 pfu.

[0173] The compositions disclosed herein may be administered by any means known in the art, including, for example, administering the compositions to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, subumbilically, intraocularly, orally, locally, by inhalation, injection, infusion, continuous infusion, localized perfusion, via catheter, lavage, in a cream, or in a lipid composition.

[0174] Any method known to those skilled in the art can be used for large-scale production of the viral vectors, packaging cells, and vector constructs described herein. For example, master seeds and working seed stocks can be prepared under GMP conditions in qualified primary CEFs or by other methods. Packaging cells can be plated onto large-surface-area flasks and grown to near-confluence, resulting in purified viral vectors. Cells can be harvested and the viral vectors released into the culture medium can be isolated and purified, or the intracellular viral vectors can be released by mechanical disruption (cell debris can be removed by large-pore depth filtration and host cell DNA can be digested with endonucleases). The viral vectors can then be purified and concentrated by tangential flow filtration, followed by diafiltration. The resulting concentrated bulk can be formulated by diluting with a buffer containing a stabilizer, filling into vials, and lyophilizing. Compositions and formulations can be stored for later use. For use, lyophilized viral vectors can be reconstituted by adding a diluent.

[0175] Certain additional agents 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, vehicles, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants can stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy. [Example]

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

[0178] Example 1: Generation of adeno-associated virus vectors containing heterologous epitopes 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 the recombinant capsid proteins. Briefly, after the codons encoding N587 of the AAV2 capsid protein, Q585 of the AAV6 VP1 capsid protein, N590 of the AAV8 VP1 capsid protein, A589 of the AAV9 VP1 capsid protein, or G453 of the AAV9 VP1 capsid protein, a sequence encoding FLAG, c-myc, or hexahistidine is inserted in frame.

[0179] Adeno-associated virus production was performed using a triple transfection method with HEK293 cells (see, for example, Erik Arden and Joseph M. Metzger, J. Biol Methods. 2016;3(2)). Cells are transfected with the appropriate vector: helper plasmid, pHelper (Agilent, Cat#240074); Plasmids encoding wild-type or modified AAV rep / cap genes (pAAV RC2 (Cell biolabs, Cat# VPK-422), such as, for example, pAAV RC2 / 6 / 9 (Cell Biolabs, Cat# VPK-426), pAAV RC8, pAAV RC2-N587myc, pAAV RC2 / 6-Q585myc, pAAVRC8-N590myc, pAAV RC9-A589myc; and A plasmid encoding the nucleotides of interest and AAV ITR sequences, such as pscAAV-CMV-eGFP, pAAV-CMVGFP (Agilent Cat#240074), pAAV-EF1a-eGFP, or pAAV-CAGG-eGFP. The cells are plated one day before PEFpro (Polyplus transfection, New York, NY)-mediated transfection using PBS.

[0180] 72 hours after transfection, the medium is collected and the cells are lysed in a buffer solution [50 mM Tris-HCl, 150 mM NaCl, and 0.5% sodium deoxycholate (Sigma, Cat# D6750-100G)]. Benzonase (Sigma, St. Louis, MO) is then added to both the medium and the cell lysate to a final concentration of 0.5 U / μl, followed by incubation at 37°C for 60 minutes. The cell lysate is centrifuged at 4000 rpm for 30 minutes. The cell lysate and medium are combined and precipitated with PEG8000 (Teknova Cat# P4340) at a final concentration of 8%. The pellet is resuspended in 400 mM NaCl and centrifuged at 10,000 g for 10 minutes. Virus in the supernatant is pelleted by ultracentrifugation at 149,000 g for 3 hours and titrated by qPCR.

[0181] For qPCR to titrate AAV genomes, AAV samples were treated with DNase I (Thermofisher Scientific, Cat# EN0525) at 37°C for 1 hour and then lysed using DNA Extract All Reagents (Thermofisher Scientific, Cat# 4403319). Primers were used directly in the AAV2 ITR to quantify encapsidated viral genomes using a QuantStudio 3 Real-Time PCR System (Thermofisher Scientific). The AAV2 ITR primer sequences were 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 and right AAV internal inverted repeat (ITR) sequences, 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 a 10-minute activation step at 95°C, 40 PCR cycles are performed, each consisting of 15 seconds at 95°C and 30 seconds at 60°C. TaqMan Universal PCR Master Mix (Thermofisher Scientific, Cat# 4304437) is used for qPCR. DNA plasmid (Agilent, Cat# 240074) is used as a standard to determine absolute titers.

[0182] Adeno-associated viral vectors containing capsids engineered with a c-myc epitope. 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. That is, 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 the plasmid pAAV RC2 (Cell Biolabs, Inc., San Diego, CA) or the plasmid pAAV RC2 / 9, and the modified pAAV RC2-N587Myc and pAAV RC9-A589Myc plasmids, respectively, were used to encode modified capsid proteins for AAV viral vectors with reduced or abolished tropism.

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

[0184] To generate pAAV RC2 / 6-Q585Myc, a gblock DNA fragment containing positions 3700 and 3940 of pAAV RC2 / 6 with the c-myc epitope sequence inserted between positions 3757 and 3758 was ordered from Integrated DNA Technologies (Coralville, Iowa). The gblock fragment was inserted 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 to generate the pAAV RC2 / 6-Q585Myc plasmid.

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

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

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

[0188] Example 2: Antibody-mediated retargeting of AAV viral vectors in vitro HepG2 is a human hepatoma cell line that expresses the liver-specific marker asialoglycoprotein receptor 1 (ASGR1). To test whether the scAAV2-N587Myc-CMV-hrGFP viral vector can infect HepG2, for example, a mixture containing scAAV2-N587Myc-CMV-eGFP and a bispecific anti-myc-ASGR1 antibody at different ratios (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, or 1:100) of the number of viral genomes (5e9 viral genomes) to the number of antibody molecules was incubated at room temperature for 30 minutes, and then added to HepG2 cells. As a control, HepG2 cells were also cultured with wild-type scAAV2-CMV-eGFP viral vector alone, scAAV2-N587Myc-CMV-eGFP viral vector alone, or scAAV2-N587Myc-CMV-eGFP viral vector containing a monospecific anti-myc antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) at a 1:8 ratio. Three days after infection, GFP expression by infected HepG2 cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 1). GFP expression was also detected in comparable percentages of HepG2 cells cultured with wild-type scAAV2-CMV-eGFP and with a 1:8 ratio of scAAV2-N587Myc-CMV-eGFP viral vector and a bispecific anti-myc-ASGR1 antibody (44.6% and 44.1%, respectively; Figures 1A and 1G, upper and lower panels). Lower GFP expression was detected by FACS in HepG2 cells cultured with a mixture of scAAV2-N587Myc-CMV-eGFP viral vector and higher or lower bispecific anti-myc-ASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) ( Fig. 1C , lower panel).In addition, we did not detect GFP 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-hASGR1 cells, which were genetically modified to express human (h)ASGR1 on the cell surface, were cultured with a mixture containing scAAV2-N587Myc-CMV-eGFP and a bispecific anti-myc-ASGR1 antibody at different ratios (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, or 1:100) of the number of viral genomes to antibody molecules. Wild-type 293T cells (which do not express hASGR1) were cultured with a mixture of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody at a 1:8 ratio, and 293T-hASGR1 cells were cultured with (a) wild-type scAAV viral vector alone, (b) scAAV2-N587Myc-CMV-eGFP viral vector alone, or (c) scAAV2-N587Myc-CMV-eGFP viral vector containing c-myc and an irrelevant bispecific anti-myc-GCGR antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY), which recognizes the glucagon receptor (GCGR) not expressed by 293T cells, at a 1:8 ratio, which served as a control. 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 (Figures 2Ai-2Ax and 2Axii), but not 293T cells (Figure 2Axi). After incubation with a mixture of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody at 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 incubated with wild-type scAAV (56.7%) (Figures 2Ai and 2Aiii–2Aix).A 1:100 ratio of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody reduced infectivity (Figure 2Ax). Incubation with scAAV2-N587Myc-CMV-eGFP alone or with scAAV-N587Myc containing an irrelevant bispecific anti-myc-GCGR antibody did not result in GFP expression by 293T-hASGR1 cells (Figures 2Aii and 2Axii).

[0190] In a similar experiment, 293T-hASGR1 cells were cultured with unmodified AAV9-CAGG-GFP viral vector alone, AAV9-A589Myc-CAGG-eGFP viral vector alone, or AAV9-A589Myc-CAGG-eGFP viral vector mixed with bispecific anti-Myc-ASGR1 antibody at different ratios. 3 days after infection, GFP expression was analyzed by FACS. After incubation with a mixture of AAV9-A589Myc-CAGG-eGFP and the bispecific anti-myc-ASGR1 antibody at ratios of 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, and 1:100, we detected GFP-positive 293T-hASGR1 cells (41.1%–91%) at levels comparable to those of 293T-hASGR1 cells cultured with wild-type AAV9-CAGG-eGFP (9.72%) (Figure 2B(i) and Figure 2B(iii)–(ix)). Incubation 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 with bivalent anti-hASGR1 antibodies at different concentrations for 1 hour at room temperature, and then cultured with a mixture containing scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibodies at a 1:8 ratio. Three days after infection, cells were fixed and analyzed by FACS as described above. Figure 3 provides data showing that entry of scAAV2-N587Myc-CMV-eGFP can be inhibited by bivalent anti-hASGR1 antibodies in a dose-dependent manner.

[0192] To determine whether sequential administration of bispecific antibodies and modified AAV can result in antibody-mediated retargeting of the modified AAV, different numbers of bispecific anti-myc-ASGR1 antibody molecules (1 x 10) were administered. 9 ~1x10 12 (numerator in the range of 2x10 5 293T-hASGR1 cells for 1 hour, and then 1x10 9 As controls, (1) 293T-hASGR1 cells were cultured with wild-type scAAV alone, i.e., in the absence of antibody, and (2) 1x10 scAAV-N587Myc viral vector was added. 11 of unrelated bispecific anti-myc-GCGR antibody molecules and 1 x 10 9 293T-hASGR1 cells continuously cultured with the scAAV2-N587Myc-CMV-eGFP viral vector, and (3) 1x10 11 of bispecific anti-myc-ASGR1 antibody molecules and 1x10 9293T cells, which do not express hASGR1, were cultured continuously 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 culture with all concentrations of anti-ASGR1 antibody molecules and scAAV2-N587Myc-CMV-eGFP (Figures 4A, 4C-4I). We did not detect GFP in 29T3-hASGR1 cells after incubation with scAAV2-N587Myc-CMV-eGFP alone (Fig. 4B), in 293T cells that do not express ASGR1 after sequential incubation with anti-myc-hASGR1 antibody and scAAV2-N587Myc-CMV-eGFP viral vector (Fig. 4J), or in 29T3-hASGR1 cells after sequential incubation with an irrelevant bispecific anti-myc-GCGR antibody and scAAV2-N587Myc-CMV-eGFP viral vector (Fig. 4K).

[0193] As described herein, the tropism of self-complementary AAV (scAAV) can be (1) inactivated, for example, by modification of the capsid protein, e.g., by insertion of a c-myc epitope, and optionally (2) redirected using a bispecific antibody, e.g., a bispecific antibody that recognizes the c-myc epitope and a ligand expressed by the target cell. To determine whether the tropism of single-stranded AAV (ssAAV) could be similarly (1) reduced or abolished, and optionally (2) redirected, 293T-hASGR1 cells were cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector generated as described in Example 1 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). Controls included 293T-hASGR1 cells cultured with wild-type ssAAV, 293T-hASGR1 cells cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector and an irrelevant bispecific anti-myc-GCGR antibody at a viral vector:antibody ratio of 1:8, and 293T cells (which do not express hASGR1) cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector and an irrelevant bispecific anti-myc-hASGR1 antibody at a viral vector:antibody ratio of 1:8. 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 vector and bispecific anti-myc-ASGR1 antibody at all ratios (Figures 5A, 5C–5I).We did not observe GFP expression by 29T3 cells cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector and bispecific anti-myc-ASGR1 antibody ( Figure 5J ), or by 293T-hASGR1 cells cultured with the ssAAV2-N587Myc-CMV-hrGFP viral vector alone or with the ssAAV-N587Myc viral vector and an irrelevant bispecific anti-myc-GCGR bispecific antibody ( Figures 5B , 5K ).

[0194] 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 the cell surface. To test whether retargeting of the scAAV2-N587Myc-CMV-eGFP viral vector can be mediated by bispecific anti-myc-GCGR antibodies via the hGCGR receptor, 293T-hGCGR cells were cultured with different mixtures containing different ratios of scAAV2-N587Myc-CMV-eGFP:bispecific anti-myc-GCGR antibodies. The scAAV2-N587Myc-CMV-eGFP viral vector was mixed with bispecific anti-myc-GCGR antibody at a ratio of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, or 1:100 at room temperature 30 minutes before addition to 293T-hGCGR cells. Three days after infection, GFP expression by infected 293T-hCGCR cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 6). A significant percentage of GFP-positive cells was detected after incubation with wild-type scAAV (Figure 6A) or with scAAV2-N587Myc-CMV-eGFP / bispecific anti-myc-GCGR antibody at 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). No GFP was 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 the 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 could 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 1:1, 1:5, 1:10, 1:100, and 1:1000 ratios at room temperature 30 minutes before addition to Jurkat cells. Three days after infection, GFP expression by infected Jurkat cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 7). After incubation with wild-type AAV6-EF1a-eGFP (Figure 7B) or with AAV6-Q585Myc-EF1a-eGFP / bispecific anti-myc-CD3 antibody at ratios of 1:1, 1:5, 1:10, and 1:100 (Figures 7D-7G), we detected a significant percentage of GFP-positive cells. However, no GFP was detected in Jurkat cells incubated with the AAV6-Q585Myc-EF1a-eGFP viral vector (Figure 7C).

[0196] Described in this example is the naturally directed inactivation of several self-complementary (sc) or single-stranded (ss) AAV serotypes, demonstrated by inactivation of scAAV2, or genetically modified ssAAV2 in which a c-myc epitope has been inserted between amino acids N587 and R588 of the VP1 capsid protein (scAAV-N587myc or ssAAV-N587myc), AAV6 in which a c-myc epitope has been 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). Additionally, this example demonstrates the ability of bispecific antibodies that recognize a c-myc epitope and a second ligand (e.g., hASGR1, hGCGR, or hCD3) expressed by target cells to retarget the tropism of modified AAV and mediate infection of target cells by pseudotyped AAV in a ligand-specific manner (Figures 1-7). Furthermore, this example demonstrates that simultaneous administration of genetically modified sc- or ss-AAV and bispecific antibodies is not required for infection; sequential administration is sufficient for ex vivo retargeting of genetically modified sc- or ss-AAV (Figure 4).

[0197] Example 3: Antibody-mediated redirection of modified viral vectors in vivo Retargeting viral vectors to the liver using different multispecific binding molecule formats To determine whether the bispecific anti-myc-ASGR1 antibody could retarget the scAAV2-N587myc-CMV-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 1 × 10 cells were injected into C57BL / 6 mice genetically modified to express hASGR1 in hepatocytes and into control wild-type C57BL / 6 mice. 11Mice were intravascularly injected with wild-type scAAV2-CMV-eGFP alone (titrated by qPCR) or with the scAAV2-N587myc-CMV-eGFP viral vector combined with bispecific anti-myc-ASGR1 at a viral genome-to-antibody molecule ratio of 1:8. Controls included mice injected with saline [250 mM NaCl] or the scAAV2-N587myc-CMV-eGFP viral vector alone. Ten days after injection, mice were sacrificed and transcardially perfused with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned on slides and immunoblotted with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West). The livers were stained with a 100-kDa antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and an Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) (Figures 8A-8C). GFP-positive cells were detected in livers from transgenic animals engineered to express ASGR1 in the liver and injected with wild-type scAAV2-CMV-eGFP or scAAV2-N587myc-CMV-eGFP combined with a 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)). No GFP was detected in any spleen or kidney samples from animals injected with saline or the scAAV2-N587myc-CMV-eGFP viral vector alone (Figures 8B and 8C), or in any liver, spleen, or kidney samples from animals injected with saline or the scAAV2-N587myc-CMV-eGFP viral vector alone (Figure 8A(ii, iii, vi, vii)), or in any liver sample from a wild-type C57BL / 6 animal injected with scAAV2-N587myc-CMV-eGFP in combination with the bispecific anti-myc-ASGR1 antibody (Figure 8A(viii)). In summary, the combination of the scAAV2-N587myc-CMV-eGFP viral vector with a bispecific anti-myc-ASGR1 antibody infected only hASGR1-expressing (hepatic) cells, strongly suggesting that the scAAV2-CMV-eGFP viral vector was inactivated by modification of the capsid protein, e.g., that the natural tropism of the scAAV viral vector could be reduced or abolished, e.g., using the c-myc epitope, and that such viral vector could be specifically reactivated, e.g., specifically retargeted, to hepatocytes, e.g., in vivo, e.g., by a bispecific anti-myc-ASGR1 antibody.

[0198] Similarly, to determine whether the bispecific anti-myc-ASGR1 antibody could retarget the ssAAV2-N587myc-CAGG-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 2.18 × 10 cells were injected into C57BL / 6 mice genetically modified to express hASGR1 in hepatocytes and into control wild-type C57BL / 6 mice. 11Mice were intravascularly injected with wild-type ssAAV2-CAGG-eGFP alone or with the ssAAV2-N587myc-CAGG-eGFP viral vector combined with bispecific anti-myc-ASGR1 at a viral genome-to-antibody ratio of 1:4 (titrated by qPCR). Control mice were injected with PBS or the ssAAV2-N587myc-CAGG-eGFP viral vector alone. Four weeks after injection, mice were sacrificed and transcardially perfused with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA). GFP-positive cells were detected in livers from transgenic animals engineered to express ASGR1 in the liver and injected with wild-type ssAAV2-CAGG-eGFP or ssAAV2-N587myc-CAGG-eGFP combined with a bispecific anti-myc-ASGR1 antibody (Figures 9E–9F, 9P–9R), as well as in livers from wild-type C57BL / 6 mice injected with wild-type ssAAV2-CAGG-eGFP (Figures 9B–9C). Strikingly, the infection efficiency of ssAAV2-N587myc-CAGG-eGFP combined with the bispecific anti-myc-ASGR1 antibody was much higher than that of WT ssAAV2-CAGG-GFP (Figures 9E–9F, 9P–9R). We did not detect or barely detect GFP in liver samples from all animals injected with saline or the ssAAV2-N587myc-CAGG-eGFP viral vector alone (Figures 9A, 9D, 9G–9L), or in liver samples collected from wild-type C57BL / 6 animals injected with ssAAV2-N587myc-CAGG-eGFP combined with the bispecific anti-myc-ASGR1 antibody (Figures 9M–9O).In summary, the combination of the ssAAV2-N587myc-CAGG-eGFP viral vector with a bispecific anti-myc-ASGR1 antibody infected only hASGR1-expressing (hepatic) cells, strongly suggesting that the ssAAV2-N587myc-CAGG-eGFP viral vector was inactivated by modification of the capsid protein, e.g., that the natural tropism of the scAAV viral vector could be reduced or abolished, e.g., using the c-myc epitope, and that such viral vector could be specifically reactivated, e.g., specifically retargeted, to hepatocytes, e.g., in vivo, e.g., by a bispecific anti-myc-ASGR1 antibody.

[0199] Further experiments using pseudotyped AAV9 viral vectors were performed in C57BL / 6 mice genetically modified to express hASGR1 in hepatocytes. 11Mice were injected with wild-type AAV9-CAGG-eGFP (titrated by qPCR) or AAV9-A589myc-CAGG-eGFP viral particles combined with a bispecific anti-myc-ASGR1 antibody at a viral genome-to-antibody ratio of 1:100. Controls included mice injected with PBS or AAV9-N587myc-CAGG-eGFP viral particles combined with an irrelevant bispecific anti-myc-hCD3 antibody. Four weeks after injection, mice were sacrificed. 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 from animals injected with wild-type AAV9-CAGG-eGFP or AAV9-N587myc-CAGG-eGFP viral particles combined with a bispecific anti-myc-ASGR1 antibody (Figures 10A and 10D). In all liver samples from animals injected with saline or AAV9-N587myc-CAGG-eGFP viral particles combined with a bispecific anti-myc-hCD3 antibody, GFP was either undetectable or barely detectable (Figures 10B and 10C). In summary, similar to AAV2, the natural tropism of AAV9 can be reduced or abolished by capsid protein modifications, e.g., by insertion of a c-myc epitope, and such modified viral vectors, e.g., AAV9-A587myc, can be retargeted to specific cell types using corresponding bispecific anti-epitope cell-specific marker-binding proteins, e.g., bispecific anti-myc-ASGR1 antibodies against hepatocytes.

[0200] Anti-hASGR1 IgG4 knob-into-hole Fc format with anti-myc scFc To determine whether various multispecific binding molecule formats can mediate AAV infection, an anti-myc scFv was fused to the C-terminus of one chain of a knob-into-hole anti-hASGR1 IgG4 antibody (see Figure 11A). Specifically, a gblock encoding the anti-myc scFv (SEQ ID NO: 28) flanked by arms homologous to 1459-1478 and 1479-1498 of pRG7078, encoding the anti-hASGR1 stealth knob IgG4 heavy chain, was obtained from Integrated DNA technologies (Skokie, Illinois). The nucleotide sequence (NA) encoding the amino acid sequences (AA) of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the scFv (SEQ ID NO: 37) encoded by SEQ ID NO: 28 is provided in Table 3. [Table 3]

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

[0202] To generate pseudotyped AAV8 viral vectors, pAAV RC8 N590myc, a gblock containing a c-myc epitope (SEQ ID NO: 22) inserted between N590 and T591 of the AAV8 VP1 capsid protein, was ordered 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 the vector using SLIC to generate pAAV RC8 N590myc (SEQ ID NO: 24).

[0203] 293T-hASGR1 cells, genetically engineered to express human (h)ASGR1 on the cell surface, were cultured with different ratios (1:0, 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50, or 1:100) of AAV8-N590Myc-CAGG-eGFP to hASGR1-IgG4-Fc / anti-myc bispecific binding molecules, or a mixture containing the AAV8-CAGG-eGFP viral genome. 3 days after infection, GFP expression was analyzed by FACS. After incubation with a mixture of AAV8-N590Myc-CAGG-eGFP and anti-hASGR1-IgG4-Fc / anti-myc bispecific molecules at ratios of 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50, or 1:100, we detected GFP-positive 293T-hASGR1 cells (2.12% to 22.2%, Figures 12D-12K), comparable to the number of 293T-hASGR1 cells incubated with wild-type AAV8-CAGG-eGFP (46.1%) (Figure 12A). Neither mock-infected cells (Figure 12B) nor incubation 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 could retarget AAV8 N590myc-CAGG-eGFP viral particles to hepatocytes expressing hASGR1 in vivo, 1 × 10 cells were injected into mice genetically modified to express hASGR1 in a C57BL / 6 hepatocyte background. 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 at a viral genome to binding protein ratio of 1:12 were injected intravascularly. Control animals were injected with AAV8 N590myc-CAGG-eGFP viral particles combined with an irrelevant anti-hCD3x-anti-myc bispecific binding molecule with a similar format to the anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecule. Three weeks after injection, mice were sacrificed and transcardially perfused with 4% PFA. Liver, kidney, and heart organs were collected and dehydrated in 15% sucrose followed by 30% sucrose. Organs were then cryosectioned onto slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa488-conjugated 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 viral particles combined with anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecules (Figures 13G-13I). No or barely detectable GFP was detected in liver samples from animals injected with AAV8 N590myc-CAGG-eGFP viral particles combined with an irrelevant anti-hCD3 / myc binding protein (Figures 13D-13F). In summary, epitope-tagged 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 intestine and / or pancreas 293T cells ("293T-ENTPD3") genetically engineered to express human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3) on the 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 an anti-myc scFv to the C-terminus of one chain of a knob-into-hole anti-hENTPD3 IgG4 antibody (anti-hENTPD3-IgG4-Fc / anti-myc) at 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 incubation with a mixture of AAV2-N587Myc-CAGG-eGFP and anti-hENTPD3-IgG4-Fc / anti-myc, we detected GFP-positive 293T-ENTPD3 cells (1.48%-16%, Figures S14C-S14J) at levels comparable to those of 293T-ENTPD3 cells incubated with wild-type AAV2-CAGG-eGFP (66%) (Figure S14A). Incubation with AAV2-N587Myc-CAGG-eGFP alone resulted in very low levels of GFP expression by 293T-ENTPD3 cells (Figure S14B).

[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 could retarget AAV2-N587myc-CAGG-eGFP viral particles to intestinal cells expressing ENTPD3 in vivo, C57BL / 6 mice were infected with 5x10 11AAV2-N587myc-CAGG-eGFP viral particles combined with anti-hENTPD3-IgG4-Fc / anti-myc binding protein at a viral genome to binding protein ratio of 1:20 (titered by qPCR) were injected intravascularly. Control mice were injected with PBS, wild-type AAV9, or AAV2-N587myc-CAGG-eGFP viral particles combined with an irrelevant binding protein molecule. Three weeks after injection, mice were sacrificed. Livers, intestines, and pancreases 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 livers from mice injected with PBS, AAV2-N587myc-CAGG-eGFP viral particles combined with an irrelevant binding molecule, or anti-hENTPD3-IgG4-Fc / anti-myc binding protein (Figures 15A(iii)–15A(iv)), respectively, indicating that the AAV2-N587myc viral 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 viral particles combined with hENTPD3-IgG4-Fc / anti-myc binding protein (Figures 15B(i)–15B(iv)). GFP was detected in pancreatic islets from mice injected with AAV2-N587myc-CAGG-eGFP viral particles combined with anti-hENTPD3-IgG4-Fc / anti-myc binding protein (Figure 15C(iv)). WT AAV9 infected both pancreatic islet and non-islet cells (Figure 15C(ii)). GFP was not detected in pancreatic samples from mice injected with saline or AAV2-N587myc-CAGG-eGFP viral particles combined with an irrelevant binding protein (Figures 7C(i) and 7C(iii)).In summary, the natural tropism of AAV can be reduced or abolished by inserting heterologous epitopes, and it can be retargeted to the same or different cells using epitope tags and multispecific binding proteins that bind to markers expressed by the cells or tissues to be retargeted.

[0207] This example demonstrates that several different serotypes of adeno-associated viral vectors can be genetically modified with heterologous epitopes (e.g., c-myc) as described herein to inactivate infectivity, and that bispecific antibodies (regardless of bispecific format) that recognize both the heterologous epitope (e.g., c-myc) and a marker expressed by the target cell can be used to retarget the viral vector for delivery to a target nucleotide.

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

[0209] Delivery of suicide genes to cells expressing a targeting ligand To test the ability of the scAAV2-N587Myc viral vector to deliver the suicide gene to specific cells, we used HER2 + Breast cancer xenograft nude mouse model (Wang et al. al. ((2010) Cancer Gene Therapy 17:559-570)) is used.

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

[0211] Cell lines: 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 carcinoma are HER2-negative human tumor cell lines, and BEAS-2b is an immortalized bronchial epithelial HER2-negative cell line (Jia LT et al. (2003) Cancer Res. 63:3257-3262; Kern JA, et al., (1993) Am J Respir Cell Mol Biol 9:448-454; Martinez-Ramirez A, et al., (2003) Cancer Genet Cytogenet. 2003, 141:138-142). All of these cell lines are obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in the media recommended by ATCC.

[0212] Mice: Obtain female nude mice, 6-8 weeks old, and house them under specific pathogen-free conditions. On day 0, mice were simultaneously inoculated with (1) 10 7BT474, SK-BR-3, Calu-3, A-673, or HeLa tumor cells were injected subcutaneously into the right flank and treated intravenously with a bispecific anti-myc-HER2 antibody and a ss- or scAAV-N587Myc viral vector carrying a reporter (e.g., EGFP) or suicide gene. Untreated animals (animals injected with tumor cells alone), animals injected with wild-type ss- or scAAV viral vectors carrying a reporter or suicide gene, animals injected with bispecific anti-myc-HER2 antibodies alone, or animals injected with ss- or scAAV-N587Myc viral vectors carrying a reporter or suicide gene alone served as controls. All animals were treated with the appropriate prodrug one day after injection and treatment. The size of each tumor was measured twice weekly using calipers, and tumor volume was calculated as length × width. 2 The calculation is made as follows: × 0.52. 3 Once the mice reach the target age, they are sacrificed, and the date of sacrifice is recorded as the date of death. Livers, spleens, kidneys, and tumors from animals injected with wild-type ss- or scAAV, or ss- or scAAV-N587Myc viral vectors carrying a reporter gene, are fixed to visualize reporter gene expression.

[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 suicide genes to cells expressing a targeting HER2 ligand using viral vectors described herein containing a heterologous epitope, e.g., c-myc, and a bispecific antibody that specifically binds to the targeting ligand and the heterologous epitope. In additional experiments, viral vectors containing a heterologous epitope described herein and a bispecific antibody that specifically binds to a heterologous epitope (e.g., c-myc) and a target receptor are used to deliver suicide genes to other cell types that express one or more other targeting ligands. Illustrative, non-limiting examples of receptors suitable for targeting include receptors that mediate endocytosis of viral vectors, e.g., carcinoembryonic antigen (CEA) (Qiu et al. Y, et al. (2012) Cancer Lett. 316:31-38), and vascular endothelial growth factor receptor (VEGFR) (Leng A, et al. (2013) Tumor Biol. 32:1103-1111, Liu T, et al. (2011) Exp Mol Pathol. 91:745-752). Additional receptors that can be targeted include epidermal growth factor receptor (EGFR) Heimberger AB, et al. (2009) Expert Opin Biol Ther.9:1087-1098), 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. al. (2012) BMC Med.;10:85), folate receptor (FR) (Duarte S, et al., (2011) J Control Release149(3):264-72), transferrin receptor (TfR) or leukocyte differentiation antigen 71 (CD71) (Habashy HO, et al., Breast Cancer Res Treat.119(2):283-93), mucin (Torres MP, et al., (2012) Curr Pharm Des.2012;18(17):2472-81), stage-specific embryonic antigen 4 (SSEA-4) (Malecki M., et al., (2012) J Stem Cell Res Ther.2(5)), tumor resistance antigen 1-60 (TRA-1-60) (Malecki M., et al., (2013) J Stem Cell Res Ther.3:134).

Claims

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

Images