Tropically modified recombinant viral vector and its use for targeted introduction of genetic material into human cells

Abstract

Provided herein are compositions and methods for retargeting recombinant viral capsid proteins / capsids / vectors in vivo using multispecific binding molecules, such as bispecific antibodies, which specifically bind to heterologous epitopes displayed by capsid proteins, and proteins expressed on target cells, for targeted delivery of target nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the Chinese invention patent application with application number 201880042990.1, application date June 27, 2018, and invention name “Recombinant viral vector with modified tropism and its use for targeted introduction of genetic material into human cells”. The original application is the national phase application PCT / US2018 / 039874, which claims the priority and benefits of the U.S. provisional application serial number 62 / 525,704 filed on June 27, 2017. Technical Field

[0003] The disclosure herein generally relates to tropism-modified recombinant viral vectors, and compositions comprising the same, which can be used to target the introduction of genetic material into cells and / or tissues. Background Art

[0004] Delivering genes to specific target cells has become one of the most important technologies in modern medicine for diagnosing and treating various chronic diseases and genetic diseases. So far, the progress of gene therapy in clinical applications has been limited by the lack of ideal gene delivery vehicles. In order to achieve treatment success, gene delivery vehicles must be able to transduce target cells while avoiding transduction of non-target cells. Specifically, when the natural tropism of the virus cannot meet the immediate treatment needs, a recombinant viral vector in which the natural tropism is eliminated or reduced and the desired tropism is successfully transformed is required. (Buchholz et al.)

[0005] In recent years, most progress in vector development has been achieved using naked viruses (e.g., viruses comprising a capsid formed by viral capsid proteins without a capsid (e.g., a lipid bilayer)), such as adeno-associated virus (AAV) and adenovirus (Ad), and enveloped viruses (e.g., viruses whose capsid is surrounded by a lipid bilayer), such as retroviruses, lentiviruses, and herpes simplex viruses. AAV-based vectors have been the focus of much research because AAV is a non-enveloped virus that is only mildly immunogenic but is able to transduce a wide range of species and tissues in vivo without evidence of toxicity.

[0006] AAV is a small, non-enveloped, single-stranded DNA virus. The AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs), and two open reading frames encoding Rep protein and Cap protein, respectively. These two ITRs are the only cis elements required 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 mainly play a role in regulating AAV replication and AAV integration into the chromosomes of host cells. The Cap reading frame encodes three structural (capsid) viral proteins (VP) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2) and 61-62 kD (VP3). More than 80% of the total proteins in AAV virions contain VP3; in mature virions, VP1, VP2 and VP3 are found in a relative abundance of approximately 1:1:10. In vitro, these three proteins spontaneously assemble into virion-like structures, such as viral capsids. Thus, it appears that viral capsid formation in infected cells occurs independently of viral DNA synthesis (reviewed by Kotin et al. (1994) Hum. Gene Ther. 5:793).

[0007] Of all the known AAV serotypes, AAV2 is probably the most well-characterized serotype, as its infectious clone was the first to be prepared. (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA 79:2077-2081). Subsequently, the complete sequences of AAV3A, AAV3B, AAV4, and AAV6 were also 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.

[0008] 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). In addition, AAV safely transduces postmitotic tissues with relatively low immunogenicity and can integrate into host chromosomes in a site-specific manner and into chromosome 19 in tissue culture cells if the Rep protein is supplied in trans. (Kotin et al. (1990) Proc. Natl. Acad. Sci. USA 87:2211-2215; Samulski et al. (1991) EMBO J. 10(12):3941-3950; Balague et al. (1997) J. Virol. 71:3299-3306; Surosky et al. (1997) J. Virol. 71:7951-7959). The integrated genome of AAV has been shown to allow long-term gene expression in many 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).

[0009] Many viruses, including AAV, infect cells via virus / ligand:cell / receptor interactions that ultimately result in the virus being internalized by the infected cell. This ligand:receptor interaction is a focus of viral vector research and can be manipulated, for example, to redirect the natural tropism of the virus from cells that naturally allow infection by wild-type virus to target cells, for example, via receptors expressed by the target cell.

[0010] In theory, retargeting any cell surface protein or marker by vector should lead to infection by target cell, because most cell surface receptors or markers are involved in or constitutive (for example, for recycling) or ligand-induced (for example, receptor-mediated) endocytic pathway. These receptors cluster in the pits coated with clathrin, enter cells via clathrin-coated vesicles, pass through the acidified endosomes in which receptors are sorted, and then recirculate to the cell surface, become stored in the cell, or degrade in lysosomes. Like this, the platform for retargeting viral vectors is often intended to eliminate the natural tropism of viral vectors, and redirect viral vectors to receptors or markers expressed alone or mainly by target cells. Many advances in targeted gene therapy using viral vectors can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral vectors, which lead to pseudotyping, expansion and / or retargeting of the natural tropism of viral vectors. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[0011] Non-genetic methods generally utilize adapters that recognize both wild-type (unmodified) viral surface proteins and target cells. Soluble pseudoreceptors (for wild-type viruses), polymers such as polyethylene glycol, and antibodies or portions thereof have been used as the viral binding domains of adapters, while natural peptide or vitamin ligands, and antibodies or portions thereof have been used for the cell binding domains of the above-mentioned adapters. In this way, retargeting of viral vectors to target cells can be achieved after the vector: adapter complex binds to proteins expressed on the surface of the target cell, such as cell surface proteins.

[0012] Such methods have been used for AAV (Bartlett et al. (1999) Nat. Biotechnol. 74:2777-2785), adenovirus (Hemminki et al. (2001) Cancer Res. 61:6377-81; van Beusechem et al. (2003) Gene Therapy 10:1982-1991; Einfeld et al. (2001) J. Virol. 75:11284-91; Glasgow et al. (2009) PLOS One 4:e8355), herpes virus (Nakano et al. (2005) Mol. Ther. 11:617-24), and paramyxovirus (Bian et al. (2005) Cancer Gene Ther. 12: 295-303; Bian et al. (2005) Int. J. Oncol. 29: 1359-69), coronavirus (Haijema et al. (2003) J. Virol. 77: 4528-43]8; Wurdinger et al. (2005) Gene Therapy 12: 1394-1404).

[0013] The more popular method has been the recombinant genetic modification of viral capsid proteins, and therefore the recombinant genetic modification of the surface of the viral capsid. In the indirect recombination method, the viral capsid is modified with a heterologous "scaffold" that is then connected to an adaptor. The adaptor binds to the scaffold and the target cell. (Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; see also WO 97 / 05266) scaffolds, such as (1) Fc binding molecules (e.g., Fc receptors, protein A, etc.), which bind to the Fc of antibody adaptors, (2) (strept) avidin, which binds to biotinylated adaptors, (3) biotin, which binds to adaptors fused to (strept) avidin, and (4) protein:protein binding pairs that form isometric peptide bonds, such as SpyCatcher, which binds to SpyTagged adaptors, which has 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. (2005) Molecular Therapy 11:856–865; Stachler et al. (2008) Molecular Therapy 16:1467–1473), and togaviruses (Quetglas et al. (2010) Virus Research 153:179–196; Ohno et al. (1997) Nature Biotechnology 15:763–767; Klimstra et al. (2005) Virology 338:9–21).

[0014] In direct recombinant targeting methods, the targeting ligand is directly inserted into or coupled to the viral capsid, i.e., the proteinaceous viral capsid is modified to express a heterologous targeting ligand. The ligand is then redirected or binds to a receptor or marker that is preferentially or exclusively expressed on the target cell. (Stachler et al. (2006) Gene Ther. 13:926–931; White et al. (2004) Circulation 109:513–519.). Direct recombination methods have been used for AAV (Park et al. (2007) Frontiers in Bioscience 13:2653-59; Girod et al. (1999) Nature Medicine 5:1052-56; Grifman et al. (2001) Molecular Therapy 3:964-75; Shi et al. (2001) Human Gene Therapy 12:1697-1711; Shi and Bartlett (2003) Molecular Therapy 7:515-525), retroviruses (Dalba et al. Current Gene Therapy 5:655-667; Tai and Kasahara (2008) Frontiers in Bioscience 13:3083-3095; Russell and Cosset (1999) Journal of Gene Medicine 1:300–311; Erlwein et al. (2002) Virology 302:333–341; Chadwick et al. (1999) Journal of Molecular Biology 285:485–494; Pizzato et al. (2001) Gene Therapy 8:1088–1096), poxviruses (Guse et al. (2011) Expert Opinion on Biological Therapy 11:595–608; Galmiche et al. (1997) Journal of General Virology 78:3019–3027; 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 herpes viruses (Shah and Breakefield (2006) Current Gene Therapy 6:361-370; Campadelli-Fiume et al. (2011) Reviews in Medical Virology 21:213–226).

[0015] The three methods each have advantages and disadvantages. The main advantage of the direct recombination method is that the specificity of the viral vector is inherent to the viral genome and can be maintained during replication. However, for this and indirect recombination methods, the ability of the genetically modified virus requires the structure of the capsid to be maintained, and the targeting ligand or scaffold is placed in a position that tolerates and appropriately displays the targeting ligand or scaffold, thus limiting the appropriate ligand or scaffold reservoir that can be used. Like this, the recombinant retargeting method is limited by the naturally occurring molecules that can be used as targeting ligands, resulting in the incorporation of other binding ligands, such as antibodies or a portion thereof. Both the non-recombinant adapter platform and the recombinant adapter platform are advantageous in terms of the flexibility of the adapter used. However, it is difficult to achieve optimal transduction efficiency with these two component systems.

[0016] Provided herein is a viral retargeting strategy that solves the problems inherent in previous recombinant retargeting strategies by inserting heterologous epitopes into viral capsids. The recombinant viral capsids of the present invention exhibit reduced to eliminated natural tropism that 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 viral vector:multispecific binding molecule ratio. Summary of the invention

[0017] Disclosed herein are recombinant viral capsid proteins, viral capsids comprising recombinant viral capsid proteins, and viral vectors comprising a target nucleotide encapsulated by the recombinant viral capsids; wherein the capsid proteins, capsids and viral vectors are genetically modified to comprise (display) heterologous epitopes, wherein the heterologous epitopes (parts thereof or in combination with viral capsid proteins) form binding pairs with antibody paratopes, and wherein the recombinant viral capsid proteins / capsids / vectors may also comprise mutations, insertions or deletions at amino acid positions involved in receptor binding (e.g., the (natural) tropism of the viral capsid proteins / capsids / vectors), such that the recombinant viral capsid proteins or viral capsids comprising the recombinant viral capsid proteins have a reduced to eliminated (natural) tropism (e.g., in the absence of a multispecific, optionally bispecific binding molecule, have a transduction efficiency that is less than the transduction efficiency of a reference viral capsid protein / capsid / vector lacking the heterologous epitope, or in the absence of a multispecific, optionally bispecific binding molecule, have an undetectable transduction efficiency). Such reduced to eliminated (natural) tropism of recombinant viral capsid protein / capsid / vector can be enhanced or restored in the presence of appropriate multispecific, optionally bispecific binding moieties. Accordingly, also described are compositions comprising: (1) a recombinant viral vector having a capsid comprising a recombinant capsid protein as described herein, and (2) a multispecific, optionally bispecific binding molecule comprising an antibody paratope and a targeting ligand, including compositions comprising a certain viral vector: multispecific binding molecule ratio; also described herein are uses thereof for directing and / or introducing genetic material into target cells. Also described are methods for retargeting recombinant viral vectors, such as methods for targeted delivery of target nucleotides to target cells, comprising contacting the recombinant viral vector with a multispecific, optionally bispecific binding molecule, and methods for preparing recombinant virus as a whole are also described.

[0018] Described herein are recombinant viral capsid proteins comprising an epitope that is heterologous to the capsid protein, wherein the epitope or a portion thereof specifically binds an antibody paratope, and wherein the recombinant viral capsid protein or a viral capsid comprising the recombinant viral capsid has a reduced to eliminated natural tropism, e.g., in the absence of a multispecific, optionally bispecific binding moiety.

[0019] In some embodiments, a heterologous epitope is inserted (displayed) by a recombinant viral capsid protein such that the insertion and / or display of the heterologous epitope reduces or eliminates the (natural) tropism of the viral capsid compared to a reference viral capsid lacking the heterologous epitope, for example, the viral capsid comprises a mutation comprising an epitope insertion at an amino acid position and / or a substitution of an amino acid by an epitope at an amino acid position, wherein the mutation reduces or eliminates the (natural) tropism of the capsid protein, for example in the absence of a multispecific, optionally bispecific binding portion. In some embodiments, a heterologous epitope is inserted (displayed) by a viral capsid protein such that, for example in the absence of a multispecific, optionally bispecific binding portion, the insertion and / or display partially reduces the (natural) tropism of the recombinant viral capsid compared to a reference viral capsid lacking the heterologous epitope, and the viral capsid further comprises further mutations (e.g., substitutions, deletions, insertions in addition to the insertion of the heterologous epitope), which further reduce and / or eliminate the (natural) tropism of the recombinant viral capsid or a recombinant viral vector comprising it, compared to a reference viral capsid lacking the mutation, for example in the absence of a multispecific, optionally bispecific binding portion.

[0020] Generally, the recombinant viral capsid protein described herein can be derived from a capsid gene, for example, encoded by a capsid gene modified to express an epitope, and / or a genetically modified non-enveloped virus that generally infects human cells, or a serotype of a non-enveloped virus that generally infects human cells, such as adenovirus, adeno-associated virus, etc. In some embodiments, the recombinant viral capsid protein described herein is derived from an AAV capsid gene, for example, encoded by a capsid gene modified to express an epitope, and / or a genetically modified adeno-associated virus (AAV) capsid protein of an AAV serotype that infects primates, optionally wherein the AAV is selected from 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, for example, a genetically modified AAV2 capsid protein, a genetically modified AAV6 capsid protein, a genetically modified AAV8 capsid protein, or a genetically modified AAV9 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, for example, encoded by an AAV2 capsid gene modified to express an epitope, and / or is a genetically modified AAV2 VP1, VP2 and / or VP3 capsid protein, and the amino acid sequence of its wild-type AAV2 VP1 protein is shown in SEQ ID NO: 1. 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 AAV6 VP1, VP2 and / or VP3 capsid protein, and the amino acid sequence of its wild-type AAV6 VP1 is shown in SEQ ID NO: 3. 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 AAV8 VP1, VP2 and / or VP3 capsid protein, and the amino acid sequence of its wild-type AAV VP1 protein is shown in SEQ ID NO: 21. In some embodiments, the recombinant viral capsid protein is derived from an AAV9 capsid gene, for example, encoded by an AAV9 capsid gene modified to express an epitope, and / or is a genetically modified AAV9 VP1, VP2 or VP3 capsid protein, and the wild-type amino acid sequence of AAV9 VP1 is shown in SEQ ID NO: 5. 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, for example, encoded by an AAV9 capsid gene modified to express an epitope, and / or is a genetically modified VP1, VP2, and / or VP3 capsid protein of AAV9.

[0021] In some embodiments, the recombinant viral capsid protein is derived from, for example, encoded by a chimeric AAV capsid gene modified to express an epitope, wherein the chimeric AAV capsid gene comprises a plurality of nucleic acid sequences, wherein each of the plurality of nucleic acid sequences encodes a portion of a capsid protein of a different AAV serotype, and wherein the plurality of nucleic acid sequences together encode a 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.

[0022] Generally, the recombinant viral capsid protein as described herein comprises a heterologous epitope inserted into and / or displayed by the recombinant capsid protein, such that the heterologous epitope itself reduces and / or eliminates the natural tropism of the recombinant capsid protein or the capsid comprising it, compared to a reference capsid lacking the heterologous epitope or a capsid comprising the reference capsid, respectively. In some embodiments, the heterologous epitope is inserted into (displayed therein) a capsid protein region of the natural tropism of the wild-type reference capsid protein, such as a capsid protein region involving cell targeting. In some embodiments, the heterologous epitope is inserted into and / or displayed by the nodular domain of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and / or displayed by the HI loop of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and / or displayed in the heparin binding site of the AAV capsid protein. In some embodiments, the heterologous epitope is inserted into and / or displayed in the heparin binding site of the AAV2 capsid protein. In some embodiments, the heterologous epitope is inserted into and / or displayed in the heparin binding site of the AAV6 capsid protein. In some embodiments, the heterologous epitope is inserted and / or displayed in the heparin binding site of the AAV8 capsid protein. In some embodiments, the heterologous epitope is inserted and / or displayed in the heparin binding site of the AAV9 capsid protein. In some embodiments, (i) the viral capsid protein is derived from the AAV2 capsid gene, and the epitope is inserted at position I453 or I587 of the AAV2 VP1 capsid protein, and / or after the corresponding positions of the AAV2 VP2 and / or VP3 capsid proteins, and / or replaces the amino acid at position I453 or I587 of the AAV2 VP1 capsid protein, and / or the amino acid at the corresponding positions of the AAV2 VP2 and / or VP3 capsid proteins; (ii) the viral capsid protein is derived from the AAV6 capsid gene, and the epitope is inserted at position I585 of the AAV6 VP1 capsid protein, and / or after the corresponding positions of the AAV6 VP2 and / or VP3 capsid proteins, and / or replaces the amino acid at position I585 of the AAV6 VP1 capsid protein, and / or after the corresponding positions of the AAV6 VP2 and / or VP3 capsid proteins. amino acid; (iii) the viral capsid is derived from the AAV8 capsid gene, and the epitope is inserted after position I590 of the AAV8VP1 capsid protein, and / or the corresponding positions of the AAV8VP2 and / or VP3 capsid proteins, and / or replaces the amino acid at position I590 of the AAV8VP1 capsid protein, and / or the amino acid at the corresponding positions of the AAV8VP2 and / or VP3 capsid proteins, or (iv) the viral capsid protein is derived from the AAV9 capsid gene, and the epitope is inserted after position I453 or I589 of the AAV9VP1 capsid protein, and / or the corresponding positions of the AAV9VP2 and / or VP3 capsid proteins, and / or replaces the amino acid at position I453 or I589 of the AAV9VP1 capsid protein, and / or the amino acid at the corresponding positions of the AAV9VP2 and / or VP3 capsid proteins.In some embodiments, the heterologous epitope is inserted immediately after an amino acid (e.g., fused to its C-terminus), wherein the amino acid is selected from G453 of AAV2 capsid protein VP1 (or the corresponding position 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 different AAVs that infect humans, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9), N587 of AAV2 capsid protein VP1 (or the corresponding position of VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N627 of AAV2 capsid protein VP2 (or the corresponding amino acids of VP1, VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N630 of AAV2 capsid protein VP1 (or the corresponding position of VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N647 of AAV2 capsid protein VP2 (or the corresponding amino acids of VP1, VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N650 of AAV2 capsid protein VP1 (or the corresponding amino acids of VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N661 of AAV2 capsid protein VP1 (or the corresponding amino acids of VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N648 of AAV2 capsid protein VP1 (or the corresponding amino acids of VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N651 of AAV2 capsid protein VP1 (or the corresponding amino acids of VP2 and / or VP3 capsid proteins encoded by the same capsid gene), N662 of AAV2 capsid protein VP1 and / or VP3 capsid proteins, or the corresponding amino acids of VP1, VP2 and / or VP3 capsid proteins of different AAVs that infect humans, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9), Q585 of AAV6 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8 and AAV9), Q586 of AAV6 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8 and AAV9 VP2 and / or VP3 capsid proteins), N590 of AAV8 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV9), G453 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV9), of different AAVs, such as 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8). In some embodiments, the heterologous epitope is inserted (e.g., fused to its C-terminus) immediately after G453 of AAV2 capsid protein VP1 (or the corresponding position 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).In some embodiments, the heterologous epitope is inserted immediately after N587 of AAV2 capsid protein VP1 (or the corresponding position 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 different AAVs that infect humans, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9) (e.g., fused to its C-terminus). In some embodiments, the heterologous epitope is inserted immediately after Q585 of AAV6 capsid protein VP1 (or the corresponding position 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8, and AAV9) (e.g., fused to its C-terminus). In some embodiments, the heterologous epitope is inserted immediately after N590 of AAV8 capsid protein VP1 (or the corresponding position 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV9) (e.g., fused to its C-terminus). In some embodiments, the heterologous epitope is inserted immediately after G453 of AAV9 capsid protein VP1 (or the corresponding position 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 different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8) (e.g., fused to its C-terminus). In some embodiments, the heterologous epitope is inserted (e.g., fused to its C-terminus) immediately after A589 of the AAV9 capsid protein VP1 (or the corresponding position of the VP2 and / or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids of the VP1, VP2 and / or VP3 capsid proteins of different AAVs that infect humans, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8). In some embodiments, the heterologous epitope is inserted and / or displayed between amino acids N587 and R588 of the AAV2 VP1 capsid protein (or the corresponding position of the VP2 and / or VP3 capsid encoded by the same capsid gene). In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprise the amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the recombinant viral capsid, the viral vector comprising the recombinant viral capsid, and / or the composition comprising the recombinant viral capsid comprise the amino acid sequence as shown in 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 an amino acid sequence encoded by the nucleic acid sequence as shown in 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 as shown in 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 as shown in SEQ ID NO: 27.

[0023] In some embodiments, in addition to the heterologous epitope, the recombinant capsid protein as described herein also comprises a second and different mutation. For example, in some embodiments, the recombinant viral capsid protein as described herein can be a genetically modified AAV2 capsid protein, comprising a heterologous epitope, and can also comprise mutations, such as R585A and / or R588A mutations. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, such as a genetically modified AAV2VP1 capsid protein, comprising a heterologous epitope inserted immediately after G453 of the AAV2VP1 protein (e.g., fused to its C-terminus), and further comprising a mutation selected from R585A and / or R5889A. In some embodiments, the recombinant viral capsid protein is derived from an AAV 2 capsid gene, such as a genetically modified AAV2VP1 capsid protein, comprising a heterologous epitope inserted immediately after N587 of the AAV2VP1 protein (e.g., fused to its C-terminus), and further comprising a mutation selected from R585A and / or R5889A. In some embodiments, the recombinant viral capsid protein is derived from an AAV9 capsid gene, such as a genetically modified AAV9VP1 capsid protein, comprising a heterologous epitope inserted immediately after G453 of the AAV9VP1 protein (e.g., fused to its C-terminus), and further comprising a W503A mutation. In some embodiments, the recombinant viral capsid protein is derived from an AAV9 capsid gene, such as a genetically modified AAV9VP1 capsid protein, comprising a heterologous epitope inserted immediately after A589 of the AAV9VP1 capsid protein (e.g., fused to its C-terminus), and further comprising a W503A mutation.

[0024] Generally, the recombinant viral capsid protein and / or the viral vector comprising the recombinant viral capsid comprises a heterologous epitope of 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 complementary site, such as an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises a length of at least 10 amino acids. 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 does not bind to metal ions, such as Ni. 2+ 、Co 2+ , Cu 2+ 、Zn 2+ , Fe 3+ In some embodiments, the heterologous epitope is not a polypeptide selected from streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A. In some embodiments, the affinity tag is selected from 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).

[0025] In some embodiments, the recombinant viral capsid protein is a genetically modified AAV2VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after G453 of the AAV2VP1 capsid protein (e.g., fused to its C-terminus). In some embodiments, the recombinant viral capsid protein (i) is derived from an AAV2 capsid gene, such as a genetically modified AAV2VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and inserted immediately after G453 of the AAV2VP1 capsid protein (e.g., fused to its C-terminus), and (iii) further comprises a mutation selected from R585A and / or R5889A. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV2VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after N587 of the AAV2VP1 capsid protein (e.g., fused to its C-terminus). In some embodiments, the recombinant viral capsid protein (i) is derived from an AAV2 capsid gene, such as a genetically modified AAV2VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and inserted immediately after N587 of the AAV2VP1 capsid protein (e.g., fused to its C-terminus), and (iii) further comprises a mutation selected from R585A and / or R588A. In some embodiments, the recombinant viral capsid protein (i) is derived from an AAV6 capsid gene, for example, a genetically modified AAV6VP1 capsid protein, and (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and inserted immediately after Q585 of the AAV6VP1 capsid protein (e.g., fused to its C-terminus). In some embodiments, the recombinant viral capsid protein is a genetically modified AAV8VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after N590 of the AAV8VP1 capsid protein (e.g., fused to its C-terminus). In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after G453 of the AAV9VP1 capsid protein (e.g., fused to its C-terminus).In some embodiments, the recombinant viral capsid protein (i) is derived from an AAV9 capsid gene, such as a genetically modified AAV9VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and inserted immediately after G453 of the AAV9VP1 capsid protein (e.g., fused to its C-terminus), and (iii) further comprises a W503A mutation. In some embodiments, the recombinant viral capsid protein is a genetically modified AAV9VP1 capsid protein and comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) inserted immediately after A589 of the AAV9VP1 capsid protein (e.g., fused to its C-terminus). In some embodiments, the recombinant viral capsid protein (i) is derived from an AAV9 capsid gene, such as a genetically modified AAV9VP1 capsid protein, (ii) comprises a heterologous epitope comprising the sequence EQKLISEEDL (SEQ ID NO: 6) and is inserted immediately after A589 of the AAV9VP1 capsid protein (e.g., fused to its C-terminus), and (iii) further comprises a W503A mutation.

[0026] In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises an EQKLISEEDL (as shown in SEQ ID NO: 6) inserted at I587 of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises an EQKLISEEDL (as shown in SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein, for example, comprising the amino acid sequence as shown in SEQ ID NO: 2.

[0027] In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV6VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted at I585 of the AAV6VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted between Q585 and S586 of the AAV6VP1 capsid protein, for example, comprising the amino acid sequence as shown in SEQ ID NO: 4.

[0028] In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV8VP1 capsid. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted at I590 of the AAV8VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted between N590 and T591 of the AAV8VP1 capsid protein, for example, comprising the amino acid sequence as shown in SEQ ID NO: 25.

[0029] In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV9VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted at I453 of the AAV9VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted between G453 and S454 of the AAV9VP1 capsid protein, for example, comprising an amino acid sequence as shown in SEQ ID NO: 26. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO: 6) inserted at I589 of the AAV9VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (as shown in SEQ ID NO:6) inserted between A589 and Q590 of the AAV9VP1 capsid protein, for example, comprises the amino acid sequence shown in SEQ ID NO:27.

[0030] 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 a first linker, an affinity tag, and a second linker from the N-terminus to the C-terminus. In some embodiments, the first linker and the second linker are each independently a polypeptide of at least 1 amino acid in length. In some embodiments, the heterologous epitope as described herein, for example, an affinity tag alone or in combination with one or more linkers, is about 5 amino acids to about 35 amino acids in length. In some embodiments, the first linker and the second linker have an equivalent length and / or comprise an equivalent amino acid sequence.

[0031] Generally, in the absence of an appropriate multispecific, optionally bispecific binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein has a natural tropism that is reduced to eliminated, such as having a reduced ability or inability to target and bind to a reference cell that naturally allows transduction, compared to a reference viral capsid (e.g., a capsid comprising a reference viral capsid protein, such as a viral capsid protein that is identical to the recombinant viral capsid protein but lacks a heterologous epitope), in comparison to that of a reference viral capsid (e.g., a capsid comprising a reference viral capsid protein, such as a viral capsid protein that is identical to the recombinant viral capsid protein but lacks a heterologous epitope). In some embodiments, and in the absence of an appropriate multispecific, optionally bispecific binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 10% compared to a reference viral capsid. In some embodiments, and in the absence of an appropriate multispecific, optionally bispecific binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 20% compared to a reference viral capsid. In some embodiments, and in the absence of an appropriate multispecific, optionally bispecific binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 30% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 40% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 50% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 60% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 70% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 75% compared to a reference viral capsid. In some embodiments, and in the absence of an appropriate multispecific, optionally bispecific binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least 80% reduction in transduction efficiency compared to a reference viral capsid.In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 85% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 90% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 95% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein shows a transduction efficiency reduction of at least 99% compared to a reference viral capsid. In some embodiments, and in the absence of appropriate multi-specific, optionally bi-specific binding molecules, transduction of control cells by a recombinant viral capsid comprising a recombinant viral capsid protein as described herein is eliminated, for example, undetectable.

[0032] In some embodiments, the viral capsid comprising a recombinant viral capsid protein as described herein is a chimeric capsid, for example, comprising a recombinant viral capsid protein comprising a heterologous epitope at a certain ratio and a reference capsid protein that does not comprise a heterologous epitope. In some embodiments, the reference capsid protein is a wild-type reference capsid protein because it comprises an amino acid sequence of a wild-type capsid protein having the same serotype as the recombinant viral capsid protein. In some embodiments, the reference capsid protein is a control reference capsid protein because it comprises an amino acid sequence of a recombinant viral capsid protein, except that the control reference capsid protein lacks a heterologous epitope. In some embodiments, the reference capsid protein is a mutated wild-type reference protein because the amino acid sequence it comprises is substantially equivalent to the amino acid sequence of a wild-type capsid protein, and the wild-type capsid protein has the same serotype as the recombinant viral capsid protein, but has a mutation that reduces the tropism of the wild-type capsid protein (e.g., insertion of an amino acid sequence, chimerism, etc.). In some embodiments, the composition described herein comprises the following, or the method described herein combines the following: a recombinant viral capsid protein and a reference capsid protein having 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.

[0033] Also disclosed herein are nucleic acids encoding the recombinant viral capsid proteins described herein, compositions comprising such nucleic acids (e.g., which can be used in methods for preparing recombinant viral capsids) and / or recombinant viral capsid proteins (e.g., compositions consisting essentially of recombinant viral capsid proteins, compositions comprising only viral vectors encapsulated by capsids comprising viral capsid proteins described herein, compositions comprising such viral vectors and multispecific, optionally bispecific binding molecules (e.g., at a certain viral vector to multispecific, optionally bispecific binding molecule (molecule: molecule) ratio), compositions comprising such viral vectors, retargeting moieties, and pharmaceutically acceptable carriers, etc.). In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence encoding the amino acid sequence of EQKLISEEDL (SEQ ID NO: 6), and a nucleotide sequence encoding at least 5 contiguous amino acids of an adenovirus or adeno-associated virus capsid protein.

[0034] In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV2 VP1 capsid protein. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted at I587 of the AAV2 VP1 capsid protein (as shown in SEQ ID NO: 6). In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted between N587 and R588 of the AAV2 VP1 capsid protein (as shown in SEQ ID NO: 6), for example, in some embodiments, the amino acid sequence encoded by the nucleic acid as described herein comprises the amino acid sequence as shown in SEQ ID NO: 2.

[0035] In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV6VP1 capsid protein. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted at I585 of the AAV6VP1 capsid protein (as shown in SEQ ID NO: 6). In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted between Q585 and S586 of the AAV6VP1 capsid protein (as shown in SEQ ID NO: 6), for example, in some embodiments, the amino acid sequence encoded by the nucleic acid as described herein comprises the amino acid sequence as shown in SEQ ID NO: 4.

[0036] In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV8VP1 capsid protein. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (as shown in SEQ ID NO: 6) inserted at I590 of the AAV8VP1 capsid protein. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (as shown in SEQ ID NO: 6) inserted between N590 and T591 of the AAV8VP1 capsid protein, for example, in some embodiments, the amino acid sequence encoded by the nucleic acid as described herein comprises the amino acid sequence as shown in SEQ ID NO: 25.

[0037] In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV9VP1 capsid protein. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted at I453 of the AAV9VP1 capsid protein (as shown in SEQ ID NO: 6). In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted between G453 and S454 of the AAV9VP1 capsid protein (as shown in SEQ ID NO: 6), for example, in some embodiments, the amino acid sequence encoded by the nucleic acid as described herein comprises an amino acid sequence as shown in SEQ ID NO: 26. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding an EQKLISEEDL inserted at I589 of the AAV9VP1 capsid protein (as shown in SEQ ID NO: 6). In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence encoding EQKLISEEDL (as shown in SEQ ID NO:6) inserted between A589 and Q590 of the AAV9VP1 capsid protein, for example, in some embodiments, the amino acid sequence encoded by the nucleic acid as described herein comprises the amino acid sequence shown in SEQ ID NO:27.

[0038] Generally, the recombinant viral vector as described herein comprises a viral capsid, and the viral capsid comprises a recombinant viral capsid protein as described herein, wherein the viral capsid encapsulates the target nucleotide. In some embodiments, the target nucleotide is under the control of a promoter selected from 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 target nucleotide is under the control of a non-human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter.

[0039] In general, the target nucleotide can be one or more genes that can encode a detectable marker, such as a reporter molecule or a therapeutic polypeptide. In some embodiments, the target nucleotide is a reporter gene. In some embodiments, the target nucleotide is a reporter gene that encodes a detectable marker selected from green fluorescent protein, luciferase, beta-galactosidase, etc. In some embodiments, the detectable marker is green fluorescent protein. In other embodiments, the target nucleotide is selected from suicide genes, nucleotides encoding antibodies or fragments thereof, nucleotides encoding CRISPR / Cas systems or a portion thereof, nucleotides encoding antisense RNA, nucleotides encoding siRNA, secretory enzymes, etc. In one embodiment, the target nucleotide encodes a multi-domain therapeutic agent, such as a protein comprising at least two domains that provide two different functions.

[0040] The compositions described herein generally comprise a viral vector comprising a recombinant viral capsid protein as described herein, for example comprising a capsid comprising a recombinant viral capsid protein, wherein the capsid encapsulates a target nucleotide. In some embodiments, the compositions described herein comprise (1) a viral vector having a capsid comprising a recombinant viral capsid protein genetically modified to comprise a heterologous epitope, (2) a multispecific, optionally bispecific binding molecule comprising (i) an antibody paratope that specifically binds to an epitope and (ii) a retargeting ligand that specifically binds to a receptor, and optionally (3) a pharmaceutically acceptable carrier.

[0041] As described herein, the antibody paratope generally comprises at least a complementary determining region (CDR) that specifically recognizes a heterologous epitope, such as a CDR3 region of a heavy chain 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, for example, the multispecific, optionally bispecific binding molecule may comprise an scFv comprising an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, a multispecific, optionally bispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a heterologous epitope, wherein the antibody (or a 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 the one or more antibody constant domains do not bind to the heterologous epitope.

[0042] In addition to the paratope (e.g., an antibody or a portion thereof comprising a paratope) of a heterologous epitope inserted into / displayed by a recombinant viral capsid protein, a multispecific, optionally bispecific binding molecule as described herein also comprises a retargeting ligand. In some embodiments, the retargeting ligand specifically binds to a receptor on the surface of a bead (e.g., for separation and / or purification of a recombinant viral capsid protein as described herein). In some embodiments, the retargeting ligand specifically binds to a cell surface protein expressed on the surface of a mammalian (e.g., human) eukaryotic cell, such as a target cell, such as a receptor, a cell surface marker, etc. In some embodiments, the retargeting ligand binds to (human) hepatocytes, (human) brain cells, (human) T cells, (human) kidney cells, (human) intestinal cells, (human) pancreatic cells, (human) cancer cells, and / or (human) cells infected by heterologous pathogens. In some embodiments, the retargeting ligand binds to a (human) hepatocyte-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.

[0043] In some embodiments, the retargeting ligand binds to a receptor expressed by (human) hepatocytes, such as an asialoglycoprotein receptor, such as hASGR1. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) neuronal cells, such as GABA, transferrin, etc. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) T cells, such as CD3, such as CD3ε. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) hematopoietic stem cells, such as 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, such as integrins. In some embodiments, the retargeting ligand binds to a receptor expressed by a (human) cancer cell, such as a tumor-associated antigen, such as adipophilin, AIM-2, ALDH1A1, α-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, β-catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD 274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, E6, E7 、FGF5、FLT3-ITD、FN1、G250 / MN / CAIX、GAGE-1,2,8、GAGE-3,4,5,6,7、GAS7、Glypican-3、GnTV、gp100 / Pme117、GPNMB、HAUS3、Hepsin、HER-2 / neu、HERV-K-MEL、HLA-A11、HLA-A2、HLA-DOB、hsp70-2、IDO1、IGF2B3、IL13Rα2、intestinal carboxylesterase、K -ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin A,MART2, MATN, MC1R, MCSP, mdm-2, ME1, Melan-A / MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, myosin, class I myosin, 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-RARα 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-βRII, 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 antigen (in lymphoma and other solid tumors), ErbB receptor, Melan A [MART1], gp 100, tyrosinase, TRP-1 / gp 75 and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder cancer, head and neck cancer and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); mucin [MUC-1] (in breast cancer, pancreatic cancer, colon cancer and prostate cancer); prostate specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon cancer, breast cancer and gastrointestinal cancer), as well as such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3TO 7. LAGE-1, NY-ESO-1 / LAGE-2, NA-88, GnTV,TRP2-INT2, etc. In some embodiments, the retargeting ligand binds E6 and / or E7. In some embodiments, the retargeting ligand binds Her2. In some embodiments, the retargeting ligand binds human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds human exonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

[0044] In some embodiments, the paratope (e.g., an antibody or a portion thereof) and the retargeting ligand are directly fused to each other. In some embodiments, the paratope (e.g., an antibody or a portion thereof) that specifically binds to a heterologous epitope and the retargeting ligand are covalently linked to each other.

[0045] In some embodiments, the multispecific binding molecule is a bispecific binding molecule, such as an antibody comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a paratope that specifically binds to a heterologous epitope, the heterologous epitope is inserted into or displayed by 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 antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain comprises a paratope that specifically binds to a heterologous epitope, the heterologous epitope is inserted into or displayed by a recombinant viral capsid protein, and the second antigen binding domain specifically binds to a receptor expressed by a target cell, wherein the first antigen binding domain is operably linked to a first heavy chain region comprising a first CH3 domain, wherein the second antigen binding domain is operably linked to a second heavy chain region comprising a second CH3 domain, wherein the first Ig CH3 domain and the second Ig C H The three 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. H The 3 domain binds protein A, while the second Ig C H The 3 domain contains a mutation that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering; H435R according to EU numbering). H The 3 domain may also contain a Y96F modification (according to IMGT; Y436F according to EU). HFurther modifications that may be found within the 3 domains include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies.

[0046] In some embodiments, the multispecific binding molecule is a bispecific binding molecule, such as a bispecific antibody comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds to an affinity tag displayed by a recombinant viral capsid protein as described herein, and wherein 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, such as a bispecific antibody comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds to an amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by a recombinant viral capsid protein as described herein, wherein 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, such as a bispecific antibody comprising a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain binds to an amino acid sequence EQKLISEEDL (SEQ ID NO: 6) displayed by a recombinant viral capsid protein as described herein, wherein the second antigen binding domain binds to hASGR1. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, such as a bispecific antibody comprising a first antigen binding domain 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 as described herein, wherein the second antigen binding domain binds to a CD3 protein, such as CD3ε. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, such as a bispecific antibody comprising a first antigen binding domain 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 as described herein, wherein the second antigen binding domain binds to an integrin. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, such as a bispecific antibody comprising a first antigen binding domain 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 as described herein, wherein the second antigen binding domain binds to an integrin, such as hGCGR. In some embodiments, the multispecific binding molecule is a bispecific binding molecule, e.g., a bispecific antibody comprising a first antigen binding domain 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 as described herein, wherein the second antigen binding domain binds ENTPD3.

[0047] Also described herein are methods of making and using recombinant viral capsid proteins, viral vectors comprising them, compositions, and the like. In some embodiments, methods of redirecting viruses such as adenoviruses, adeno-associated viruses, and the like; delivering diagnostic / therapeutic cargoes to target cells, and the like, include combining a recombinant viral vector comprising a recombinant viral capsid protein as described herein, such as 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 an epitope and (ii) a retargeting ligand that specifically binds to a receptor. Such methods may include producing a recombinant viral vector as a first step, for example, culturing a packaging cell under conditions sufficient to produce a viral vector, wherein the packaging cell comprises a plasmid encoding a capsid protein comprising an epitope. When delivering a diagnostic / therapeutic cargo to a target cell, the methods described herein may include contacting the target cell with a combination of a viral vector comprising a capsid comprising a recombinant viral capsid displaying a heterologous epitope, and a multispecific binding molecule, wherein the multispecific binding molecule comprises i) an antibody paratope that specifically binds to the epitope, and (ii) a retargeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro. In other embodiments, the target cell is in a subject, such as a human.

[0048] In some embodiments, the compositions described herein comprise a recombinant viral vector and a multispecific binding molecule, or the methods described herein combine a recombinant viral vector and a multispecific binding molecule, wherein the recombinant viral vector comprises a target nucleotide encapsulated by a capsid, and the capsid comprises a recombinant capsid protein as described herein, and its molecule: molecule ratio restores the transduction efficiency of the viral vector to a ratio similar to that of a reference viral vector. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) ranges from 1:0.5 to 1:100. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) ranges from 1:4 to 1:20. In some embodiments, the ratio of the recombinant viral vector to the bispecific binding molecule (molecule: molecule) ranges from 1:8 to 1:15. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is 1:4. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is 1:8. In some embodiments, the ratio of the recombinant viral vector to the 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.

[0049] Also described herein are methods for inactivating viral capsids and / or producing viral vectors, the methods generally comprising (a) inserting a nucleic acid encoding a heterologous protein into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising a heterologous protein, and / or (b) culturing packaging cells under conditions sufficient to produce viral vectors, wherein the packaging cells comprise nucleotide sequences. In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising a target nucleotide. In some embodiments, the method further comprises separating a self-complementary adeno-associated viral vector from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating a single-stranded adeno-associated viral vector from the cell lysate. In some embodiments, the method further comprises (a) removing cell debris, (b) treating the supernatant containing the viral vector with DNase I and MgCl2, (c) concentrating the viral vector, (d) purifying the viral vector, and (e) any combination of (a)-(d). Also provided herein are viral vectors prepared according to the methods described herein, and packaging cells that can be used to produce viral vectors as described herein, e.g., packaging cells comprising a plasmid encoding the recombinant capsid protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0051] Figure 1 Provided are immunofluorescence microscopy images (upper panels) or histograms (lower panels) obtained from fluorescence-activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by HepG2 cells incubated with: (A) wild-type scAAV2-CMV-eGFP viral vector alone; (B) scAAV2N587Myc-CMV-hrGFP viral vector alone; scAAV2N587Myc viral vector mixed with a 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; (L) or scAAV N587Myc viral vector mixed with a monospecific anti-Myc antibody at a ratio of 1:8.

[0052] Figure 2AProvided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with: wild-type scAAV2 viral vector alone (i), scAAV2-N587Myc-CMV-eGFP viral vector alone (ii), scAAV2-N587Myc-CMV-eGFP viral vector mixed with a 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 an irrelevant bispecific anti-Myc-GCGR antibody at a ratio of 1:8 (xii). Also shown is GFP expression by 29T3 cells incubated with scAAV N587Myc viral vector mixed with a bispecific anti-Myc-ASGR1 antibody at a ratio of 1:8 (xi). For each experiment, 2x10 5 cells and 5x10 9 Viral vector.

[0053] Figure 2B Provided are histograms obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with: unmodified AAV9-CAGG-GFP viral vector alone (i), AAV9-A589Myc-CAGG-eGFP viral vector alone (ii), AAV9-A589Myc-CAGG-eGFP viral vector mixed with a 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). For each experiment, 2x10 5 cells and 1x10 10 Viral vector (titration by qPCR).

[0054] Figure 3Provided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (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 vectors mixed with bispecific anti-Myc-ASGR1 antibodies at a ratio of 1:8 (L). 293T3-hASGR1 cells incubated with wild-type scAAV viral vectors only (A) or scAAV2-N587Myc-CMV-eGFP viral vectors only (B) served as controls. For each experiment, 2x10 5 cells and 5x10 9 Viral vector (as titrated by qPCR).

[0055] Figure 4 Provided are images of immunofluorescence microscopy evaluating green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with wild-type scAAV viral vector alone (A), scAAV2-N587Myc-CMV-eGFP viral vector alone (B), or with 1×10 9 (C), 2x10 9 (D), 4x10 9 (E), 8x10 9 (F), 2x10 10 (G), 1x10 11 (H), 1x10 12 (I) bispecific anti-Myc-ASGR1 antibody, followed by 1x10 9 scAAV2-N587Myc-CMV-eGFP viral vectors were sequentially incubated. Also shown is the 11 Anti-myc-ASGR1 antibody molecules, followed by 1x10 9 293T cells were sequentially incubated with scAAV2-N587Myc-CMV-eGFP viral vector (J), and with 1x10 11 Unrelated bispecific anti-Myc-GCGR antibody molecules followed by 1x10 9 Immunofluorescence microscopy images of 293T-hASGR1 cells (K) sequentially incubated with scAAV2-N587Myc-CMV-eGFP viral vectors.

[0056] Figure 5 Provided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with: wild-type ssAAV viral vector alone (A), ssAAV2-N587Myc-CMV-hrGFP viral vector alone (B), ssAAV2-N587Myc-CMV-hrGFP viral vector mixed with a 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 ssAAVN587Myc viral vector mixed with an irrelevant bispecific anti-Myc-GCGR antibody at a ratio of 1:8 (K). Also shown is GFP expression by 29T3 cells incubated with ssAAV N587Myc viral vector mixed with a bispecific anti-Myc-ASGR1 antibody at a ratio of 1:8 (J). For each experiment, 2x10 5 cells and 5x10 9 Viral vector.

[0057] Figure 6 Provided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by 29T3-hGCGR cells incubated with: wild-type scAAV viral vector alone (A), scAAV2N587Myc-CMV-eGFP viral vector alone (B), scAAV2N587Myc-CMV-eGFP viral vector mixed with a 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 scAAV2N587Myc-CMV-eGFP viral vector mixed with an irrelevant monospecific anti-Myc antibody (Regeneron Pharmaceuticals, Tarrytown, NY) at a ratio of 1:8 (L). For each experiment, 2×10 5 cells and 5x10 9 Viral vector.

[0058] Figure 7Provided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by Jurkat cells alone (A) or incubated with (B): wild-type scAAV6-EF1-eGFP viral vector alone (B), AAV6-Q585Myc-EF1a-eGFP viral vector alone (C), 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). For each experiment, 2x10 5 cells and 1x10 9 Viral vector.

[0059] Fig. 8A Immunofluorescence microscopy images of the liver are provided. Figure 8B Immunofluorescence microscopy images of spleen are provided. Figure 8C Immunofluorescence microscopy images of kidneys are provided. All samples were obtained from C57BL / 6 mice genetically modified to express human ASGR1 in hepatocytes (i-iv) or wild-type C57BL / 6 mice (v-viii) ten days after intravenous injection with: 1x10 11 Wild-type scAAV2-CMV-eGFP (i,v), saline (ii,vi), 1x10 11 scAAV2-N587myc-CMV-eGFP viral vector, or scAAV2-N587myc-CMV-eGFP viral vector with bispecific anti-myc ASGR1 antibody (iv, viiii).

[0060] Fig. 9 Provided are immunofluorescence microscopy images of liver samples obtained from C57BL / 6 mice genetically modified to express human ASGR1 on hepatocytes (DF, JL, PR) or wild-type C57BL / 6 mice (AC, GI, MO) four weeks after intravenous injection with: 2.18x10 11 Wild-type ssAAV2-CAGG-eGFP (B, C, E, F), saline (A, D), 2.18x10 11 ssAAV2-N587myc-CAGG-eGFP viral vector (GI, JL), or ssAAV2-N587myc-CAGG-eGFP viral vector with bispecific anti-myc-ASGR1 antibody (MO, PR). Each image represents one mouse.

[0061] Fig.10 Provided are images of immunofluorescence microscopy of liver samples taken from C57BL / 6 mice transgenically modified to express human ASGR1 via hepatocytes ten days after intravenous injection with: (A) wild-type AAV9, (B) 250 nM NaCl, (C) AAV9A589myc-CAGG-eGFP viral particles in combination with a bispecific anti-myc hCD3 antibody, or (D) AAV9A589myc-CAGG-eGFP viral particles in combination with a bispecific anti-myc ASGR1 antibody.

[0062] Fig.11 Illustrative, not to scale, and non-limiting exemplary multispecific binding molecule formats useful in some embodiments of the invention are provided.

[0063] Fig.12 Provided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by 29T3-hASGR1 cells incubated with: (A) wild-type AAV8 viral vector alone, (C) AA8-N590-myc viral vector alone, or pAAV RC8N590myc viral vector mixed with bispecific anti-hASGR1-IgG4-Fc / anti-myc bispecific molecules 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. Also shown is GFP expression by mock-transfected 29T3-hASGR1 cells (B). For each experiment, 2x10 5 cells and 1x10 9 Viral vector.

[0064] Fig.13 Provided are images of immunofluorescence microscopy of liver samples taken from C57BL / 6 mice transgenically modified to express human ASGR1 via hepatocytes ten days after intravenous injection with: (A)-(C) wild-type AAV8, (D)-(F) AA8-N590-myc viral vector and control bispecific binding molecules, or (G)-(I) AAV8N590myc-CAGG-eGFP viral particles in combination with anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecules.

[0065] Fig.14Provided are dot plots obtained from fluorescence activated cell sorting (FACS) evaluating green fluorescence (GFP) expression by 29T3-h ENTPD3 cells incubated 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 bispecific anti-hENTPD3-IgG4-Fc / anti-myc bispecific molecules 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. For each experiment, 2x10 5 cells and 1x10 9 Viral vector.

[0066] Fig.15A Immunofluorescence microscopy images of liver samples are provided. Fig. 15B Immunofluorescence microscopy images of intestinal samples are provided, and Fig. 15C Immunofluorescence microscopy images of pancreatic samples are provided. All samples were obtained from wild-type C57BL / 6 mice ten days after intravenous injection with: PBS (15A(i), 15B(i), and 15C(i)), 5x10 11 Wild-type AAV9 (15A(ii), 15B(ii), and 15C(ii)) with 1x10 3 5x10 of irrelevant bispecific IgG4-Fc / anti-myc binding protein 11 AAV2-N587myc-CAGG-eGFP viral vector (15A(iii), 15B(iii) and 15C(iii)), or with 1x10 13 5x10 of bispecific hENTPD3-IgG4-Fc / anti-myc binding protein 11 AAV2-N587myc-CAGG-eGFP viral vector (15A(iv), 15B(iv) and 15C(iv)). DETAILED DESCRIPTION

[0067] A common problem with adapter approaches utilizing either unmodified viral capsids or scaffold-modified viral capsids has been 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 adenoviral vectors containing fibers with an RGD-4C sequence genetically incorporated into the HI loop of the carboxyl-terminal knob domain and demonstrate the utility of the HI loop of the fiber knob as an optimal site for incorporating short peptide ligands. See, e.g., U.S. Pat. No. 7,297,542; see also Beatty and Curiel (2012) Adv. Cancer. Res. 115:39-67. Similarly, insertion of ligand peptides into AAV capsid proteins results in capsids that are capable of displaying ligands on the surface of the capsid and mediating transduction through interaction of the ligand with its receptor, thereby redirecting viral tropism through genetic capsid modification (Girod et al. (1999) Nat. Med. 5(9):1052-6, 1438 (including errata) (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). In particular, it has been demonstrated that the insertion of an integrin-binding Arg-Gly-Asp (RGD) motif at insertion site I-587 of the AAV capsid protein VP1 enables the AAV viral vector to be expressed via α vβ1 integrin transduces cells (Girod et al. (1999), supra). In contrast, although the insertion of the 14 amino acid peptide L14 after amino acid R447 (I-447) resulted in capsids that were still recognized by the conformation-sensitive antibody A20, such recombinant viral vectors were unable to transduce cells expressing the L-14 receptor (Girod et al., 1999; cf. Wu et al. (2000) (reporting the insertion of a hemagglutinin (HA) peptide at position I-447 and the successful transduction of cells expressing the HA peptide). Insertion of the Myc epitope between T448 and N449 is recognized by anti-Myc antibodies and is therefore present on the surface of the capsid, but results in an inactivated viral vector. (Grifman et al., 2001). In contrast, again for the NGR motif at N Successful retargeting has been reported by inserting c-myc after N587, rather than inserting c-myc after N587. (Grifman et al., 2001). U.S. Pat. No. 9,624,274 describes I-453 of the AAV capsid protein as a suitable insertion site for heterologous epitopes. Although these studies demonstrate successful insertion and display of heterologous peptides, such as epitopes, by AAV capsid proteins, none of these studies provide any expectation that multispecific binding molecules, such as bispecific binding molecules, such as bispecific antibodies, that specifically bind to heterologous peptides and cell surface proteins can retarget the modified viral vector to cells expressing cell surface proteins and restore their transduction efficiency.

[0068] Disclosed herein are recombinant viral capsid proteins modified with heterologous epitopes, which can be used in combination with multispecific binding molecules, the multispecific binding molecules comprising a complementary position to a specific binding epitope, such as an Fv domain, and a ligand that binds to a receptor expressed on the surface of a target cell. As shown herein, a viral vector having a capsid formed by a capsid protein as described herein is contacted with a multispecific binding molecule at a certain ratio to restore the transduction efficiency of the viral capsid to a level comparable to that of a wild-type virus, see, for example, Example 2. In general, the capsid proteins modified with heterologous epitopes as described herein can be derived from non-enveloped viruses, such as, but not limited to, adenovirus (Ad) and adeno-associated virus (AAV).

[0069] Although the present invention has been particularly shown and described with reference to many embodiments, those skilled in the art will understand that changes in form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the present invention, and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims.

[0070] Although some preferred methods and materials are now described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications cited herein are incorporated herein by reference for their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs.

[0071] definition

[0072] Unless defined otherwise, 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.

[0073] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to one of ordinary skill in the art upon reading this disclosure.

[0074] The term "antibody" includes immunoglobulin molecules, which are composed of four polypeptide chains interconnected by disulfide bonds: two heavy (H) chains and two light (L) chains. Each heavy chain contains a heavy chain variable domain (V H ) and the heavy chain constant region (C H The heavy chain constant region contains at least three domains, C H 1. C H 2. C H 3 and optional C H 4. Each light chain contains a light chain variable domain (C H ) and the light chain constant region (C L ). The heavy chain variable domain and the light chain variable domain can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy chain variable domain and light chain variable domain contains three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2 and LCDR3. The usual tetrameric antibody structure contains two identical antigen binding domains, each of which is bound by a V H and V L The domains are combined to form a H and C L The domains together form the antibody Fv region. Single-domain antibodies contain a single antigen-binding domain, e.g., V HOr VL. The term "antibody" encompasses monoclonal antibodies, multispecific (e.g., bispecific) antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv (sdFv), intracellular antibodies, miniantibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to 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 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication 2009 / 0060910. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or subclass.

[0075] The antigen binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to an antigenic epitope, is also called a "paratope." It is a small region of the antibody Fv region (having 5 to 10 amino acids), part of the antigen binding fragment (Fab region), and may contain portions of the antibody heavy and / or light chains. When the paratope binds to the epitope with high affinity, the paratope specifically binds to the epitope. The term "high affinity" refers to an antibody that has about 10 amino acids for its target epitope. -9 M or less (e.g., about 1x10 -9 M, 1x10 -10 M, 1x10 -11 M or about 1x10 -12 M) K D In one embodiment, the surface plasmon resonance (SPR) of the nanostructured carbon nanotubes is performed by surface plasmon resonance (SPR), such as BIACORE. TM To measure K D In another embodiment, K is measured by ELISA D .

[0076] The phrase "complementarity determining region" or the term "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of the immunoglobulin gene of an organism, which usually (i.e., in wild-type animals) appears between two framework regions in the light chain or heavy chain variable region of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). CDR can be, for example, a germline sequence or a rearranged sequence or an unrearranged sequence, and is, for example, encoded by a naive or mature B cell or T cell. CDR can be somatically mutated (e.g., different from the sequence encoded in the germline of the animal), humanized and / or modified with amino acid substitutions, additions or deletions. In some cases (e.g., for CDR3), CDR can be encoded by two or more sequences (e.g., germline sequences), which are not adjacent (e.g., in unrearranged nucleic acid sequences), but are adjacent in the B cell nucleic acid sequence, for example, due to splicing or connecting sequences (e.g., VDJ recombination to form heavy chain CDR3).

[0077] "Epitope" is a part of a macromolecule that is recognized by the immune system, particularly by antibodies, B cells or cytotoxic T cells. Although it is generally believed that epitopes are derived from non-self proteins, sequences that can be recognized that are derived from a host are also classified as epitopes. An epitope has at least 4 amino acids, preferably 4 to 30 amino acids, more preferably 5 to 20 amino acids, particularly 5 to 15 amino acids in length. An epitope can be linear or three-dimensional, and the three-dimensional epitope is usually formed by amino acids that are far away from each other in the primary protein structure, but become closely related in the secondary structure and / or tertiary structure. The epitope specifically recognized by a B cell is called a B cell epitope.

[0078] The phrase "inverted terminal repeats" or "ITRs" includes symmetrical nucleic acid sequences in the genome of the adeno-associated virus that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as origins of replication for viral DNA synthesis and are essential cis components for the generation of AAV integration vectors.

[0079] The phrase "light chain" includes immunoglobulin light chain sequences from any organism, and unless otherwise specified, includes human kappa and lambda light chains and VpreB and alternative light chains. Unless otherwise specified, the light chain variable domain generally includes three light chain CDRs and four framework (FR) regions. Generally, the full-length light chain includes a variable domain and a light chain constant region from the amino terminus to the carboxyl terminus, and the variable domain includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The light chain variable domain is encoded by a light chain variable region gene sequence, which generally contains V and J segment repertoires derived from the germline. L and J LSegment. The sequences, positions 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, those that do not selectively bind to a first epitope or a second epitope, which are selectively bound by an epitope-binding protein in which they appear. Light chains also include such light chains that bind and recognize or help a heavy chain or another light chain bind and recognize one or more epitopes that are selectively bound by an epitope-binding protein in which they appear. Common light chains or universal light chains include those derived from human Vκ1-39Jκ genes or human Vκ3-20Jκ genes, and include somatically mutated (e.g., affinity matured) forms thereof. Exemplary human V L Segments include human Vκ1-39 gene segments, human Vκ3-20 gene segments, human Vλ1-40 gene segments, human Vλ1-44 gene segments, human V2-8 gene segments, human V 2-14 gene segments, and human V 3-21 gene segments, and include somatically mutated (e.g., affinity-matured) forms thereof. A light chain may be prepared comprising a variable domain from one organism (e.g., human or rodent, such as rat or mouse; or bird, such as chicken), and a constant region from the same or different organism (e.g., human or rodent, such as rat or mouse; or bird, such as chicken).

[0080] The term "about" or "approximately" includes within statistically significant ranges of values. Such ranges may be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The permissible variations encompassed by the term "about" or "approximately" depend on the particular system under study and can be readily appreciated by those of ordinary skill in the art.

[0081] The term "affinity tag" includes a polypeptide sequence that is a member of a specific binding pair, for example, it specifically binds to another polypeptide sequence, such as an antibody paratope, with high affinity. Exemplary and non-limiting affinity tags include hexahistidine tags, FLAG tags, Strep II tags, streptavidin binding peptide (SBP) tags, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S tags, HA tags, and c-Myc tags. (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; the references are incorporated herein by reference).

[0082] The term "capsid protein" includes proteins that are part of the viral capsid. For adeno-associated viruses, capsid proteins are generally referred to as VP1, VP2 and / or VP3, and are each encoded by a single cap gene. For AAV, these three AAV capsid proteins are produced in an overlapping manner from the cap open reading frame (ORF) via alternate mRNA splicing and / or alternate translation start codon usage, although all three proteins use a common stop codon. Warrington et al. (2004) J. Virol. 78: 6595, incorporated herein by reference in its entirety. The VP1 of AAV2 is generally translated from the ATG start codon (amino acid M1) on the 2.4-kb mRNA, while the VP2 and VP3 of AAV2 start from a smaller 2.3-kb mRNA, using a weaker ACG start codon for the production of VP2 (amino acid T138), and read-through translation to the next available ATG codon (amino acid M203) for the production of the most abundant capsid protein VP3. Warrington, supra; Rutledge et al. (1998) J. Virol. 72:309-19, incorporated herein by reference in its entirety. The amino acid sequences of the capsid proteins of adeno-associated viruses are well known in the art and are generally conserved, particularly with respect to parvoviruses. See Rutledge et al., supra. For example, Rutledge et al. (1998) supra, in Figure 4 B provides an amino acid sequence alignment for the VP1, VP2 and VP3 capsid proteins of AAV2, AAV3, AAV4 and AAV6, wherein the start site for each of the VP1, VP2 and VP3 capsid proteins is indicated by an arrow, and the variable domains are framed. Accordingly, although the amino acid positions provided herein may be provided with respect to the VP1 capsid protein of AAV, and the amino acid positions provided herein that are not further specified refer to the AAV2 sequence of the major coat protein VP1 as shown in SEQ ID NO: 1, the skilled person can separately and easily determine the position of the same amino acid within the VP2 and / or VP3 capsid protein of AAV, as well as the corresponding position of the amino acid in different serotypes. In addition, the skilled person can exchange domains between capsid proteins of different AAV serotypes for the formation of "chimeric capsid proteins".

[0083] Domain swapping between two AAV capsid protein constructs for generating "chimeric AAV capsid proteins" has been described, see, e.g., Shen et al. (2007) Mol. Therapy 15(11): 1955-1962, which is incorporated herein by reference in its entirety. "Chimeric AAV capsid proteins" include AAV capsid proteins that contain amino acid sequences, e.g., domains, from two or more different AAV serotypes and are capable of forming and / or forming AAV-like viral capsids / viral particles. Chimeric AAV capsid proteins are encoded by chimeric AAV capsid genes, e.g., nucleotides containing multiple (e.g., at least two) nucleic acid sequences, each of which is equivalent to a portion of a capsid gene encoding a capsid protein of a different AAV serotype, and the multiple together encode a functional chimeric AAV capsid protein. Reference to a chimeric capsid protein associated with a particular AAV serotype indicates that the capsid protein contains one or more domains of a capsid protein from that serotype, and one or more domains of a capsid protein from a different serotype. For example, an AAV2 chimeric capsid protein includes a capsid protein comprising one or more domains of an AAV2 VP1, VP2 and / or VP3 capsid protein and one or more domains of a VP1, VP2 and / or VP3 capsid protein of a different AAV.

[0084] A "chimeric capsid" comprises at least two sets of VP1, VP2 and / or VP3 proteins, wherein each set is encoded by a different cap gene.

[0085] In some embodiments, the chimeric capsid described herein comprises a recombinant VP1, VP2 and / or VP3 protein encoded by a cap gene, wherein the cap gene is genetically modified by the insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprises a VP1, VP2 and / or VP3 protein encoded by a reference cap gene, such as a wild-type reference cap gene encoding a wild-type VP1, VP2 and / or VP3 protein having the same AAV serotype as the recombinant VP1, VP2 and / or VP3 protein, a control reference cap gene encoding a VP1, VP2 and / or VP3 protein that is identical to the recombinant VP1, VP2 and VP3 protein but lacks the heterologous epitope, a mutated wild-type reference cap gene encoding a substantially wild-type VP1, VP2 and / or VP3 protein having the same AAV serotype as the recombinant VP1, VP2 and / or VP3 protein but having a mutation (e.g., insertion, substitution, deletion), wherein the mutation preferably reduces the tropism of the wild-type VP1, VP2 and VP3 protein. In some embodiments, the reference capsid protein is a chimeric reference protein comprising 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 reference cap gene encodes a chimeric VP1, VP2 and / or VP3 protein.

[0086] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences from any organism, including immunoglobulin heavy chain constant region sequences. Unless otherwise indicated, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Fragments of a heavy chain include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has (from N-terminus to C-terminus) CDRs following the variable domain. H 1 domain, hinge, C H 2 domains and C H Functional fragments of the heavy chain include fragments that are capable of specifically recognizing an epitope (e.g., with a K in the micromolar, nanomolar, or picomolar range). D The heavy chain variable domain is encoded by a variable region nucleotide sequence, which generally comprises a V domain derived from a germline. H , D H and J H V of the segment reservoir H , D H and J H Segments. The sequences, locations and nomenclature for the V, D and J heavy chain segments of various organisms can be found in the IMGT database, which is accessible via the Internet at the URL "imgt.org" on the World Wide Web (www).

[0087] 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 monomeric or homodimeric immunoglobulin molecules comprising immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region that is incapable of binding to a light chain because the heavy chain constant region generally lacks a functional C H 1 domain. Accordingly, the terms "heavy chain-only antibody", "heavy chain-only antigen binding protein", "single domain antigen binding protein", "single domain binding protein" and the like encompass both: (i) a monomeric single domain antigen binding protein comprising one of the immunoglobulin-like chains comprising a heavy chain antigen binding protein lacking a functional C H 1 domain, or (ii) a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains, each of which comprises a variable domain that lacks a functional C H In various aspects, the homodimeric single-domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which comprises a variable domain that lacks a functional C H In addition, each immunoglobulin-like chain of the single-domain antigen-binding protein comprises an equivalent variable domain operably linked to an equivalent heavy chain constant region (C H ) gene sequence, the variable domain can be 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, wherein the heavy chain constant region (CH) gene sequence is included in the C of a heavy chain constant region gene (eg, IgG, IgA, IgE, IgD or a combination thereof). H 1 coding sequence (and optionally the hinge region). Single domain antigen binding proteins comprising a variable domain derived from a heavy chain gene segment may be referred to as a “V H Single domain antibodies" or "V H Single domain antigen binding proteins”, see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated by reference in its entirety. A single domain antigen binding protein comprising a variable domain derived from a light chain gene segment may be referred to as or “VL Single domain antigen binding proteins", see, e.g., U.S. Publication No. 20150289489, which is incorporated by reference in its entirety.

[0088] The phrase "light chain" includes immunoglobulin light chain sequences from any organism, and unless otherwise specified, includes human kappa (κ) and lambda (λ) light chains and VpreB and alternative light chains. Unless otherwise specified, the light chain variable domain generally includes three light chain CDRs and four framework (FR) regions. Generally, the full-length light chain includes a variable domain and a light chain constant region from the amino terminus to the carboxyl terminus, and the variable domain includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and a light chain constant region amino acid sequence. The light chain variable domain is encoded by a light chain variable region nucleotide sequence, which generally includes a light chain V derived from a light chain V and J gene segment reservoir present in the germline. L and light chain J L Gene segment. The sequences, positions and nomenclature of the light chain V and J gene segments of various organisms can be found in the IMGT database, which can be accessed via the Internet at the URL "imgt.org" on the World Wide Web (www). Light chains include, for example, those that do not selectively bind to the first epitope or the second epitope, which are selectively bound by the epitope binding protein in which they appear. The light chain also includes such a light chain that binds and recognizes or helps the heavy chain to bind and recognize, one or more epitopes selectively bound by the epitope binding protein in which they appear. The light chain also includes such a light chain that binds and recognizes or helps the heavy chain to bind and recognize, one or more epitopes selectively bound by the epitope binding protein in which they appear. Common light chains or universal light chains include those derived from human Vκ1-39Jκ5 genes or human Vκ3-20Jκ1 genes, and include somatic mutations thereof (e.g., affinity matured) forms.

[0089] As used herein, the phrase "operably connected" includes the physical juxtaposition of components or elements (e.g., in three-dimensional space) that interact directly or indirectly with each other, or otherwise coordinate with each other to participate in biological events, and the juxtaposition achieves or allows such interaction and / or coordination. As just one example, when a control sequence (e.g., an expression control sequence) in a nucleic acid is positioned relative to a coding sequence so that its presence or absence affects the expression and / or activity of the coding sequence, it is said to be "operably connected" to the coding sequence. In many embodiments, "operable connection" involves the covalent connection of related components or elements to each other. However, it will be readily understood by those skilled in the art that in some embodiments, covalent connection is not required to achieve effective operable connection. For example, in some embodiments, nucleic acid control sequences that are operably connected to the coding sequence they control are adjacent to the target nucleotide. Alternatively or additionally, in some embodiments, one or more such control sequences act in trans or at a distance to control the target coding sequence. In some embodiments, the term "expression control sequence" as used herein refers to a polynucleotide sequence that is necessary and / or sufficient for achieving the expression and processing of the coding sequence to which they are connected. In some embodiments, the expression control sequence may be or include appropriate transcription initiation, termination, promoter and / or enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and / or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are preferentially or exclusively active in a particular host cell or organism or type thereof. To cite just one example, in prokaryotes, control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, in many embodiments, control sequences typically include promoters, enhancers, and / or transcription termination sequences. One of ordinary skill in the art will understand from the context that in many embodiments, the term "control sequence" refers to components whose presence is necessary for expression and processing, and in some embodiments, includes components whose presence is favorable for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0090] 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 can be a reference and / or control virus for comparative studies. Recombinant capsid proteins include capsid proteins comprising heterologous epitopes, which can be inserted into and / or displayed 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 can simply be inserted between two given amino acids of the capsid protein. The insertion of amino acids can also be carried out together with the deletion of a given amino acid of the capsid protein at the insertion site, for example, 1 or more capsid protein amino acids are replaced by 5 or more heterologous amino acids).

[0091] The terms "multispecific binding molecules" and "bispecific binding molecules" and the like generally and respectively refer to binding molecules comprising at least two and only two non-identical binding components, wherein each binding component specifically binds to a different epitope - either on two different molecules (e.g., different epitopes on two different immunogens), or on the same molecule (e.g., different epitopes on the same immunogen). Generally, one of the binding components of the bispecific binding molecules herein specifically binds to a heterologous epitope displayed by a viral capsid protein, and the second binding component is specific for a protein expressed primarily and / or preferentially by a target cell, such as a cell surface marker, such as a T cell marker (e.g., CD3, CD28, etc.). Bispecific binding molecules can be prepared, for example, by combining binding components that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding binding components that recognize different epitopes (e.g., light chain or heavy chain variable sequences) can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, the same or different light chain constant regions, or the heavy chain constant region and light chain constant region, respectively, and such sequences can be expressed in cells as multispecific antigen-binding proteins in a form similar to a Fab structure, a scFab structure, a diabody structure, a scFv structure, a scFv-Fc structure, a scFv-zipper structure, a tetrameric structure similar to a conventional antibody including a cognate universal light chain, a tetrameric structure comprising a conventional bivalent antibody including a cognate universal light chain and / or additional binding components (e.g., scFv, scFv-zipper, scFab, etc.) attached to one or two heavy chains (e.g., at the N-terminus and / or C-terminus) and / or one or two light chains (e.g., at the N-terminus and / or C-terminus). Various formats of multispecific, and in particular bispecific, binding molecules are well known, see, for example, Brinkmann and Konterman (2017) mAbs 9: 182-212, which is incorporated herein by reference in its entirety.

[0092] An exemplary multispecific binding molecule has two heavy chains, each having a heavy chain CDR followed by (N-terminus to C-terminus) a C H 1 domain, hinge, C H 2 domains and C H3 domains, the immunoglobulin light chain either does not confer epitope binding specificity but can bind to each heavy chain (e.g., a common light chain), or can bind to each heavy chain and can bind to one or more epitopes bound by the heavy chain epitope binding region, or can bind to each heavy chain and enable one or both heavy chains to bind to one or two epitopes. In some embodiments, the multispecific binding molecule comprises: (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region, the first heavy chain constant region comprising a first CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, and (2) an immunoglobulin heavy chain variable domain, wherein the second immunoglobulin heavy chain variable domain is operably linked to a second heavy chain constant region, the second heavy chain constant region comprising a second CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the first heavy chain variable domain or the second heavy chain variable domain (with or without The first heavy chain constant region is associated with the second constant chain region in a manner that provides for easier separation of the multispecific binding protein, for example, wherein the first heavy chain constant region and the second heavy chain constant region form a knobs-into-hole (KIH) format, or wherein the second CH3 amino acid sequence comprises 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, which is incorporated herein by reference in its entirety).In some embodiments, the multispecific binding molecule comprises: (1) an immunoglobulin heavy chain variable domain operably linked to a first heavy chain constant region, the first heavy chain constant region comprising a first CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, and (2) an immunoglobulin heavy chain variable domain, wherein the second immunoglobulin heavy chain variable domain is operably linked to a second heavy chain constant region, the second heavy chain constant region comprising a second CH3 amino acid sequence of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the first heavy chain variable domain and the second heavy chain variable domain (with or without a cognate light chain ) binds to the same or different antigens, wherein the first heavy chain constant region or the second heavy chain constant region is modified to further comprise an additional binding domain (e.g., an scFv or Fv that binds to a heterologous epitope as described herein, e.g., wherein the additional binding domain is appended to the C-terminus or N-terminus of one or both heavy chains), and wherein the first heavy chain constant region is bound to the second constant chain region in a manner that provides for easier separation of the multispecific binding protein, e.g., wherein the first heavy chain constant region and the second heavy chain constant region form a knob-into-hole (KIH) format, or wherein 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 in which the second CH3 amino acid sequence comprises reduced or eliminated binding to protein A, the second CH3 amino acid sequence comprises an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). In one embodiment, the second CH3 amino acid sequence further comprises a Y96F modification (according to IMGT exon numbering; H436F according to EU). In another embodiment, the second CH3 amino acid sequence comprises both an H95R modification (according to IMGT exon numbering; H435R according to EU numbering) and a Y96F modification (according to IMGT exon numbering; H436F according to EU). In some embodiments, the second CH3 amino acid sequence is from a modified human IgG1 and further comprises a mutation selected from D16E, L18M, N44S, K52N, V57M and V82I (IMGT; D356E, L38M, N384S, K392N, V397M and V422I according to EU). In some embodiments, the second CH3 amino acid sequence is from a modified human IgG2 and further comprises a mutation selected from N44S, K52N and V82I (IMGT: N384S, K392N and V422I according to EU).In some embodiments, the second CH3 amino acid sequence is from a modified human IgG4 and further comprises a mutation selected from Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (IMGT: Q355R, N384S, K392N, V397M, R409K, E419Q and V422I according to EU). 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 above-mentioned modification types.

[0093] In various embodiments, the Fc domain is modified to have altered Fc receptor binding, which in turn affects effector function. In some embodiments, the modified heavy chain constant region (CH) comprising the Fc domain is chimeric. Like this, the chimeric CH region combines CH domains derived from more than one immunoglobulin isotype. For example, the chimeric CH region comprises part or all of the CH2 domain derived from human IgG1, human IgG2 or human IgG4 molecules, which is combined with part or all of the CH3 domain derived from human IgG1, human IgG2 or human IgG4 molecules. In some embodiments, the chimeric CH region contains a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues from positions 216 to 227 according to EU numbering; amino acid residues from positions 226 to 240 according to Kabat numbering), combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region (amino acid residues from positions 228 to 236 according to EU numbering; amino acid residues from positions 241 to 249 according to Kabat numbering). In some embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4, and amino acid residues derived from the lower hinge of human IgG2.

[0094] 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., of an antibody). For examples of proteins comprising chimeric CH regions and having altered effector functions, see WO2014022540, which is incorporated herein in its entirety.

[0095] The term "target cell" includes any cell in which expression of the target nucleotide is desired. Preferably, the target cell displays proteins such as receptors on its surface that allow the cell to be targeted with a retargeting ligand, as described below. Preferably, the target protein, such as a receptor, is specific to the target cell, for example, a "cell-specific marker", "cell-specific antigen", etc. The terms "cell-specific marker", "cell-specific antigen", "organ-specific marker", "tissue-specific marker", etc. refer to and include proteins whose expression is enriched by cells, tissues and / or organs for which it is a specific marker. In the context of protein expression, "enrichment" refers to and includes the following: cell / tissue / organ specific proteins are primarily, preferentially or exclusively expressed or overexpressed by cells / tissues / organs 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 a reservoir of cell / tissue / organ specific proteins is also provided. See, www.proteinatlas.org ; See also Uhlen et al. (2010) Nat. Biotech. 28:1248-50, which is incorporated herein by reference in its entirety.

[0096] The terms "transduction" or "infection" and the like refer to the introduction of nucleic acids into target cells by viral vectors. The term efficiency related to transduction and the like, for example, "transduction efficiency" refers to the fraction (e.g., percentage) of cells expressing the target nucleotide after incubation with a set number of viral vectors containing the target nucleotide. Well-known methods for determining transduction efficiency include fluorescence-activated cell sorting of cells transduced with a fluorescent reporter gene, PCR for expression of the target nucleotide, and the like.

[0097] As used herein, the term "wild type" includes entities having structures and / or activities as found in nature in a "normal" (in contrast to mutant, diseased, altered, etc.) state or background. One of ordinary skill in the art will appreciate that a wild-type viral vector, such as a wild-type capsid protein, can be used as a reference viral vector in comparative studies. Generally, a reference viral capsid protein / capsid / vector is equivalent to a test viral capsid protein / capsid / vector, but has a change for the effect to be tested. For example, in order to determine, for example, the effect of inserting a heterologous epitope into a test viral vector on transduction efficiency, the transduction efficiency of the test viral vector (in the absence or presence of an appropriate multispecific binding molecule) can be compared with the transduction efficiency of a reference viral vector (in the absence or presence of an appropriate multispecific binding molecule, if desired), the reference viral vector being equivalent to the test viral vector in each case (e.g., additional mutations, target nucleotides, number of viral vectors and target cells, etc.), except for the presence of a heterologous epitope.

[0098] Recombinant virus capsid protein, virus vector and nucleic acid

[0099] In some embodiments, the recombinant viral capsid protein as described herein is an Ad capsid protein, such as a capsid protein of an Ad serotype selected from Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, and Ad7. In some embodiments, the recombinant viral capsid protein is derived from an Ad2 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from an Ad5 capsid gene. In some embodiments, the recombinant Ad viral capsid protein as described herein is contained in a fiber protein domain, such as a heterologous epitope at the carboxyl terminus of the fiber protein, a fiber node, and / or the HI loop of the fiber node.

[0100] In some embodiments, the recombinant viral capsid protein described herein is derived from an adeno-associated virus (AAV) capsid gene, for example, a genetically modified capsid protein of an AAV serotype selected from 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, for example, a genetically modified AAV2VP1 capsid protein, and the amino acid sequence of the wild type thereof is shown in SEQ ID NO: 1, respectively. In some embodiments, the recombinant viral capsid protein is derived from an AAV8 capsid gene, for example, a genetically modified AAV8VP1 capsid protein, and the amino acid sequence of the wild type thereof is shown in SEQ ID NO: 21. In some embodiments, the recombinant viral capsid protein is derived from an AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein, and its wild-type amino acid sequence is shown in SEQ ID NO: 5. In some embodiments, the recombinant viral capsid protein is derived from an 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.

[0101] Generally, the recombinant viral capsid protein as described herein comprises a heterologous epitope inserted into and / or displayed by the capsid protein, so that the heterologous epitope reduces and / or eliminates the natural tropism of the capsid protein or the capsid comprising it. In some embodiments, the heterologous epitope is inserted into a capsid protein region related to the natural tropism of the wild-type reference capsid protein, such as a capsid protein region related to a cell receptor. In some embodiments, the heterologous epitope is inserted into and / or displayed by the nodular domain of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and / or displayed by the HI loop of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted after the amino acid position selected from the following: G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, Q585 of AAV6 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the heterologous epitope is inserted and / or displayed between amino acids N587 and R588 of the AAV2VP1 capsid. 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 as shown in SEQ ID NO:2. 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 as shown in 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 as shown in 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 as shown in 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 as shown in SEQ ID NO:27. Additional suitable insertion sites identified through use of 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, 1261, 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 as described herein can be an AAV2 capsid protein comprising a heterologous epitope inserted into a position selected from the group consisting of: I-1, I-34, I-138, I-139, I-161, I261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I 591, I-657, I-664, I-713, I-716, and combinations thereof. Additional 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 as described herein can be an AAV2 capsid protein, which comprises a heterologous epitope inserted into a position selected from the following: I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5) and combinations thereof.

[0102] The nomenclature I-### used herein refers to an insertion site with the ### designated amino acid number relative to the VP1 protein of the AAV capsid protein, however, such an insertion may be located directly at the N-terminus or C-terminus of the given amino acid within 5 amino acids, preferably at the N-terminus or C-terminus of one amino acid in a sequence within 3, more preferably 2, and especially 1 amino acid of the given amino acid, preferably at the C-terminus. In addition, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene, and by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the reference AAV capsid gene, the corresponding positions (and mutations thereof) can be easily identified for the VP2 and VP3 capsid proteins encoded by the capsid gene.

[0103] Accordingly, since the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons, an insertion in the corresponding position of the encoding nucleic acid of one of these sites of the cap gene results in an insertion into VP1, VP2 and / or VP3. Thus, for AAV2, for example, according to this nomenclature, an insertion between amino acids 1 and 138 is inserted only into VP1, an insertion between 138 and 203 is inserted into VP1 and VP2, and an insertion between 203 and the C-terminus is inserted into VP1, VP2 and VP3, which is of course also the case with respect to insertion site 1-587. Thus, the present invention encompasses structural genes of AAV having corresponding insertions in the VP1, VP2 and / or VP3 proteins.

[0104] In addition, due to the large members of at least large segments of highly conserved and closely related family members, corresponding insertion sites for AAVs other than the listed AAVs can be identified by performing amino acid alignments or by comparing capsid structures. For exemplary alignments of different AAV capsid proteins, see, e.g., Rutledge et al. (1998) J. Virol. 72:309-19 and U.S. Pat. No. 9,624,274, each of which is incorporated herein by reference in its entirety.

[0105] In some compositions consisting of recombinant viral capsids disclosed herein (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 contain an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions consisting of recombinant viral capsids disclosed herein (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 contain c-myc. In some compositions consisting of recombinant viral capsids disclosed herein (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 at the I-447 site, wherein the heterologous epitope does not contain L14 or HA.

[0106] 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, wherein the heterologous epitope comprises an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions comprising a recombinant viral capsid (e.g., further comprising a multispecific binding molecule) disclosed herein, the viral capsid is a VP1 capsid, the heterologous epitope comprises c-myc, and the heterologous epitope is inserted between T448 and N449 or between N587 and R588. In some compositions comprising a recombinant viral capsid (e.g., further comprising a multispecific binding molecule) disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein having a heterologous epitope inserted at the I-447 site, wherein 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, wherein the heterologous epitope comprises c-myc. U.S. Pat. No. 9,624,274 describes I-453 of the AAV capsid protein as a suitable insertion site for a heterologous epitope.

[0107] In some embodiments, the insertion (display) of a heterologous epitope eliminates the natural tropism of the viral vector, for example, in the absence of an appropriate multispecific binding molecule, transduction of cells and / or target cells that are naturally permissive to infection by a wild-type reference viral vector is undetectable. In some embodiments, for example, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector compared to transduction of cells that are naturally permissive to infection by a wild-type reference viral vector. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 10%. In some embodiments, the insertion (display) 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 a heterologous epitope reduces the natural tropism of the viral vector by at least 90%. In these embodiments in which the insertion (display) of a heterologous epitope does not eliminate the natural tropism of the recombinant viral capsid, the natural tropism of such recombinant viral capsid can be eliminated by a second and different mutation. For example, in one embodiment, a recombinant viral capsid protein as described herein can be derived from an AAV9 capsid gene, comprising a heterologous epitope, and can also include a mutation, such as a W503A mutation.

[0108] This off-targeting of the virus from its natural host cell is important, especially if systemic or local or localized administration of the viral vector is contemplated, because uptake of the viral vector by the natural host cells limits the effective dose of the virus. In the case of AAV2 and AAV6, HSPGs are reported to be the major receptors for viral uptake in a large number of cells, especially hepatocytes. For AAV2, HSPG binding activity depends on a set of 5 basic amino acids: R484, R487, R585, R588 and K532 (Kern et al., (2003) J Virol. 77 (20): 11072-81). Recently, it was reported that the amino acid substitution K531E of lysine to glutamate resulted in suppression of the ability of AAV6 to bind heparin or HSPG ((Wu et al., 2006) J. of Virology 80 (22): 11393-11397). Accordingly, in the case where HSPG is the main receptor for viral vector binding to HSPG, a preferred point mutation is one that reduces the transduction activity of the viral vector mediated by the natural receptor with respect to a given target cell by at least 50%, preferably at least 80%, and especially at least 95%.

[0109] Thus, further mutations preferred for viral vectors that bind to HSPGs are mutations that deplete or replace basic amino acids such as R, K or H, preferably R or K, involved in HSPG binding of the respective virus by non-basic amino acids, such as A, D, G, Q, S and T, preferably A, or amino acids present at corresponding positions of different but highly conserved AAV serotypes that lack such basic amino acids at this position. Thus, preferred amino acid substitutions are R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A or R588T, especially R585A and / or R588A for AAV2, and K531A or K531E for AAV6. A particularly preferred embodiment of the present invention is such a capsid protein mutant of AAV2, which additionally contains two point mutations R585A and R588A, because these two point mutations are sufficient to largely eliminate the HSPG binding activity. These point mutations enable efficient off-targeting from HSPG-expressing cells which, for targeting purposes, increases the specificity of the respective mutant virus for its new target cells.

[0110] One embodiment of the present invention is a multimeric structure comprising a recombinant viral capsid protein 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 comprising heterologous epitopes as described herein. They can form regular viral capsids (empty viral particles) or viral vectors (capsids encapsulating target nucleotides). The formation of a viral vector capable of packaging a viral genome is a highly preferred feature of the recombinant viral capsid described herein as a viral vector.

[0111] One embodiment of the present invention is a nucleic acid encoding a capsid protein as described above. The nucleic acid is preferably a vector comprising the nucleic acid sequence claimed for protection. Nucleic acids, especially vectors, are necessary for recombinant expression of the capsid protein of the present invention.

[0112] A further embodiment of the present invention is the use of at least one recombinant viral capsid protein and / or nucleic acid encoding the same, preferably at least one multimeric structure (eg, viral vector), for the manufacture of a gene transfer vector and for use as a gene transfer vector.

[0113] Heterologous epitope

[0114] Typically, the recombinant viral capsid protein and / or the viral vector comprising the recombinant viral capsid comprises a heterologous epitope that enables the viral vector to be retargeted, for example, via a multispecific binding molecule. In some embodiments, the heterologous epitope is a B cell epitope, for example, between about 1 amino acid and about 35 amino acids in length, and forms a binding pair with an antibody complementary site, such as an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises an affinity tag.

[0115] A large number of tags are known in the art. (See, for example, 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 systems" Applied Microbiology and Biotechnology 60: 523-533, and references therein). Affinity tags include, but are not limited to, binding to immobilized divalent cations (e.g., Ni 2+), a polyhistidine tag (e.g., a His-6, His-8, or His-10 tag) that binds to immobilized avidin (e.g., on a polypeptide sequence that is biotinylated 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 a domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to a corresponding antibody), a FLASH tag (a high affinity tag coupled to a specific arsenic-based moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), a protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, a 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 from Covalys ( www.covalys.com ) SNAP tags are commercially available. In some embodiments, the heterologous epitopes disclosed herein comprise affinity tags that are recognized only by the antibody paratope. In some embodiments, the heterologous epitopes disclosed herein comprise affinity tags that are recognized by both the antibody paratope and other specific binding pairs.

[0116] 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 does not bind to a metal ion, such as Ni. 2+ 、Co 2+ , Cu 2+ 、Zn 2+ , Fe 3+ In some embodiments, the heterologous epitope is not a polypeptide selected from streptavidin, Strep II, HA, L14, 4C-RGD, LH and protein A.

[0117] In some embodiments, the affinity tag is selected from 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.

[0118] In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises an amino acid sequence EQKLISEEDL (as shown in SEQ ID NO: 6) flanked by and / or operably linked to at least 5 contiguous amino acids of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises an EQKLISEEDL (as shown in SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid protein as described herein comprises an amino acid sequence as shown in SEQ ID NO: 2. In some embodiments, the recombinant viral capsid protein as described herein comprises an amino acid sequence as shown in SEQ ID NO: 4. In some embodiments, the recombinant viral capsid, a viral vector comprising a recombinant viral capsid, and / or a composition comprising a recombinant viral capsid comprises an amino acid sequence encoded by a nucleic acid sequence as shown in 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 as shown in 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 as shown in SEQ ID NO: 27.

[0119] 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 a first linker, an affinity tag, and a second linker from the N-terminus to the C-terminus. In some embodiments, the first linker and the second linker are each independently at least one amino acid in length. In some embodiments, the first linker and the second linker are identical.

[0120] Generally, heterologous epitopes, such as affinity tags, as described herein, alone 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 (alone or in combination with one or more linkers) is at least 5 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 6 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 7 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 8 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 9 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 10 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 11 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 12 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 13 amino acids in length. In some embodiments, a heterologous epitope (alone or in combination with one or more linkers) is 14 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 15 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 16 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 17 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 18 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 19 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 20 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 21 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 22 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 23 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 24 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 25 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 26 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 27 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 28 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 29 amino acids in length.In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 30 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 31 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 32 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 33 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 34 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 35 amino acids in length.

[0121] Retargeting part

[0122] In the absence of a multispecific binding molecule, specifically a multispecific binding molecule comprising: (i) an antibody paratope that specifically binds an epitope and (ii) a retargeting ligand that specifically binds a receptor, which can be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell, a viral vector as described herein has reduced to eliminated transduction capacity. Accordingly, a multispecific binding molecule comprising (i) an antibody paratope that specifically binds an epitope and (ii) a retargeting ligand that specifically binds a receptor retargets the viral vector. Such "retargeting" or "redirection" can include situations where a wild-type viral vector targets several cells within a tissue and / or several organs within an organism, the broad targeting of the tissue or organ is reduced to eliminated by the insertion of a heterologous epitope, and retargeting to a more specific cell in a tissue or a more specific organ in an organism is achieved with a multispecific binding molecule. Such retargeting or redirection can also include situations where a wild-type viral vector targets a tissue, the targeting of the tissue is reduced to eliminated by the insertion of a heterologous epitope, and retargeting to a completely different tissue is achieved with a multispecific binding molecule. The antibody paratope as described herein generally contains at least a complementary determining region (CDR) that specifically recognizes a heterologous epitope, such as a CDR3 region of a heavy chain and / or light chain variable domain. In some embodiments, the multispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a heterologous epitope. For example, the multispecific 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 binding molecule may comprise an Fv region, for example, the multispecific binding molecule may comprise an scFv, which comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the multispecific binding molecule as described herein comprises an antibody paratope that specifically binds to c-myc.

[0123] In some embodiments, a multispecific binding molecule as described herein comprises an antibody paratope that specifically binds to c-myc, the paratope comprising a scFv, a heavy chain and light chain variable domain of a scFv, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences, encoded by a nucleic acid sequence as set forth in SEQ ID NO: 28, e.g., a scFv comprising an amino acid sequence as set forth in SEQ ID NO: 37. In some embodiments, a multispecific binding molecule as described herein comprises an Fv or scFv encoded by a nucleic acid sequence as set forth in SEQ ID NO: 28.

[0124] Accordingly, the present invention includes antibodies, antigen-binding fragments of antibodies, and multispecific binding proteins that specifically bind to c-myc, wherein the antibodies, antibody fragments, and multispecific binding proteins comprise a paratope 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 comprise a paratope that specifically binds to c-Myc, wherein the paratope comprises a heavy chain complementary determining region 1 (HCDR1) comprising SEQ ID NO: 31, a HCDR2 comprising SEQ ID NO: 32, a HCDR3 comprising SEQ ID NO: 33, a light chain complementary determining region 1 (LCDR1) comprising SEQ ID NO: 34, a LCDR2 comprising SEQ ID NO: 35, and a LCDR3 comprising SEQ ID NO: 36.

[0125] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising a HCVR comprising the amino acid sequence as set forth in 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.

[0126] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising a LCVR comprising the amino acid sequence as set forth in 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.

[0127] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising a HCVR and LCVR amino acid sequence pair (HCVR / LCVR) comprising the HCVR amino acid sequence as set forth in SEQ ID NO: 29 paired with the LCVR amino acid sequence as set forth in SEQ ID NO: 30. In some embodiments, the HCVR / LCVR amino acid sequence pair is selected from SEQ ID NO: 29 / 30.

[0128] The present invention provides antibodies, antigen-binding fragments thereof and / or multispecific binding proteins comprising a paratope, wherein the paratope comprises a heavy chain CDR1 (HCDR1), wherein the HCDR1 comprises the amino acid sequence as shown in 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.

[0129] The present invention provides antibodies, antigen-binding fragments thereof and / or multispecific binding proteins comprising a paratope, wherein the paratope comprises a heavy chain CDR2 (HCDR2), wherein the HCDR2 comprises the amino acid sequence as shown in 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.

[0130] The present invention provides antibodies, antigen-binding fragments thereof and / or multispecific binding proteins comprising a paratope, wherein the paratope comprises a heavy chain CDR3 (HCDR3), wherein the HCDR3 comprises the amino acid sequence as shown in 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.

[0131] The present invention provides antibodies, antigen-binding fragments thereof and / or multispecific binding proteins comprising a paratope, wherein the paratope comprises a light chain CDR1 (LCDR1), wherein the LCDR1 comprises the amino acid sequence as shown in 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.

[0132] The present invention provides antibodies, antigen-binding fragments thereof and / or multispecific binding proteins comprising a paratope, wherein the paratope comprises a light chain CDR2 (LCDR2), wherein the LCDR2 comprises the amino acid sequence as shown in 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.

[0133] The present invention provides antibodies, antigen-binding fragments thereof and / or multispecific binding proteins comprising a paratope, wherein the paratope comprises a light chain CDR3 (LCDR3), wherein the LCDR3 comprises the amino acid sequence as shown in 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.

[0134] The present invention provides antibodies, antigen-binding fragments thereof, and / or multispecific binding proteins comprising a paratope comprising a HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising the HCDR3 amino acids as set forth in SEQ ID NO: 33 paired with the LCDR3 amino acid sequence as set forth in SEQ ID NO: 36. In some embodiments, the HCDR3 / LCDR3 amino acid sequence pair is as set forth in SEQ ID NO: 33 / 36.

[0135] 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 a nucleotide sequence as shown in SEQ ID NO: 28. In certain embodiments, the set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences is shown in SEQ ID NO: 31-32-33-34-35-36.

[0136] In a related embodiment, the invention provides an antibody, antigen-binding fragment thereof, and / or multispecific binding protein comprising a paratope comprising a set of six CDRs contained within a set of HCVR / LCVR amino acid sequence pairs as set forth in SEQ ID NO: 29 / 30 (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3). 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 a given HCVR and / or LCVR amino acid sequence 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, the Kabat definition is based on sequence variability, the Chothia definition is based on the position 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 can also be used to identify CDR sequences within antibodies.

[0137] The present invention also provides a nucleic acid molecule encoding an anti-myc antibody or a portion thereof.

[0138] In addition to the paratope (e.g., antibody or a portion thereof) that specifically binds to a heterologous epitope inserted into / displayed by a recombinant viral capsid protein, a multispecific binding molecule as described herein also comprises a retargeting ligand. In some embodiments, the retargeting ligand binds to a protein expressed on the surface of a cell, such as a cell surface protein on a (human) eukaryotic cell, such as a target cell. There are a large number of suitable cell surface proteins, such as cell surface receptors, that can be targeted by a retargeting ligand, and a retargeting ligand, such as an antibody or a portion thereof, is already available. Such structures include, but are not limited to, 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 factor, endothelial growth factor, epidermal growth factor, fibroblast growth factor, glial derived neurotrophic factor, glial growth factor, gro-β / mip 2. Hepatocyte growth factor, insulin-like growth factor, interferon (α-IFN, β-IFN, γIFN, shared IFN), interleukin (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte growth factor, leukemia inhibitory factor, macrophage / monocyte chemotactic activating factor, nerve growth factor, neutrophil activation protein 2, platelet-derived growth factor, stem cell factor, transforming growth factor, tumor necrosis factor, vascular endothelial growth factor, lipoprotein including other or other type 1 Transmembrane receptors (e.g., PRLR, G protein-coupled receptors such as GCGR, ion channels such as Nav1.7, ASIC1 or ASIC2; cell adhesion molecules; transport molecules for metabolites such as amino acids; antigen receptors for B and T lymphocytes (e.g., B cell receptors and related proteins (e.g., CD19, CD20, etc.) and T cell receptors and related proteins (e.g., CD3, CD4, CD8, etc.); tetraspanins (e.g., CD63). The recombinant viral capsids described herein allow specific infection of cell types by using multispecific binding molecules that contain heavy targeting ligands that bind to surface antigens of differentiated cells as targets of the viral vector complex.

[0139] In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) hepatocytes, i.e., a liver-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) brain cells, a brain cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) hematopoietic cells, i.e., a hematopoietic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) T cells, i.e., a T cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) B cells, i.e., a B cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) dendritic cells, i.e., a dendritic cell-specific marker. In some embodiments, the retargeting ligand binds to a protein expressed primarily (e.g., exclusively) by (human) macrophages, i.e., a macrophage-specific marker. In some embodiments, the retargeting ligand binds to a protein that is primarily (e.g., exclusively) expressed by (human) NK cells, i.e., an NK cell-specific marker. In some embodiments, the retargeting ligand binds to a protein that is primarily (e.g., exclusively) expressed by (human) kidney cells, i.e., a kidney-specific marker. In some embodiments, the retargeting ligand binds to a receptor that is primarily (e.g., exclusively) expressed by (human) pancreatic cells, i.e., a pancreatic-specific marker. In some embodiments, the retargeting ligand binds to a receptor that is primarily (e.g., exclusively) expressed by (human) intestinal cells, i.e., an intestinal-specific marker. In some embodiments, the retargeting ligand binds to a protein that is primarily (e.g., exclusively) expressed by (human) cancer cells, i.e., a tumor-associated antigen. In some embodiments, the retargeting ligand binds to a protein that is primarily (e.g., exclusively) expressed by (human) cells infected with heterologous pathogens. Proteins that (1) are specifically expressed by cells / tissues / organs, or whose expression is enriched in cells / tissues / organs, and (2) are recognized by antigen-binding proteins that can be used as retargeting ligands as described herein are well known and can also be found in 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 and non-limiting organ-specific markers, and cells / tissues / organs expressing such markers, for which antigen-binding proteins useful as retargeting ligands can be obtained.

[0140] Table 1: Exemplary tissue-specific markers

[0141]

[0142]

[0143] In some embodiments, the retargeting ligand binds to a receptor expressed by (human) liver cells, such as an asialoglycoprotein receptor, such as 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, such as CD3, such as 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, such as integrins. In some embodiments, the retargeting ligand binds to a receptor expressed by (human) cancer cells, such as tumor-associated antigens, such as E6 and E7. In some embodiments, the retargeting ligand binds to a human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds to human ENTPD3.

[0144] In some embodiments, the retargeting ligand binds to a tumor-associated antigen expressed by a tumor cell. Non-limiting examples of specific tumor-associated antigens include, for example, lipophilin, AIM-2, ALDH1A1, α-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, β-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, cell Cyclin-A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, Glypican-3, GnTV, gp100 / Pme117, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HL A-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Rα2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammillary bead Protein 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, class I myosin, 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-RARα 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-βRII, 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 antigen (in lymphoma and other solid tumors), ErbB receptor, Melan A [MART1], gp 100, tyrosinase, TRP-1 / gp 75 and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder cancer, head and neck cancer and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); mucin [MUC-1] (in breast cancer, pancreatic cancer, colon cancer and prostate cancer); prostate specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon cancer, breast cancer and gastrointestinal cancer), as well as such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3TO 7. LAGE-1, NY-ESO-1 / LAGE-2, NA-88, GnTV, TRP2-INT2. In some embodiments, the tumor-associated antigen is ErbB2 / Her2. In some embodiments, the tumor-associated antigen is E6 and / or E7.

[0145] 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.

[0146] 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 case of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first antigen-binding domain and a second antigen-binding domain, the CDR of the first antigen-binding domain can be named with the prefix "A1", and the CDR of the second antigen-binding domain can be named with the prefix "A2". Therefore, the CDR of the first antigen-binding domain can be referred to as A1-HCDR1, A1-HCDR2, and A1-HCDR3 in this article; and the CDR of the second antigen-binding domain can be referred to as A2-HCDR1, A2-HCDR2, and A2-HCDR3 in this article.

[0147] The first antigen-binding domain and the second antigen-binding domain can be directly or indirectly connected to each other to form the bispecific antigen binding molecules of the present invention.Alternately, the first antigen-binding domain and the second antigen-binding domain can be connected to the multimeric domain separated separately.The combination of a multimeric domain and another multimeric domain promotes the combination between two antigen-binding domains, thereby forming a bispecific antigen binding molecule.As used herein, "multimeric domain" is any macromolecule, protein, polypeptide, peptide or amino acid, which has the ability to be combined with the second multimeric domain of the same or similar structure or structure.For example, the multimeric domain can be a polypeptide comprising an immunoglobulin CH3 domain.The non-limiting example of multimeric components is the Fc part of an immunoglobulin (comprising CH2-CH3 domain), for example, selected from the Fc domain of any allotype IgG in isotype IgG1, IgG2, IgG3 and IgG4 and each isotype group.

[0148] The bispecific antigen binding molecules of the present invention generally comprise two multimeric domains, such as two Fc domains, each of which is individually part of a separate antibody heavy chain. The first multimeric domain and the second multimeric domain can have the same IgG isotype, such as IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first multimeric domain and the second multimeric domain can have different IgG isotypes, such as IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0149] In certain embodiments, the multimeric domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length, which comprises at least one cysteine ​​residue. In other embodiments, the multimeric domain is a cysteine ​​residue or a short peptide containing cysteine. Other multimeric domains include peptides or polypeptides comprising or consisting of: a leucine zipper, a spiral loop motif, or a coiled coil motif.

[0150] Any bispecific antibody form or technology can be used to prepare bispecific antigen binding molecules of the present invention.For example, an antibody or fragment thereof with a first antigen binding specificity can be functionally connected (for example, by chemical coupling, genetic fusion, non-covalent binding or other means) with one or more other molecular entities, for example, another antibody or antibody fragment with a second antigen binding specificity to produce a bispecific antigen binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domains (DVD)-Ig, Quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) antibodies, leucine zippers, Duobody, IgG1 / IgG2, dual-acting Fab (DAF)-IgG, and Mab2 bispecific formats (for a review 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).

[0151] The present invention also includes a bispecific antigen binding molecule comprising a first Ig CH3 domain and a second Ig CH3 domain, wherein the first Ig CH3 domain and the second Ig CH3 domain differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds protein A, and the second Ig CH3 domain contains a mutation that reduces or eliminates protein A binding, such as an H95R modification (according to IMGT exon numbering; H435R according to EU numbering). The second CH3 may also contain a Y96F modification (according to IMGT; Y436F according to EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M and V82I (by IMGT; D356E, L358M, N384S, K392N, V397M and V422I by EU) in the case of IgG1 antibodies; N44S, K52N and V82I (IMGT; N384S, K392N and V422I by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) in the case of IgG4 antibodies; see, e.g., WO 2010 / 151792.

[0152] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may include part or all of a CH2 sequence derived from a human IgG1, human IgG2, or human IgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgG1, human IgG2, or human IgG4. A chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may include an "upper hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region, which is combined with a "lower hinge" sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that may be included in any antigen-binding molecule described herein includes from N-terminus to C-terminus: [IgG4CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG4CH3]. Another example of a chimeric Fc domain that can be included in any antigen binding molecule described herein comprises, from N-terminus to C-terminus: [IgG1CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4CH2]-[IgG1CH3]. These and other examples of chimeric Fc domains that can be included in any antigen binding molecule of the present invention are described in PCT Application No. WO2014 / 022540, which is incorporated by reference in its entirety. Chimeric Fc domains and variants thereof having these general structural arrangements can have altered Fc receptor binding, which in turn affects Fc effector functions.

[0153] Methods of use and preparation

[0154] A further embodiment of the recombinant viral capsid protein described herein is its use for delivering a target nucleotide, such as a reporter gene or a therapeutic gene, to a target cell. Generally, the target nucleotide can be a transfer plasmid, which can generally include 5' and 3' reverse terminal repeat (ITR) sequences flanking a reporter gene or a therapeutic gene (when contained in an AAV vector, it can be under the control of a viral or non-viral promoter). In one embodiment, the target nucleotide is a transfer plasmid comprising the following from 5' to 3': 5' ITR, a promoter, a gene (e.g., a reporter gene and / or a therapeutic gene) and 3' ITR.

[0155] Non-limiting examples of useful promoters include, for example, cytomegalovirus (CMV) promoters, spleen focus forming virus (SFFV) promoters, elongation factor 1 alpha (EF1a) promoters (1.2 kb EF1a promoters or 0.2 kb EF1a promoters), chimeric EF1a / IF4-promoters and phosphoglycerate kinase (PGK) promoters. Internal enhancers may also be present in viral constructs to increase the expression of the gene of interest. For example, CMV enhancers (Karasuyama et al. 1989. J. Exp. Med. 169: 13, which are incorporated herein by reference in their entirety) may be used. In certain embodiments, CMV enhancers may be used in combination with chicken 13-actin promoters.

[0156] Various reporter genes (or detectable parts) can be encapsulated in the polymer structure comprising recombinant virus capsid protein described herein.Exemplary reporter genes include, for example, beta-galactosidase (encoded 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 its combination.The method described herein confirms the construction of the targeting vector of the reporter gene encoding green fluorescent protein, however, after reading the present disclosure, it will be understood by the technician that non-human animals described herein can be generated in the absence of reporter genes or with any reporter gene known in the art.

[0157] Various therapeutic genes can also be encapsulated in a polymeric structure comprising the recombinant viral capsid protein described herein, for example as part of a transfer vector. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or a portion thereof, antisense RNA, siRNA, shRNA, etc.

[0158] A further embodiment of the present invention is a method for preparing a recombinant capsid protein, the method comprising the following steps:

[0159] a) expressing a nucleic acid encoding a recombinant capsid protein under appropriate conditions, and

[0160] b) isolating the expressed capsid protein of step a).

[0161] A further embodiment of the present invention is a method for changing the tropism of a virus, the method comprising the following steps: (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 a heterologous epitope, and / or (b) culturing a packaging cell under conditions sufficient for producing a viral vector, wherein the packaging cell comprises the nucleotide sequence. A further embodiment of the present invention is a method for displaying a heterologous epitope on the surface of a capsid protein, the method comprising the following steps: a) expressing a nucleic acid according to the present invention under suitable conditions, and b) isolating the capsid protein expressed in step a).

[0162] In some embodiments, the packaging cell further comprises a helper plasmid and / or a transfer plasmid comprising the target nucleotide. In some embodiments, the method further comprises isolating a self-complemented adeno-associated viral vector from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral vector from the cell lysate. In some embodiments, the method further comprises (a) removing cell 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).

[0163] Packaging cells useful for producing the viral vectors described herein include, for example, animal cells that are permissive for the virus, or cells modified to be permissive for the virus; or packaging cell constructs, for example, using a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines for producing viral vectors described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells containing SV40 large T antigen (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raj i (CCL-86), glioblastoma-astrocytoma epithelioid cell line U87-MG (HTB-14), T lymphoma cells 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 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, CAP-T cells, etc.

[0164] L929 cells, the FLY virus packaging cell system outlined in Cosset et al. (1995) J Virol 69, 7430-7436, NSO (murine 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., High 5)), or bacterial cells (including but not limited to E. coli).

[0165] For additional packaging cells and systems, packaging techniques and vectors for packaging nucleic acid genomes into pseudotyped viral vectors, see, for example, Polo et al., Proc Natl Acad Sci USA, (1999) 96: 4598-4603. Packaging methods include the use of packaging cells that permanently express viral components, or transient transfection of cells with plasmids.

[0166] Further embodiments include methods of redirecting viruses and / or delivering reporter genes or therapeutic genes to target cells, the methods including methods for transducing cells in vitro or in vivo, the methods comprising the steps of contacting the target cells with a combination of a viral vector comprising a capsid and a multispecific binding molecule, the capsid comprising a recombinant viral capsid displaying a heterologous epitope, wherein the multispecific binding molecule comprises i) an antibody paratope that specifically binds to the epitope, and (ii) a retargeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the compositions described herein or the methods described herein combine recombinant viral vectors and multispecific binding molecules at a molecule: molecule ratio that restores the transduction efficiency of the viral vector to that of a wild-type control viral vector. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) ranges from 1:0.5 to 1:100. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) ranges from 1:4 to 1:20. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) ranges from 1:8 to 1:15. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is 1:4. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is 1:8. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is 1:15. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is 1:20. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is less than 1:100. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is less than 1:50. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is less than 1:20. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is less than 1:15. In some embodiments, the ratio of the recombinant viral vector to the multispecific binding molecule (molecule: molecule) is less than 1:10.

[0167] In some embodiments, the target cell is in vitro. In other embodiments, the target cell is in a subject, such as a human.

[0168] Target cells

[0169] Using recombinant viral vectors as disclosed herein, a wide variety of cells can be targeted for delivery of a target nucleotide. Target cells are generally selected based on the target nucleotide and the desired effect.

[0170] In some embodiments, the nucleotide of interest can be delivered to enable a target cell to produce a protein that complements a defect in an organism, such as an enzyme defect or an immune defect, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that normally produce a protein in an animal are targeted. In other embodiments, cells in an area where the protein would be most beneficial are targeted.

[0171] In other embodiments, the target nucleotide, such as a gene encoding siRNA, can inhibit the expression of a specific gene in a target cell. The target nucleotide can, for example, inhibit the expression of a gene involved in the life cycle of a pathogen. Thus, cells susceptible to or infected by a pathogen can be targeted. In other embodiments, the target nucleotide can inhibit the expression of a gene responsible for producing a toxin in a target cell.

[0172] In other embodiments, the nucleotide of interest may encode a toxic protein that kills the cell in which it is expressed. In this case, tumor cells or other unwanted cells may be targeted.

[0173] In yet other embodiments, a nucleotide of interest encoding a protein to be collected (eg, a therapeutic protein) can be used and targeted to cells capable of producing and secreting the protein.

[0174] Once the specific target cell group where the target nucleotide expression is needed is identified, a target receptor specifically expressed on the target cell group is selected. The target receptor can be expressed exclusively on the cell group, or expressed to a greater extent on the cell group than on other cell groups. The more specific the expression, the more specifically the target cell can be targeted. Depending on the context, the required specific amount of the marker (and therefore the gene delivery) can vary. For example, in order to introduce a toxic gene, high specificity is most preferred to avoid killing non-targeted cells. For the expression of a protein for harvest or the expression of a secretory product where an overall effect is needed, less marker specificity may be required.

[0175] As discussed 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 a binding partner, such as a ligand, for the target receptor is already known, it can be used as an affinity molecule. However, if the binding molecule is unknown, antibodies to the target receptor can be generated using standard procedures. Then, the antibody can be used as a retargeting ligand as part of a multispecific binding molecule.

[0176] Thus, target cells can be selected based on a variety of factors including, for example, (1) the specific application (e.g., therapy, expression of a protein to be harvested, and conferring disease resistance) and (2) expression of a marker with a desired specific amount.

[0177] The target cell is not limited in any way and includes germline cells and cell lines and somatic cells and cell lines. The target cell can be a stem cell derived from any origin. When the target cell is a germline cell, the target cell is preferably selected from a single cell embryo and an embryonic stem cell (ES).

[0178] Pharmaceutical compositions, dosage forms and administration

[0179] A further embodiment provides a medicament comprising at least one recombinant viral capsid protein according to the invention and an appropriate multispecific binding molecule and / or a nucleic acid according to the invention, preferably at least one multimeric structure according to the invention. Preferably, such medicament is a useful gene transfer vector.

[0180] Also disclosed herein are pharmaceutical compositions comprising the viral vectors described herein and a pharmaceutically acceptable carrier and / or excipient. In addition, disclosed herein are pharmaceutical dosage forms comprising the viral vectors described herein.

[0181] As discussed herein, the viral vectors described herein can be used in a variety of therapeutic applications (in vivo and ex vivo), and as research tools.

[0182] Pharmaceutical compositions based on 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 by, for example, injection, inhalation, or isolation (by mouth or nose), or by oral, buccal, parenteral or rectal administration, or by direct administration to a tumor.

[0183] The pharmaceutical composition can be formulated for various modes of administration, including systemic, local or localized administration. Techniques and formulations can be found, for example, in Remrnington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous. For the purpose of injection, the pharmaceutical composition can be formulated in a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition can be formulated in solid form and redissolved or suspended just before use. Lyophilized forms of the pharmaceutical composition are also suitable.

[0184] For oral administration, the pharmaceutical composition can take the form of, for example, tablets or capsules, which are prepared by conventional means with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinyl pyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silicon dioxide); 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 they can be present as dry products formulated with water or other suitable vehicles before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (e.g. lecithin or gum arabic); non-aqueous vehicles (e.g. ationd oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g. methylparaben or propylparaben or sorbic acid). Where appropriate, these preparations may also contain buffer salts, flavoring agents, coloring agents and sweeteners.

[0185] The pharmaceutical composition can be formulated for parenteral administration by injection, e.g., by push injection or continuous infusion. Injectable preparations can be present in unit dosage form, e.g., in ampoules or multidose containers, with optionally added preservatives. The pharmaceutical composition can also be formulated as a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain other agents, including suspending agents, stabilizers, and / or dispersants.

[0186] In addition, pharmaceutical compositions can also be formulated as depot preparations. These long-acting preparations can be applied by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. Therefore, for example, the compound can be formulated with a suitable polymeric material or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or formulated as a slightly soluble derivative, such as a slightly soluble salt. Other suitable delivery systems include microspheres, which provide the possibility of local non-invasive delivery of drugs over an extended period of time. This technology can include microspheres with a pre-capillary size, which can be injected into any selected part of an organ via a coronary catheter without causing inflammation or ischemia. The therapeutic agent administered is slowly released from the microspheres and absorbed by the surrounding cells present in the selected tissue.

[0187] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated 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 can be used to promote penetration. Transmucosal administration can occur using nasal sprays or suppositories. For topical administration, as generally known in the art, the viral vectors described herein can be formulated into ointments, salves, gels or creams. Cleansing fluids can also be used topically to treat injuries or inflammations to accelerate healing.

[0188] Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions; formulations including 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 form must be sterile and must be fluid. It must be stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing), and must be preserved against the contaminating effects of microorganisms (e.g., bacteria and fungi).

[0189] If the formulations disclosed herein are used as therapeutic agents to enhance the immune response in a subject, the therapeutic agent can be formulated as a composition in neutral form or in salt form. Pharmaceutically acceptable salts include acid addition salts (formed by free amino groups of proteins) and are formed by inorganic acids such as hydrochloric acid or phosphoric acid, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed by free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.

[0190] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), a suitable mixture thereof, and a vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required viral vector size in the case of a dispersion, and using a surfactant. Prevention of microbial action can be achieved by various antibacterial and antifungal agents known in the art. In many cases, isotonic agents such as sugar or sodium chloride are preferably included. Prolonged absorption of the injectable composition can be achieved by using an agent that delays absorption in the composition, such as aluminum monostearate and gelatin.

[0191] Sterile injectable solutions can be prepared by incorporating the active compound or construct in the required amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0192] After formulation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulation is easily administered in a variety of dosage forms, such as the type of injectable solution described above, but also slow-release capsules or microparticles and microspheres and the like can be used.

[0193] For example, for parenteral administration in aqueous solution, when necessary, solution should be appropriately buffered, and liquid diluent is first made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous and intraperitoneal administration. Under this background, according to the present disclosure, those skilled in the art will know the sterile aqueous medium that can be adopted. For example, a dosage can be dissolved in 1ml isotonic NaCl solution, or added in 1000ml subcutaneous infusion fluid, or injected at the infusion site of proposal.

[0194] In any case, the person responsible for administration will determine the appropriate dosage for an individual subject. For example, a subject may be administered a viral vector described herein on a daily or weekly basis for a period of time, or on a monthly, biennial, or annual basis, depending on need or exposure to pathogenic organisms or conditions in the subject (e.g., cancer).

[0195] In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, intradermal or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; liposomal formulations; timed release capsules; biodegradable and any other form currently used.

[0196] Intranasal or inhalable solutions or sprays, aerosols or inhalants may also be used. Nasal solutions may be aqueous solutions designed to be applied to the nasal passages in drops or sprays. Nasal solutions may be prepared so that they are similar in many respects to nasal secretions. Therefore, nasal aqueous solutions are generally isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives similar to those used in eye preparations, and appropriate drug stabilizers when necessary, may be included in the preparations. Various commercial nasal preparations are known and may include, for example, antibiotics and antihistamines, and are used to prevent asthma.

[0197] Oral formulations can include excipients, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release preparations or powders. In certain limited embodiments, oral pharmaceutical compositions will include inert diluents or assimilable edible carriers, or they can be enclosed in hard or soft shell gelatin capsules, or they can be compressed into tablets, or they can be directly incorporated with the food in the diet. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc.

[0198] Tablets, lozenges, pills, capsules, and the like may also contain the following: binders, such as gum tragacanth, gum arabic, corn starch, or gelatin; excipients, such as dicalcium phosphate; disintegrants, such as corn starch, potato starch, alginic acid, and the like; lubricants, such as magnesium stearate; and sweeteners, such as sucrose, lactose, or saccharin, or flavorings, such as peppermint, wintergreen oil, or cherry flavoring, may be added. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the above-mentioned types of materials. Various other materials may be present as coatings, or otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. Elixir syrups may contain the active compound, sucrose as a sweetener, methyl and propyl parabens as preservatives, dyes, and flavorings, such as cherry or orange flavoring.

[0199] Further embodiments disclosed herein may relate to a kit for use with the method and composition. The kit may also include a suitable container, such as a vial, a tube, a small or ultra-microcentrifuge tube, a test tube, a flask, a bottle, a syringe or other container. When additional components or reagents are provided, the kit may contain one or more additional containers, in which such reagents or components may be placed. The kit herein will generally also include means for accommodating viral vectors and any other reagent containers in tight constraints for commercial sale. Such containers may include injection or blow molding plastic containers in which the required vials are retained. Optionally, one or more additional activating agents may be required for the composition, such as anti-inflammatory agents, antiviral agents, antifungal agents or antibacterial agents or antitumor agents.

[0200] Dosage range and frequency of administration can be changed according to the properties and medical conditions of the viral vector, as well as the parameters of the specific patient and the route of administration used. In certain embodiments, the viral vector composition can be applied to a subject with a range of about 1x105 plaque forming units (pfu) to about 1x1015 pfu dosage, depending on the mode of administration, route of administration, the properties of the disease and the condition of the subject. In some cases, the viral vector composition can be applied to a dosage of about 1x108 pfu to about 1x1015 pfu or about 1x1010 pfu to about 1x1015 pfu or about 1x108 pfu to about 1x1012 pfu. More accurate dosage can also depend on the subject it is applied to therein. For example, if the subject is a teenager, a lower dosage may be required, and if the subject is an adult subject, a higher dosage may be required. In certain embodiments, more accurate dosage can depend on the weight of the subject. In certain embodiments, for example, an adolescent human subject may receive a dose of about 1 x 108 pfu to about 1 x 1010 pfu, while an adult human subject may receive a dose of about 1 x 1010 pfu to about 1 x 1012 pfu.

[0201] The compositions disclosed herein can be administered by any means known in the art. For example, the compositions can include intravenous, intratumoral, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intravitreal, intravaginal, intrarectal, local, intratumoral, intramuscular, intrathecal, subcutaneous, subconjunctival, intracapsular, mucosal, intrapericardial, umbilical, intraocular, oral, topical, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion, via catheter, via lavage, in cream or in lipid composition and administered to the subject.

[0202] Any method known to those skilled in the art can be used for the large-scale production of viral vectors as herein described, packaging cells and vector constructs. For example, master seeds and working seed banks can be prepared under GMP conditions, in qualified primary CEF or by other methods. Packaging cells can be plated on large surface area flasks, grow to close to converge, and purify viral vectors. Cells can be harvested, and the viral vectors released into the culture medium can be separated and purified, or the intracellular viral vectors (cell debris can be removed by macroporous depth filtration, and host cell DNA is digested with endonucleases) can be released by mechanical destruction. Viral viral vectors can be purified subsequently, and concentrated by tangential flow filtration, followed by diafiltration. The resulting concentrated block can be prepared by diluting with a buffer containing a stabilizer, filled in a vial and lyophilized. Compositions and preparations can be stored for later use. In order to use, lyophilized viral vectors can be reconstructed by adding a diluent.

[0203] Certain additional agents used in combination therapy may be formulated and administered by any means known in the art.

[0204] Compositions as disclosed herein may also include adjuvants, such as aluminum salts and other mineral adjuvants, tonicity agents, bacterial derivatives, vehicles, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants may stimulate Th1 or Th2 immunity. Compositions and methods as disclosed herein may also include adjuvant therapy.

[0205] Other implementations:

[0206] Embodiment 1. A recombinant viral capsid protein comprising an epitope that is heterologous to the capsid protein,

[0207] wherein the heterologous epitope or a portion thereof specifically binds to the antibody paratope, and

[0208] The viral capsid protein forms a recombinant viral capsid with reduced or eliminated natural tropism.

[0209] Embodiment 2. A recombinant viral capsid protein according to embodiment 1, wherein the epitope is at least 1 amino acid in length.

[0210] Embodiment 3. The recombinant viral capsid protein according to embodiment 1 or embodiment 2 comprises substitutions, insertions or deletions at amino acid positions related to the natural tropism of the viral capsid, so that the recombinant viral capsid protein forms a viral capsid with reduced to eliminated natural tropism.

[0211] Embodiment 4. A recombinant viral capsid protein according to embodiments 1-3, wherein the viral capsid protein is derived from an adeno-associated virus (AAV) capsid gene, optionally wherein the heterologous epitope is inserted into a position selected from the following: I587 of AAV2, I585 of AAV6, I590 of AAV8, I453 of AAV9, I589 of AAV9, and any corresponding amino acid of an AAV serotype that infects primates, optionally wherein the AAV serotype that infects primates is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9.

[0212] Embodiment 5. A recombinant viral capsid protein according to embodiment 4, wherein the adeno-associated virus is AAV2.

[0213] Embodiment 6. A recombinant viral capsid protein according to embodiment 4, wherein the adeno-associated virus is AAV6.

[0214] Embodiment 7. A recombinant viral capsid protein according to embodiment 4, wherein the adeno-associated virus is AAV8.

[0215] Embodiment 8. A recombinant viral capsid protein according to embodiment 4, wherein the adeno-associated virus is AAV9.

[0216] Embodiment 9. A recombinant viral capsid protein according to any one of embodiments 1-8, wherein

[0217] (i) the viral capsid protein is a genetically modified AAV2 VP1 capsid protein, and the epitope is inserted after the amino acid at position I453 or I587 and / or replaces the amino acid at position I453 or I587;

[0218] (ii) the viral capsid protein is a genetically modified AAV6 VP1 capsid protein, and the epitope is inserted after the amino acid at position I585 and / or replaces the amino acid at position I585;

[0219] (iii) the viral capsid is a genetically modified AAV8 VP1 capsid protein, and the epitope is inserted after the amino acid at position I590 and / or replaces the amino acid at position I590.

[0220] (iv) the viral capsid protein is a genetically modified AAV9VP1 capsid protein, and the epitope is inserted after the amino acid at position I453 or I589 and / or replaces the amino acid at position I453 or I589.

[0221] Embodiment 10. A recombinant viral capsid protein according to any one of embodiments 1-9, wherein the viral capsid protein is encoded by an AAV2 capsid gene modified to express an epitope flanked between amino acids N587 and R588 of the AAV2 VP1 capsid protein.

[0222] Embodiment 11. A recombinant viral capsid protein according to any one of embodiments 1-9, wherein the viral capsid protein is derived from the AAV6 capsid gene and the epitope is inserted immediately after amino acid Q585.

[0223] Embodiment 12. A recombinant viral capsid protein according to any one of embodiments 1 to 9, wherein the viral capsid protein is derived from an AAV8 capsid gene, and wherein the epitope is inserted immediately after amino acid N590 of the AAV8VP1 capsid protein.

[0224] Embodiment 13. A recombinant viral capsid protein according to any one of embodiments 1-9, wherein the viral capsid protein is derived from an AAV9 capsid gene, the epitope is inserted into the capsid protein after the amino acid at position G453 or A589 of the AAV9VP1 capsid protein, and / or replaces the amino acid at position G453 or A589 of the AAV9VP1 capsid protein, and wherein the capsid protein further comprises additional mutations.

[0225] Embodiment 14. A recombinant viral capsid protein according to embodiment 13, wherein the additional mutation is W503A.

[0226] Embodiment 15. A recombinant viral capsid protein according to any one of embodiments 1-14, wherein the epitope comprises the amino acid sequence EQKLISEEDL, the amino acid sequence or a portion thereof specifically binds to the antibody paratope, and optionally.

[0227] Embodiment 16. A recombinant viral capsid protein according to any one of embodiments 1-15, wherein the epitope comprises the amino acid sequence EQKLISEEDL, and the amino acid sequence or a portion thereof specifically binds to the antibody paratope, and

[0228] The amino acid sequence EQKLISEEDL is flanked by at least 5 contiguous amino acids of an AAV capsid protein and is operably linked thereto.

[0229] Embodiment 17. A recombinant viral capsid protein according to embodiment 16, wherein the viral capsid protein comprises the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence shown in SEQ ID NO:25, the amino acid sequence shown in SEQ ID NO:26, or the amino acid sequence shown in SEQ ID NO:27.

[0230] Embodiment 18. The recombinant viral capsid protein of any one of embodiments 1 to 17, wherein in the absence of a multispecific binding molecule comprising the antibody paratope, the recombinant viral capsid protein or a viral capsid comprising the recombinant viral capsid protein is unable to infect a target cell, optionally wherein in the absence of a multispecific binding molecule comprising the antibody paratope, the transduction efficiency of the viral capsid protein or a viral capsid comprising the recombinant viral capsid protein is:

[0231] (i) a reduction of at least 10%,

[0232] (ii) a reduction of at least 20%,

[0233] (iii) a reduction of at least 30%,

[0234] (iv) a reduction of at least 40%,

[0235] (v) a reduction of at least 50%,

[0236] (vi) a reduction of at least 60%,

[0237] (vii) a reduction of at least 70%,

[0238] (viii) a reduction of at least 80%,

[0239] (ix) a reduction of at least 90%, or

[0240] (x) Elimination.

[0241] Embodiment 19. An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant viral capsid protein according to any one of embodiments 1-18.

[0242] Embodiment 20. An isolated nucleic acid according to embodiment 19, wherein the nucleotide sequence encodes a recombinant viral capsid protein, which comprises the amino acid sequence shown in SEQ ID NO:2, the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence shown in SEQ ID NO:25, the amino acid sequence shown in SEQ ID NO:26, or the amino acid sequence shown in SEQ ID NO:27.

[0243] Embodiment 21. A recombinant viral vector comprising a target nucleotide encapsulated by a recombinant viral capsid according to embodiment 20, optionally wherein the recombinant viral capsid is a chimeric capsid.

[0244] Embodiment 22. A recombinant viral vector according to embodiment 21, wherein the target nucleotide is under the control of a promoter selected from a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter and any combination thereof.

[0245] Embodiment 23. A recombinant viral vector according to embodiment 22, wherein the target nucleotide is under the control of a non-human promoter.

[0246] Embodiment 24. A recombinant viral vector according to embodiment 23, wherein the target nucleotide is flanked by AAV ITR sequences.

[0247] Embodiment 25. A recombinant viral vector according to any one of embodiments 21-24, wherein the target nucleotide is a reporter gene.

[0248] Embodiment 26. The recombinant viral vector of embodiment 25, wherein the reporter gene encodes green fluorescent protein.

[0249] Embodiment 27. A recombinant viral vector according to any one of embodiments 21-24, wherein the target nucleotide is selected from a suicide gene, a nucleotide encoding an antibody or a fragment thereof, a nucleotide encoding a CRISPR / Cas system or a portion thereof, a nucleotide encoding an antisense RNA, a nucleotide encoding an siRNA, and a combination thereof.

[0250] Embodiment 28. A composition comprising (a) a recombinant viral vector according to any one of embodiments 21-27, and (b) a multispecific binding molecule comprising an antibody paratope that specifically binds to the epitope, optionally wherein the multispecific binding molecule also comprises a retargeting ligand that specifically binds to a receptor expressed on a target cell, wherein the multispecific binding molecule is optionally a bispecific binding molecule.

[0251] Embodiment 29. A composition according to embodiment 28, wherein the viral vector and the multispecific binding molecule are present in a ratio of 1:4.

[0252] Embodiment 30. A composition according to embodiment 28 or 29, wherein the antibody paratope is an Fv domain.

[0253] Embodiment 31. A composition according to embodiment 30, wherein the Fv domain is directly fused to the heavy chain constant domain.

[0254] Embodiment 32. A composition according to any one of embodiments 28-31, wherein the retargeting ligand is an antibody or a portion thereof.

[0255] Embodiment 33. A composition according to any one of embodiments 28-32, wherein the multispecific binding molecule is a bispecific antibody, and

[0256] wherein the paratope and the retargeting ligand each comprise a different Fv domain fused to a first heavy chain constant domain and a second heavy chain constant domain.

[0257] Embodiment 34. A composition according to embodiment 33, wherein the first heavy chain and the second heavy chain bind to protein A with different binding affinities.

[0258] Embodiment 35. A composition according to any one of embodiments 28-32, wherein the multispecific binding molecule is a bispecific antibody, and

[0259] wherein the retargeting ligand comprises a tetrameric antibody structure comprising two identical immunoglobulin heavy chains and two identical light chains, and

[0260] wherein the paratope that binds to the heterologous epitope is appended to the C-terminus or N-terminus of one or both heavy chains and / or is appended to the C-terminus or N-terminus of one or both heavy chains.

[0261] Embodiment 36. A composition according to embodiment 35, wherein the paratope is a scFv, optionally wherein the scFv comprises the amino acid sequence as shown in SEQ ID NO:37.

[0262] Embodiment 37. A composition according to any one of embodiments 28-36, wherein the heterologous epitope comprises the amino acid sequence EQKLISEEDL or a portion thereof, and wherein the antibody paratope specifically binds to the amino acid sequence EQKLISEEDL or a portion thereof.

[0263] Embodiment 38. A composition according to any one of embodiments 28-37, wherein the retargeting ligand specifically binds to a cell surface protein that is a cell surface marker.

[0264] Embodiment 39. The composition of embodiment 38, wherein the cell surface marker is asialoglycoprotein 1 (ASGR1).

[0265] Embodiment 40. The composition of embodiment 38, wherein the cell surface marker is CD3.

[0266] Embodiment 41. The composition of embodiment 38, wherein the cell surface marker is ENTPD3.

[0267] Embodiment 42. The composition of any one of embodiments 28-41, further comprising a pharmaceutically acceptable carrier.

[0268] Embodiment 43. A method for directing a recombinant viral vector according to any one of embodiments 21-27 to a target cell, comprising:

[0269] contacting the recombinant viral vector with a multispecific binding molecule,

[0270] 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 a target cell.

[0271] Embodiment 44. A method according to embodiment 43, wherein the target cell is in vivo.

[0272] Embodiment 45. A method according to embodiment 43 or embodiment 44, wherein the contacting is performed ex vivo.

[0273] Embodiment 46. A method for delivering a target nucleotide to a target cell expressing a cell surface protein, comprising contacting the target cell with a composition according to any one of embodiments 28-42, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein.

[0274] Embodiment 47. A method according to embodiment 46, wherein the target cell is in vitro.

[0275] Embodiment 48. A method according to embodiment 46, wherein the target cell is in a subject.

[0276] Embodiment 49. A method according to embodiment 48, wherein the subject is human.

[0277] Embodiment 50. A method according to any one of embodiments 46-49, wherein the target cell is a human cell.

[0278] Embodiment 51. A method according to any one of embodiments 46-50, wherein the target cell is selected from a liver cell, a brain cell, a T cell, a kidney cell, an intestinal cell, a pancreatic cell, a cancer cell, and a cell infected with a heterologous pathogen.

[0279] Embodiment 52. A method according to any one of embodiments 46-51, wherein the target cell is a human hepatocyte.

[0280] Embodiment 53. The method of any one of embodiments 46-52, wherein the cell surface protein is human asialoglycoprotein receptor 1 (hASGR1).

[0281] Embodiment 54. A method according to any one of embodiments 46-51, wherein the target cell is a human T cell.

[0282] Embodiment 55. A method according to embodiment 54, wherein the cell surface protein is CD3.

[0283] Embodiment 56. The method of any one of embodiments 46-51, wherein the cell surface protein is human glucagon receptor (hGCGR).

[0284] Embodiment 57. A method according to any one of embodiments 46-50, wherein the target cell is an intestinal cell.

[0285] Embodiment 58. A method according to any one of embodiments 46-50, wherein the target cell is a pancreatic cell.

[0286] Embodiment 59. A method according to embodiments 57 and 58, wherein the cell surface protein is ENTPD3.

[0287] Embodiment 60. A method for inactivating viral capsid protein, comprising

[0288] (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 the heterologous epitope, and

[0289] (b) culturing packaging cells under conditions sufficient to produce the viral vector, wherein the packaging cells comprise the nucleotide sequence.

[0290] Embodiment 61. A method of producing a viral vector, comprising culturing packaging cells under conditions sufficient to produce the viral vector, wherein the packaging cells comprise a plasmid encoding the recombinant capsid protein according to any one of embodiments 1-18.

[0291] Embodiment 62. A method according to embodiment 60 or embodiment 61, wherein the packaging cell further comprises a helper plasmid and / or a transfer plasmid comprising the target nucleotide.

[0292] Embodiment 63. A method according to any one of embodiments 60-62, further comprising isolating a self-complementary adeno-associated viral vector from the culture supernatant.

[0293] Embodiment 64. A method according to any one of embodiments 60-63, which also includes lysing the packaging cells and isolating the single-stranded adeno-associated virus vector from the cell lysate.

[0294] Embodiment 65. A method according to any one of embodiments 60-64, further comprising

[0295] a. Remove cell debris,

[0296] b. Treat the supernatant containing the viral vector with DNase I and MgCl2,

[0297] c. Concentrated viral vector,

[0298] d. purifying the viral vector, and

[0299] Any combination of ea-d.

[0300] Embodiment 66. A viral vector prepared according to the method of any one of embodiments 60-65.

[0301] Embodiment 67. A packaging cell for producing a viral vector, wherein the viral vector comprises a plasmid encoding a capsid protein according to any one of embodiments 1-18.

[0302] Embodiment 68. A binding molecule comprising a paratope that binds to SEQ ID NO:6, wherein the paratope comprises the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and / or LCDR3 sequence of a scFv encoded by the nucleic acid sequence shown in SEQ ID NO:28.

[0303] Embodiment 69. A binding molecule according to embodiment 68, wherein the antibody paratope is an Fv domain.

[0304] Embodiment 70. A binding molecule according to embodiment 69, wherein the Fv domain is directly fused to the heavy chain constant domain.

[0305] Embodiment 71. A binding molecule according to any one of embodiments 68-70, wherein the binding molecule is a bispecific antibody and further comprises a retargeting ligand, and

[0306] wherein the paratope and the retargeting ligand each comprise a different Fv domain fused to a first heavy chain constant domain and a second heavy chain constant domain.

[0307] Embodiment 72. A binding protein according to embodiment 71, wherein the first heavy chain and the second heavy chain bind to protein A with different binding affinities.

[0308] Embodiment 73. A binding protein according to any one of embodiments 68-69, wherein the binding molecule is a bispecific antibody and further comprises a retargeting ligand, and

[0309] wherein the retargeting ligand comprises a tetrameric antibody structure comprising two identical immunoglobulin heavy chains and two identical light chains, and

[0310] wherein the paratope is appended 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.

[0311] Embodiment 74. The binding protein of embodiment 73, wherein the paratope is a scFv, optionally wherein the scFv comprises the amino acid sequence shown in SEQ ID NO:37.

[0312] Embodiment 75. The binding protein of any one of embodiments 69-72, further comprising a retargeting ligand which is an antibody or a portion thereof.

[0313] Embodiment 76. A binding protein according to embodiment 75, wherein the retargeting ligand specifically binds to a cell surface protein that is a cell surface marker.

[0314] Embodiment 77. The binding protein of embodiment 76, wherein the cell surface marker is asialoglycoprotein 1 (ASGR1).

[0315] Embodiment 78. The binding protein of embodiment 76, wherein the cell surface marker is CD3.

[0316] Embodiment 79. The binding protein of embodiment 76, wherein the cell surface marker is GCGR.

[0317] Embodiment 80. The binding protein of embodiment 76, wherein the cell surface marker is ENTPD3.

[0318] Embodiment 81. A composition comprising (a) a recombinant viral vector according to any one of embodiments 21-27, and (b) a binding protein according to any one of embodiments 68-81.

[0319] Embodiment 82. A composition according to embodiment 81, wherein the viral vector and the multispecific binding molecule are present in a ratio of 1:4.

[0320] Embodiment 83. The composition of embodiment 81 or embodiment 82, further comprising a pharmaceutically acceptable carrier.

[0321] Examples

[0322] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0323] Example 1: Generation of adeno-associated viral vectors with heterologous epitopes

[0324] AAV capsid proteins were modified using PCR to contain one of several heterologous epitopes: FLAG, c-myc, hexa-histidine, etc., to generate plasmids encoding recombinant capsid proteins. Briefly, sequences encoding FLAG, c-myc, or hexa-histidine were inserted in frame after the codons encoding: N587 of AAV2 capsid protein, Q585 of AAV6 VP1 capsid protein, N590 of AAV8 VP1 capsid protein, A589 of AAV9 VP1 capsid protein, or G453 of AAV9 VP1 capsid protein.

[0325] Adeno-associated virus production was performed using a triple transfection method with HEK293 cells (see, e.g., Erik Arden and Joseph M. Metzger, J Biol Methods. 2016; 3(2)). The cells were plated one day before PEFpro (Polyplus Transfection, New York, NY)-mediated transfection with the appropriate vector:

[0326] Helper plasmid pHelper (Agilent, catalog #240074);

[0327] Plasmids encoding wild-type or modified AAV rep / cap genes (pAAV RC2 (Cell biolabs, catalog #VPK-422), such as pAAV RC2 / 6 / 9 (Cell Biolabs, catalog #VPK-426), pAAV RC8, pAAV RC2-N587myc, pAAV RC2 / 6-Q585myc, pAAVRC8-N590myc, pAAV RC9-A589myc, etc.; and

[0328] A plasmid encoding the nucleotide of interest and AAV ITR sequences, such as pscAAV CMV-eGFP, pAAV-CMVGFP (Agilent catalog #240074), pAAV-EF1a-eGFP, or pAAV-CAGG-eGFP.

[0329] Seventy-two hours after transfection, the culture medium was collected, and the cells were lysed in a buffer [50mM Tris-HCl, 150mM NaCl, and 0.5% sodium deoxycholate (Sigma, catalog # D6750-100G)]. Next, benzonase (Sigma, St. Louis, MO) was added to both the culture medium and the cell lysate to a final concentration of 0.5U / μl, and then incubated at 37°C for 60 minutes. The cell lysate was spun down at 4000rpm for 30 minutes. The cell lysate and culture medium were combined and precipitated with PEG 8000 (T Teknova catalog # P4340) at a final concentration of 8%. The mass was resuspended in 400mM NaCl and centrifuged at 10000g for 10 minutes. The virus in the supernatant was formed into a mass by ultracentrifugation at 149,000g for 3 hours, and titrated by qPCR.

[0330] For qPCR titration of AAV genomes, AAV samples were treated with DNase I (Thermofisher Scientific, catalog #EN0525) for one hour at 37°C and lysed using DNA Extract All Reagents (Thermofisher Scientific catalog #4403319). Encapsidated viral genomes were quantified using primers for AAV2 ITR using the QuantStudio 3 Real-Time PCR System (Thermofisher Scientific). The sequences of the AAV2 ITR primers 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 internal inverted repeat (ITR) sequence of AAV and the right internal inverted repeat (ITR) sequence of AAV, respectively. The sequence of the AAV2 ITR probe is 5′-6-FAM-CACTCCCTCTCTGCGCGCTCG-TAMRA-3′ (SEQ ID NO: 11) (Aurnhammer C., Haase M., Muether N. et al., 2012, Hum. Gene Ther. Methods 23, 18–28). After an activation step at 95° C. for 10 minutes, two-step PCR cycles were performed at 95° C. for 15 minutes and at 60° C. for 30 seconds, for a total of 40 cycles. TaqMan Universal PCR Master Mix (Thermofisher Scientific, catalog #4304437) was used in qPCR. DNA plasmid (Agilent, catalog #240074) was used as a standard for determining absolute titers.

[0331] An adeno-associated viral vector containing a capsid containing a c-myc epitope is produced. In this example, the c-myc epitope (EQKLISEEDL; SEQ ID NO: 6) is inserted between amino acids N587 and R588 of the AAV2VP1 capsid protein, or between amino acids A589 and Q590 of the AAV9VP1 capsid protein, i.e., the nucleotide sequence encoding the c-myc epitope (GAA CAAAAA CTC ATC TCA GAA GAG GAT CTG; SEQ ID NO: 12) is inserted into plasmid pAAV RC2 (Cell Biolabs, Inc., San Diego, CA) or plasmid pAAV RC2 / 9, and the modified pAAV RC2-N587Myc pAAVRC9-A589Myc plasmids, respectively, are each used to encode a modified capsid protein for an AAV viral vector with reduced or eliminated tropism.

[0332] To generate pAAV RC2-N587Myc, the primers shown in Table 2 were used to generate a first polymerase chain reaction (PCR) product comprising (from 5' to 3') a BsiW1 restriction site, a nucleotide sequence between positions 3050 and 3773 of pAAV RC2, and a c-myc epitope overhang nucleotide sequence, and a second PCR product comprising (from 5' to 3') a c-myc epitope overhang nucleotide sequence, a nucleotide sequence between positions 3774 to 4370 of pAAV RC2, and a Pme1 restriction site. The pAAVRC2N587Myc plasmid (i.e., a pAAV RC2 plasmid modified to encode a c-myc epitope between amino acids N587 and R588 of the VP1 capsid protein) was generated by digesting pAAV RC2 with BsiW1 (New England Biolabs, R0553L) and Pme1 (New England Biolabs, R0560L) and inserting the two PCR products via ligation-dependent cloning as described in (2012) Methods Mol. Biol. 52:51-9.

[0333] To generate pAAV RC2 / 6-Q585Myc, a gblock DNA fragment containing positions 3700 and 3940 of pAAV RC2 / 6 with a c-myc-epitope sequence inserted between 3757 and 3758 was ordered from Integrated DNA Technologies (Coralville, Iowa). The pAAV RC2 / 6-Q585Myc plasmid was generated via ligation-dependent cloning by inserting the gblock fragment into pAAV RC2 / 6 digested with Mscl (New England Biolabs, catalog #R0534L) and AflII (New England Biolabs, catalog #R0520L) as described in (2012) Methods Mol. Biol. 52:51-9.

[0334] To generate pAAV RC9-A589Myc, the primers shown in Table 2 were used to generate a first polymerase chain reaction (PCR) product comprising (from 5' to 3') a BsiW1 restriction site, a nucleotide sequence between positions 3052 and 3779 of pAAV RC9, and a c-myc epitope overhang nucleotide sequence, and a second PCR product comprising (from 5' to 3') a c-myc epitope overhang nucleotide sequence, a nucleotide sequence between positions 3779 to 4404 of pAAV RC9, and a Pme1 restriction site.

[0335] The pAAV RC9A589Myc plasmid (i.e., a pAAV RC2 / 9 plasmid modified to encode the 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 inserting the two PCR products via ligation-dependent cloning as described in (2012) Methods Mol. Biol. 52:51-9.

[0336] Table 2

[0337]

[0338]

[0339] The GFP fragment was introduced into the pscAAV MCS vector (Cell Biolabs, catalog #VPK-430) by using BamHI and NotI restriction sites to generate pscAAV-CMV-eGFP. The pAAV-EF1a-eGFP plasmid and pAAV-CAGG-eGFP were prepared de novo by Thermofisher Scientific (Waltham, MA).

[0340] Example 2: Antibody-mediated retargeting of AAV viral vectors in vitro

[0341] HepG2 is a human liver cancer cell line that expresses the liver-specific marker asialoglycoprotein receptor 1 (ASGR1). To test whether the scAAV2N587Myc-CMV-hrGFP viral vector can infect HepG2 cells, for example, via a bispecific antibody that recognizes both c-myc and ASGR1 (anti-myc-ASGR1 antibody), a mixture of scAAV2N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody containing different ratios of the number of viral genomes (5e9 viral genomes) to the number of antibody molecules (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50, or 1:100) was incubated at room temperature for half an hour and then added to HepG2 cells. HepG2 cells were also incubated with the following at a ratio of 1:8 as controls: wild-type scAAV2-CMV-eGFP viral vector alone, scAAV2-N587Myc-CMV-eGFP viral vector alone, or scAAV2-N587Myc-CMV-eGFP viral vector with a monospecific anti-myc antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY). 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 a significant percentage of HepG2 cells when incubated with wild-type scAAV2-CMV-eGFP, and when incubated with scAAV2-N587Myc-CMV-eGFP viral vector and bispecific anti-myc-ASGR1 antibody at a ratio of 1:8 ( Figure 1A and 1G, upper and lower panels, 44.6% and 44.1%, respectively). In HepG2 cells incubated with a mixture of scAAV2-N587Myc-CMV-eGFP viral vector and higher or lower bispecific anti-myc-ASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY), lower GFP expression was detected by FACS ( Figure 1 C, lower panel). In addition, no GFP was detected in HepG2 cells infected with scAAV-N587Myc-CMV-eGFP in the absence of anti-myc-ASGR1 antibody or in HepG2 cells incubated with scAAV2-N587Myc-CMV-eGFP and a monospecific anti-myc antibody.

[0342] Similarly, 293T-hASGR1 cells, which are 29T3 cells genetically modified to express (h)ASGR1 on the cell surface, were incubated with a mixture comprising scAAV2-N587Myc-CMV-eGFP and a bispecific anti-myc-ASGR1 antibody at different ratios of viral genomes to the number of antibody molecules (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, or 1:100). Wild-type 293T cells (which do not express hASGR1) incubated with a mixture of scAAV2-N587Myc-CMV-eGFP and a bispecific anti-myc-ASGR1 antibody at a ratio of 1:8, and 293T-hASGR1 cells incubated with the following at a ratio of 1:8 served as controls: (a) wild-type scAAV viral vector alone, (b) scAAV2-N587Myc-CMV-eGFP viral vector alone, or (c) scAAV2-N587Myc-CMV-eGFP viral vector and an irrelevant bispecific anti-myc-GCGR antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) that recognizes c-myc and glucagon receptor (GCGR) and is not expressed by 293T cells. Three days after infection, 293T-hASGR1 or 293T cells were stained with human anti-hASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY), followed by APC-conjugated goat anti-human antibody (Jackson ImmunoResearch laboratories Inc, catalog #109-136-098, West Grove, PA), and GFP expression was analyzed by FACS. Figure 2Ai-2Ax and 2Axii), but not 293T cells ( Figure 2A hASGR1 expression was detected on the surface of 293T-hASGR1 cells. After incubation with scAAV2N587Myc-CMV-eGFP and a bispecific anti-myc-ASGR1 antibody cocktail 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 at levels (56.9% to 68.3%) comparable to those of 293T-hASGR1 cells incubated with wild-type scAAV (56.7%). Figure 2A i and 2Aiii-2Aix). The ratio of scAAV2N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody 1:100 reduced infectivity ( Figure 2A x). Incubation with scAAV2-N587Myc-CMV-eGFP alone or scAAV-N587Myc with an irrelevant bispecific anti-myc-GCGR antibody did not result in GFP expression by 293ThASGR1 cells ( Figure 2A ii and 2Axii).

[0343] In a similar experiment, 293T-hASGR1 cells were incubated with cells incubated with different ratios of unmodified AAV9-CAGG-GFP viral vector alone, AAV9-A589Myc-CAGG-eGFP viral vector alone, or AAV9-A589Myc-CAGG-eGFP viral vector mixed with a bispecific anti-Myc-ASGR1 antibody. Three days after infection, GFP expression was analyzed by FACS. After incubation with AAV9A589Myc-CAGG-eGFP and a bispecific anti-myc-ASGR1 antibody cocktail at ratios of 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, and 1:100, GFP-positive 293T-hASGR1 cells were detected at levels (41.1% to 91%) comparable to those of 293T-hASGR1 cells incubated 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))

[0344] The ability of a bivalent anti-hASGR1 antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) to block infection of 293T-hASGR1 cells was tested. 293T-hASGR1 cells were incubated with the bivalent anti-hASGR1 antibody at different concentrations for 1 hour at room temperature and then incubated with a mixture containing scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody at a ratio of 1:8. Three days after infection, cells were fixed and analyzed by FACS as described above. Figure 3 The data presented show that the entry of scAAV2-N587Myc-CMV-eGFP could be blocked by the bivalent anti-hASGR1 antibody in a dose-dependent manner.

[0345] To determine whether sequential administration of bispecific antibodies and modified AAVs could result in antibody-mediated retargeting of modified AAVs, different numbers of bispecific anti-myc-ASGR1 antibody molecules (ranging from 1×10 9 Up to 1x10 12 molecule) add 2x10 5 293T hASGR1 cells for one hour, after which 1x10 9 The controls included (1) 293T-hASGR1 cells incubated with wild-type scAAV alone, i.e., in the absence of any antibody, and (2) 1x10 11 Unrelated bispecific anti-myc-GCGR antibody molecules and 1x10 9 293T-hASGR1 cells were sequentially incubated with scAAV2-N587Myc-CMV-eGFP viral vector, and (3) with 1x10 11 Bispecific anti-myc-ASGR1 antibody molecules and 1x10 9 293T cells that do not express hASGR1 were sequentially incubated with scAAV-N587Myc viral vector. Two days after infection, GFP expression by infected 293T-hASGR1 cells was observed by microscopy ( Figure 4 ). GFP expression by 293T-hASGR1 cells was observed after incubation with wild-type scAAV alone and after sequential incubation with anti-myc-ASGR1 antibody molecules and scAAV2-N587Myc-CMV-eGFP at all concentrations ( Figure 4 A, 4C-4I). In 29T3-hASGR1 cells incubated with scAAV2N587Myc-CMV-eGFP alone ( Figure 4B), in 293T cells not expressing ASGR1 after sequential incubation with anti-myc-hASGR1 antibody and scAAV2-N587Myc-CMV-eGFP viral vector ( Figure 4 J), or in 29T3hASGR1 cells after sequential incubation with an irrelevant bispecific anti-myc-GCGR antibody and the scAAV2N587Myc-CMV-eGFP viral vector ( Figure 4 K), no GFP was detected.

[0346] As described herein, the tropism of self-complementary AAV (scAAV) can be (1) inactivated, for example, by modification of the capsid protein, such as by insertion of a c-myc epitope, and optionally, (2) redirected using a bispecific antibody (e.g., a bispecific antibody that recognizes a c-myc epitope and a ligand expressed by a target cell). To determine whether the tropism of single-stranded AAV (ssAAV) can be similarly (1) reduced or eliminated, and optionally (2) redirected, 293T-hASGR1 cells were incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors generated as described in Example 1 in the absence or presence of bispecific anti-myc-hASGR1 antibodies 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 incubated with wild-type ssAAV, 293T-hASGR1 cells incubated with 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) incubated with ssAAV2-N587Myc-CMV-hrGFP viral vector and a 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 incubated with wild-type ssAAV, as well as a mixture of ssAAV2-N587Myc-CMV-hrGFP viral vector and bispecific anti-myc-ASGR1 antibody at all ratios ( Figure 5 A, 5C-5I). 29T3 cells ( Figure 5J), or 293T-hASGR1 cells incubated with ssAAV2N587Myc-CMV-hrGFP viral vector alone, or 293T-hASGR1 cells incubated with ssAAV N587Myc viral vector and an irrelevant bispecific anti-myc-GCGR bispecific antibody ( Figure 5 B, 5K), no GFP expression was observed.

[0347] Human (h) glucagon receptor (GCGR) is not normally expressed by 293T cells. However, 293T-hGCGR is a stable 293T cell line that has been genetically modified to express hGCGR on the cell surface. To test whether retargeting of the scAAV2N587Myc-CMV-eGFP viral vector can be mediated by the hGCGR receptor via a bispecific anti-myc-GCGR antibody, 293T-hGCGR cells were incubated with different mixtures containing different ratios of scAAV2N587Myc-CMV-eGFP: bispecific anti-myc-GCGR antibodies. Before being added to 293T hGCGR cells, the scAAV2N587Myc-CMV-eGFP viral vector was mixed with the 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 for half an hour at room temperature. Three days after infection, GFP expression by infected 293T-hCGCR cells was confirmed by fluorescence activated cell sorting (FACS). Figure 6 ). Compared with wild-type scAAV ( Figure 6 A) or scAAV2N587Myc-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 ( Figure 6 After incubation with scAAV2N587myc-CMV-eGFP viral vector alone or with monospecific anti-myc antibody, a significant percentage of GFP-positive cells was detected. No GFP was detected in 293T-hGCGR cells incubated with scAAV2N587myc-CMV-eGFP viral vector alone or with scAAV2N587Myc-CMV-eGFP viral vector with monospecific anti-myc antibody ( Figure 6 B and 6L).

[0348] Jurkat is a human acute T-cell leukemia cell line that expresses human (h) CD3. To test whether retargeting of the AAV6Q585Myc-EF1a-eGFP viral vector can be mediated by the hCD3 receptor via a bispecific anti-myc-CD3 antibody, Jurkat cells were incubated with different mixtures containing different ratios of AAV6Q585Myc-EF1a-eGFP: bispecific anti-myc-CD3 antibodies. Before being added to Jurkat cells, the AAV6-Q585Myc-EF1a-eGFP viral vector was mixed with the bispecific anti-myc-CD3 antibody at a ratio of 1:1, 1:5, 1:10, 1:1000 at room temperature for half an hour. Three days after infection, GFP expression ( Figure 7 ). Compared with wild-type AAV6-EF1a-eGFP ( Figure 7 B) or AAV6Q585Myc-EF1a-eGFP / bispecific anti-myc-CD3 antibody at 1:1, 1:5, 1:10, and 1:100 ratios ( Figure 7 After incubation with AAV6Q585Myc-EF1a-eGFP viral vector, a significant percentage of GFP-positive cells was detected. In Jurkat cells incubated with AAV6Q585Myc-EF1a-eGFP viral vector, no GFP was detected ( Figure 7 C).

[0349] Described in this example is the natural tropism inactivation of several self-complementary (sc) or single-stranded (ss) AAV serotypes, as demonstrated by the inability of scAAV2 or ssAAV2 genetically modified with a c-myc epitope inserted between amino acids N587 and R588 of the VP1 capsid protein (scAAV-N587myc or ssAAV-N587myc), AAV6 with a c-myc epitope inserted between Q585 and S586, and AAV9 to infect cells normally infected by wild-type AAV ( Figure 1 A-1B, 2A-2B, 3A-3B, 4A-4B, 5A-5B, 6A-6B and 7). Also demonstrated in this example is the ability of bispecific antibodies that recognize the c-myc epitope and a second ligand expressed by the target cell (e.g., hASGR1, hGCGR or hCD3) to retarget the tropism of the modified AAV and mediate infection of the target cell AAV by the pseudotyped AAV in a ligand-specific manner ( Figure 1-7 In addition, this example demonstrates that simultaneous administration of a genetically modified sc- or ss-AAV and a bispecific antibody is not necessary for infection, i.e., sequential administration is sufficient to retarget the genetically modified sc- or ss-AAV in vitro ( Figure 4 ).

[0350] Example 3: Antibody-mediated redirection of modified viral vectors in vivo

[0351] Retargeting of viral vectors to the liver using different multispecific binding molecule formats

[0352] To determine whether the bispecific anti-myc-ASGR1 antibody can retarget the scAAV2N587myc-CMV-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 1x10 11 Mice genetically modified to express hASGR1 in their hepatocytes on a C57BL / 6 background, as well as control wild-type C57BL / 6 mice, were injected intravascularly with wild-type scAAV2-CMV-eGFP alone, or scAAV2N587myc-CMV-eGFP viral vectors at a 1:8 ratio of viral genome to antibody molecule, in combination with a bispecific anti-myc-ASGR1 antibody (titrated by qPCR). Controls included mice injected with saline [250 mM NaCl] or scAAV2N587myc-CMV-eGFP viral vector alone. Ten days after injection, mice were sacrificed and perfused transcardially with 4% PFA. Organs of liver, kidney, and heart were collected and dehydrated in 15% sucrose, followed by 30% sucrose. The organs were then cryosectioned on slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). Figures 8A-8C In livers from transgenic animals modified to express ASGR1 in the liver and injected with wild-type scAAV2-CMV-eGFP, or scAAV2-N587myc-CMV-eGFP in combination with a bispecific anti-myc-ASGR1 antibody ( Fig. 8A (i) and 8A(iv)), and in livers from wild-type C57BL / 6 mice injected with wild-type scAAV2-CMV-eGFP ( Fig. 8A (v)), GFP-positive cells were detected. In any spleen or kidney samples ( Figure 8B and 8C ), in liver, spleen, or kidney samples from any animal injected with saline or scAAV2N587myc-CMV-eGFP viral vector alone ( Fig. 8A (ii, iii, vi, vii)), in liver samples taken from wild-type C57BL / 6 animals injected with scAAV2-N587myc-CMV-eGFP in combination with a bispecific anti-myc-ASGR1 antibody ( Fig. 8A (viii)), no GFP was detected in either case. In conclusion, the combination of scAAV2N587myc-CMV-eGFP viral vector and bispecific anti-myc-ASGR1 antibody only infects those (hepatic) cells expressing hASGR1, strongly suggesting that the scAAV2-CMV-eGFP viral vector is inactivated by modification of the capsid protein, e.g., the natural tropism of scAAV viral vectors can be reduced to eliminated, e.g., with the c-myc epitope, and such viral vectors can be specifically reactivated, e.g., specifically retargeted, e.g., to hepatocytes in vivo, e.g., by bispecific anti-myc-ASGR1 antibodies.

[0353] Similarly, to determine whether the bispecific anti-myc-ASGR1 antibody could retarget the ssAAV2N587myc-CAGG-eGFP viral vector to hepatocytes expressing hASGR1 in vivo, 2.18x10 11 Mice genetically modified to express hASGR1 in their hepatocytes on a C57BL / 6 background, as well as control wild-type C57BL / 6 mice, were injected intravascularly with wild-type ssAAV2-CAGG-eGFP alone, or ssAAV2N587myc-CAGG-eGFP viral vectors at a 1:4 ratio of viral genome to antibody molecule, in combination with a bispecific anti-myc-ASGR1 antibody (titrated by qPCR). Controls included mice injected with PBS or ssAAV2N587myc-CAGG-eGFP viral vector alone. Four weeks after injection, mice were sacrificed and perfused transcardially with 4% PFA. Organs of liver, kidney, and heart were collected and dehydrated in 15% sucrose, followed by 30% sucrose. The organs were then cryosectioned on slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). In livers from transgenic animals modified to express ASGR1 in the liver and injected with wild-type ssAAV2-CAGG-eGFP, or ssAAV2-N587myc-CAGG-eGFP in combination with a bispecific anti-myc-ASGR1 antibody ( Fig. 9 E-9F, 9P-9R), and in livers from wild-type C57BL / 6 mice injected with wild-type ssAAV2-CAGG-eGFP ( Fig. 9B-9C), GFP-positive cells were detected. Surprisingly, 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 ( Fig. 9 E-9F, 9P-9R). In liver samples from any animal injected with saline or ssAAV2N587myc-CAGG-eGFP viral vector alone ( Fig. 9 A, 9D, 9G-9L)), in liver samples taken from wild-type C57BL / 6 animals injected with ssAAV2N587myc-CAGG-eGFP in combination with a bispecific anti-myc-ASGR1 antibody ( Fig. 9 M-90), no or almost no GFP was detected. In conclusion, the combination of ssAAV2N587myc-CAGG-eGFP viral vector and bispecific anti-myc-ASGR1 antibody infects only those (liver) cells expressing hASGR1, strongly suggesting that the ssAAV2N587myc-CAGG-eGFP viral vector is inactivated by modification of the capsid protein, for example, the natural tropism of scAAV viral vectors can be reduced to eliminated, for example, with the c-myc epitope, and such viral vectors can be specifically reactivated, for example, by bispecific anti-myc-ASGR1 antibodies, for example, specifically retargeted, for example, to liver cells in vivo.

[0354] Further experiments were performed with pseudotyped AAV9 viral vectors using mice genetically modified to express hASGR1 in hepatocytes on a C57BL / 6 background. 11 These mice were injected intravascularly with wild-type AAV9-CAGG-eGFP, or AAV9A589myc-CAGG-eGFP viral vectors in combination with a bispecific anti-myc-ASGR1 antibody at a 1:100 ratio of viral genome to antibody molecules (titrated by qPCR). Controls included mice injected with PBS, or AAV9 N587myc-CAGG-eGFP viral particles in combination with an irrelevant bispecific anti-myc hCD3 antibody. Four weeks after injection, mice were sacrificed. The livers were fixed with 10% formalin and sent to HistoWiz.Inc (New York, NY) for GFP staining. GFP-positive cells ( Fig.10A and 10D). GFP was not detected or barely detected in liver samples from any animal injected with saline or AAV9 N587myc-CAGG-eGFP viral particles in combination with a bispecific anti-myc hCD3 antibody ( Fig.10 B and 10C). In summary, similar to AAV2, the natural tropism of AAV9 can be reduced or eliminated by modification of the capsid protein (e.g., by insertion of the c-myc epitope), and such modified viral vectors, such as AAV9-A589myc, can be retargeted to specific cell types using corresponding bispecific anti-epitope-cell-specific marker binding proteins, such as bispecific anti-myc-ASGR1 antibodies against hepatocytes.

[0355] Anti-myc scFc attached to anti-hASGR1 IgG4 knob-in-hole Fc format

[0356] To determine whether various multispecific binding molecule formats could mediate AAV infection, an anti-myc scFv was fused to the C-terminus of one chain of the knob-into-hole anti-hASGR1 IgG4 antibody (see Fig.11 Left). Specifically, a gblock encoding an anti-myc scFv (SEQ ID NO: 28) flanked by homology arms to 1459-1478 and 1479-1498 of pRG7078 encoding an anti-hASGR1 stealth knob IgG4 heavy chain was provided by Integrated DNA Technologies (Skokie, Illinois). The encoding nucleotide sequences (NA) of the amino acid (AA) sequences of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the scFv (SEQ ID NO: 37) encoded by SEQ ID NO: 28 are provided in Table 3.

[0357] Table 3: SEQ ID NOs related to scFv as shown in SEQ ID NO: 37

[0358] scFv HCVR LCVR HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 AA 37 29 30 31 32 33 34 35 36 NA 38 39 40

[0359] The gblock was cloned into pRG7078 encoding the anti-hASGR1 stealth knob IgG4 heavy chain to prepare pRG7078 encoding the anti-hASGR1 stealth knob IgG4 anti-myc scFv fusion polypeptide "anti-hASGR1-IgG4-Fc / anti-myc bispecific". The pRG7078 plasmid modified with c-myc, the unmodified pRG7078 anti-hASGR1 IgG4 stealth hole star, and the plasmid containing the antibody light chain were transfected into 293T cells and cultured with OPTI-MEM (Catalog 31985070, Thermo Fisher). Four days after transfection, the culture medium was collected and the anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecules were purified using a protein A column (Catalog #89948, Thermo Fisher).

[0360] To generate the pseudotyped AAV8 viral vector pAAV RC8N590myc, a gblock with the c-myc epitope (SEQ ID NO: 22) inserted between N590 and T591 of the VP1 capsid protein of AAV8 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 make pAAV RC8N590myc (SEQ ID NO: 24).

[0361] 293T-hASGR1 cells (which are 293T cells genetically modified to express (h)ASGR1 on the cell surface) were incubated with either a mixture comprising different ratios of AAV8-N590Myc-CAGG-eGFP and anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecules (1:0, 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50 or 1:100) or AAV8CAGG eGFP viral genome. Three days after infection, GFP expression was analyzed by FACS. Three days after infection, GFP expression was analyzed by FACS. GFP-positive 293T-hASGR1 cells (2.12% to 22.2%; Fig.12D-12K), which was comparable to 293T-hASGR1 cells incubated with wild-type AAV8-CAGG-eGFP (46.1%) ( Fig.12 A). Mock-infected cells ( Fig.12 B) and incubation with AAV8-N590Myc-CAGG-eGFP alone did not result in GFP expression by 293T-hASGR1 cells ( Fig.12 C).

[0362] To determine whether the anti-hASGR1-IgG4-Fc / anti-myc bispecific binding protein can retarget AAV8N590myc-CAGG-eGFP viral particles to hepatocytes expressing hASGR1 in vivo, 1x10 11 Mice genetically modified to express hASGR1 on a C57BL / 6 background were injected intravascularly with wild-type AAV8-CAGG-eGFP (titrated by qPCR), or AAV8N590myc-CAGG-eGFP virus particles in combination with anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecules at a ratio of 1:12 of viral genome to binding protein molecules. Control animals were injected with AAV8N590myc CAGG-eGFP virus particles in combination with an irrelevant anti-hCD3xanti-myc bispecific binding molecule (which has a similar format to the anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecule). Three weeks after injection, mice were sacrificed and perfused transcardially with 4% PFA. Organs of liver, kidney, and heart were collected and dehydrated in 15% sucrose, followed by 30% sucrose. The organs were then cryosectioned on slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA) and Alexa-488-conjugated anti-chicken secondary antibody (Jackson ImmunoResearch Labs, Inc. West Grove, PA). Fig.13 A-13C), or AAV8N590myc-CAGG-eGFP virus particles combined with anti-hASGR1-IgG4-Fc / anti-myc bispecific binding molecules ( Fig.13 In the livers of those animals injected with AAV8N590myc-CAGG-eGFP virus particles combined with an irrelevant anti-hCD3 / myc binding protein, GFP-positive cells were detected. In the liver samples from any animal injected with AAV8N590myc-CAGG-eGFP virus particles combined with an irrelevant anti-hCD3 / myc binding protein, GFP was not detected or was barely detected ( Fig.13D-13F). In summary, epitope-tagged AAV8 (e.g., AAV8N590myc) can be retargeted to specific cell types using bispecific binding molecules that specifically bind to both an epitope and a cell-specific marker expressed by the target cell type.

[0363] Retargeting of viral vectors to the intestine and / or pancreas

[0364] 293T cells genetically modified to express human exonucleoside triphosphate diphosphohydrolase 3 (hENTPD3) on the cell surface ("293T-ENTPD3") were incubated with a mixture comprising different ratios of AAV2-N587Myc-CAGG-eGFP or AAV2-CAGG-GFP viral genomes and a bispecific binding molecule formed by fusing 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, GFP-positive 293T-ENTPD3 cells were detected at levels (1.48% to 16%; Fig.14 C-14J) and 293T-ENTPD3 cells incubated with wild-type AAV2-CAGG-eGFP (66%) were comparable ( Fig.14 A). Incubation with AAV2-N587Myc-CAGG-eGFP alone resulted in very low levels of GFP expression by 293T ENTPD3 cells ( Fig.14 B).

[0365] It was determined that ENTPD3 is expressed in the intestinal mucosa (data not shown). To determine whether anti-hENTPD3-IgG4-Fc / anti-myc binding protein can retarget AAV2N587myc-CAGG-eGFP viral particles to intestinal cells expressing ENTPD3 in vivo, 5x10 11C57BL / 6 mice were injected intravascularly with AAV2N587myc-CAGG-eGFP viral particles combined with anti-hENTPD3-IgG4-Fc / anti-myc binding protein at a 1:20 ratio of viral genome to binding protein molecule (titrated by qPCR). Control mice were injected with PBS, wild-type AAV9, or AAV2N587myc-CAGG-eGFP viral particles combined with irrelevant binding protein molecules. Three weeks after injection, mice were sacrificed. The liver, intestine, and pancreas were fixed with 10% formalin and sent to HistoWiz.Inc (New York, NY) for GFP staining. GFP-positive cells ( Fig. 15B (ii)), but not in livers from mice injected with PBS, or AAV2N587myc-CAGG-eGFP viral particles in combination with an irrelevant binding molecule or anti-hENTPD3-IgG4-Fc / anti-myc binding protein ( Fig.15A (iii)-15A(iv)), indicating that the AAV2N587myc 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 AAV2N587myc-CAGG-eGFP viral particles combined with hENTPD3-IgG4-Fc / anti-myc binding protein ( Fig. 15B (i)-15B(iv)). GFP was detected in pancreatic islets of mice injected with AAV2N587myc-CAGG-eGFP viral particles combined with anti-hENTPD3-IgG4-Fc / anti-myc binding protein. ( Fig. 15C (iv)). WT AAV9 infects both islet cells and non-islet cells ( Fig. 15C (ii)). GFP was not detected in pancreatic samples from mice injected with saline, or AAV2N587myc-CAGG-eGFP viral particles in combination with an irrelevant binding protein. ( Figure 7 C(i) and 7C(iii)). In summary, the natural tropism of AAV can be reduced or eliminated by inserting a heterologous epitope and retargeting the same or other cells using a multispecific binding protein that binds to the epitope tag as well as a marker expressed by the retargeted cell or tissue.

[0366] This example demonstrates that adeno-associated viral vectors of several different serotypes can be genetically modified with a heterologous epitope (e.g., c-myc) as described herein to inactivate infectivity, and that bispecific antibodies (regardless of the bispecific format) that recognize both the heterologous epitope (e.g., c-myc) and a marker expressed by target cells can be used to retarget the viral vector to deliver the target nucleic acid.

[0367] Example 4: Use of Genetically Modified AAV-N587Myc Viral Vectors to Deliver Therapeutic Cargo to Specific Cells way

[0368] This example demonstrates the ability of the AAV-N587Myc viral vector to specifically deliver therapeutic cargo to targeted cell types, such as one or more suicide genes, biotherapeutics (e.g., antibodies), CRISPR / Cas gene editing systems, shRNA, etc. Specifically, this example describes the delivery of suicide genes, antibody coding sequences, or CRISPR / Cas gene editing systems to cells expressing targeting ligands.

[0369] Suicide gene delivery to cells expressing targeting ligands

[0370] To test the ability of the scAAV2-N587Myc viral vector to deliver suicide genes to specific cells, the HER2 + Xenograft nude mouse model of breast cancer.

[0371] Viral vectors: scAAV2-N587Myc or ssAAV2-N587Myc viral vectors (scAAV2-N587Myc-EGFP or ssAAV2-N587MycEGFP) carrying the reporter EGFP gene were generated as described in Example 1. scAAV2-N587Myc or ssAAV2-N587Myc viral vectors carrying a suicide gene (SG) were similarly generated. Briefly, 293T17 cells were transfected with: (1) pAd Helper and (2) pAAV RC2 (encoding wild-type capsid) or pAAV RC2-N587Myc vector (encoding capsid modified with a c-myc epitope) as described above, and (3) pAAV vector carrying a suicide gene, such as a cytosine deaminase gene, a herpes simplex virus thymidine kinase gene, under the control of a promoter, such as CMV. ssAAV-N587MycSG or scAAV N587SG viral vectors were isolated and titered as described in Example 1.

[0372] 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, while BEAS-2b is an immortalized bronchial epithelial HER2-negative cell line (Jia LT et al. (2003) Cancer Res. 63:3257-3262; Kern JA et al., (1993) Am J Respir Cell Mol Biol 9:448-454; Martinez-Ramirez A et al., (2003) Cancer Genet Cytogenet. 2003; 141:138-142). All of these cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in the culture medium recommended by ATCC.

[0373] Mice: Female nude mice were obtained at 6 to 8 weeks of age and housed under specific pathogen-free conditions. On day 0, mice were simultaneously (1) injected with 10 7 BT474, SK-BR-3, Calu-3, A-673 or HeLa tumor cells were injected subcutaneously and (2) treated intravenously with a bispecific anti-myc-HER2 antibody and an ss- or scAAV-N587Myc viral vector carrying a reporter gene (e.g., EGFP) or a suicide gene. Serving as controls were untreated animals (animals injected with tumor cells alone), animals injected with wild-type ss- or scAAV viral vectors carrying a reporter gene or a suicide gene, animals injected with a bispecific anti-myc-HER2 antibody alone, or animals injected with an ss- or scAAV-N587Myc viral vector carrying a reporter gene or a suicide gene alone. All animals were treated with appropriate prodrugs 1 day after injection and treatment. The size of each tumor was measured twice a week using a caliper and the tumor volume was calculated as length × width 2 ×0.52. At onset, tumor ulceration, tumor diameter 15 mm or tumor volume 1000 mm 3The mice were sacrificed at 4 hrs and the date of sacrifice was recorded as the date of death. The liver, spleen, kidney and tumors of animals injected with wild-type ss- or scAAV, or ss- or scAAV-N587Myc viral vectors carrying the reporter gene were fixed and the expression of the reporter gene was visualized.

[0374] Although 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 use of a viral vector as described herein comprising a heterologous epitope such as c-myc and a bispecific antibody that specifically binds to the targeting ligand and the heterologous epitope to deliver the suicide gene to cells expressing a targeted HER2 ligand. In additional experiments, a viral vector as described herein comprising a heterologous epitope and a bispecific antibody that specifically binds to a heterologous epitope (e.g., c-myc) and a target receptor were used to deliver the suicide gene to another cell type expressing one or more other target ligands. Illustrative and non-limiting examples of receptors suitable for targeting include those that mediate endocytosis of viral vectors, such as carcinoembryonic antigen (CEA) (Qiu Y et al. (2012) Cancer Lett. 316:31-38), and vascular endothelial growth factor receptor (VEGFR) (Leng A et al. (2013) Tumour Biol. 32:1103–1111; Liu T et al. (2011) Exp Mol Pathol. 91:745–752). Additional receptors that can be targeted include: epidermal growth factor receptor (EGFR) (Heimberger AB et al. (2009) Expert Opin Biol Ther. 9:1087-1098), cluster of differentiation 44s (CD44s) (Heider KH et al. (2004) Cancer Immunol Immunother. 53:567-579), cluster of differentiation 133 (CD133 also known as AC133) (Zhang SS et al. (2012) BMC Med.; 10:85), folate receptor (FR) (Duarte S et al., (2011) J Control Release 149(3):264-72), transferrin receptor (TfR) or cluster of differentiation 71 (CD71) (Habashy HO et al., Breast Cancer Res Treat. 119(2):283-93), mucin (Torres MP et al., (2012) Curr Pharm. Des. 2012; 18(17): 2472-81), stage-specific 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

1. A composition comprising (i) a recombinant adeno-associated virus (AAV) vector comprising AAV capsid encapsulating the target nucleotide, wherein the AAV capsid comprises a recombinant AAV capsid protein modified to comprise a heterologous epitope, wherein the heterologous epitope consists of the amino acid sequence EQKLISEEDL, and wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express the heterologous epitope; (ii) a multispecific binding molecule comprising (a) an antibody paratope that specifically binds to the amino acid sequence EQKLISEEDL; and (b) a retargeting ligand that specifically binds to a cell surface protein or marker, wherein the AAV capsid exhibits: - a tropism that is reduced to eliminated in the absence of the multispecific binding molecule; and - an altered tropism compared to a reference AAV capsid when combined with said multispecific binding molecule, wherein said reference AAV capsid comprises a reference AAV capsid protein that is identical to said recombinant AAV capsid protein but lacks said heterologous epitope.

2. The composition of claim 1, wherein the recombinant AAV capsid protein further comprises a mutation in addition to the heterologous epitope.

3. The composition of claim 1, wherein the heterologous epitope is inserted such that the insertion partially reduces the tropism of the AAV capsid compared to the reference AAV capsid lacking the heterologous epitope, and the recombinant AAV capsid protein further comprises a mutation in addition to the insertion of the heterologous epitope, which further reduces and / or eliminates the tropism of the AAV capsid compared to the reference AAV capsid lacking the mutation, wherein the mutation comprises a substitution, a deletion, an insertion in addition to the insertion of the heterologous epitope.

4. The composition of claim 1, wherein the altered tropism when combined with the multispecific binding molecule refers to restoration and redirection of the tropism of the AAV capsid compared to the tropism of the reference AAV capsid.

5. The composition according to claim 1, wherein (i) the heterologous epitope comprises a linker, and / or (ii) the heterologous epitope is flanked by and / or operably linked to at least 5 contiguous amino acids of the recombinant AAV capsid protein.

6. The composition of claim 1, wherein the target nucleotide: (i) is under the control of a non-human promoter; or (ii) flanked by AAV ITR sequences; or (iii) under the control of a non-human promoter and flanked by AAV ITR sequences.

7. The composition of claim 6, wherein the non-human promoter is 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.

8. The composition of claim 1, wherein the target nucleotide is (iv) a reporter gene; or (v) a therapeutic gene selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or a fragment thereof, a nucleotide encoding a CRISPR / Cas system or a portion thereof, a nucleotide encoding an antisense RNA, a nucleotide encoding an siRNA, and a combination thereof.

9. The composition of claim 8, wherein the reporter gene encodes green fluorescent protein.

10. The composition of claim 1, wherein the multispecific binding molecule is a bispecific binding molecule.

11. The composition of claim 1, wherein the antibody paratope is an Fv domain.

12. The composition of claim 11, wherein the Fv domain is fused directly to the heavy chain constant domain.

13. The composition of claim 1, wherein the retargeting ligand is an antibody or a portion thereof.

14. The composition of claim 13, wherein the antibody is a bispecific antibody, wherein the antibody paratope and the retargeting ligand each comprise a different Fv domain fused to a first heavy chain constant domain and a second heavy chain constant domain.

15. The composition of claim 14, wherein the first heavy chain and the second heavy chain bind to Protein A with different binding affinities.

16. A composition according to claim 13, wherein the retargeting ligand comprises a tetrameric antibody structure, which comprises two identical immunoglobulin heavy chains and two identical immunoglobulin light chains, and wherein the antibody paratope that binds to the heterologous epitope is attached to the C-terminus or N-terminus of one or both immunoglobulin heavy chains and / or to the C-terminus or N-terminus of one or both immunoglobulin light chains.

17. The composition of claim 16, wherein the antibody paratope is a scFv.

18. The composition of claim 17, wherein the scFv comprises the amino acid sequence shown in SEQ ID NO:

37.

19. The composition of claim 1, wherein the cell surface protein is a receptor; and / or wherein the retargeting ligand binds to a receptor expressed on the surface of a mammalian or human cell.

20. The composition of claim 1, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9, and / or wherein the heterologous epitope is inserted after a position selected from the group consisting of I-453 of AAV2, I-587 of AAV2, I-585 of AAV6, I-590 of AAV8, I-453 of AAV9, I-589 of AAV9, and / or replaces the position.

21. The composition of claim 1, wherein the AAV capsid is a chimeric capsid comprising a reference AAV capsid protein of the same serotype at a certain ratio, wherein the reference AAV capsid protein does not comprise the heterologous epitope.

22. The composition of claim 1, wherein the recombinant AAV capsid protein is derived from a chimeric AAV capsid gene modified to express the heterologous epitope, wherein the chimeric AAV capsid gene comprises a plurality of nucleic acid sequences, wherein each of the plurality of nucleic acid sequences encodes a portion of a capsid protein of a different AAV serotype, and wherein the plurality of nucleic acid sequences together encode a chimeric AAV capsid protein.

23. A pharmaceutical composition comprising (i) the recombinant AAV vector and (ii) the multispecific binding molecule of claim 1, and (iii) a pharmaceutically acceptable carrier.

24. Use of the composition according to any one of claims 1 to 22 or the pharmaceutical composition according to claim 23 in the preparation of a medicament for delivering the target nucleotide to a target cell expressing a cell surface protein or marker.

25. The use according to claim 24, wherein: (a) the delivering is to a human subject; or (b) The target cell is a human cell.

26. The use according to claim 25, wherein the target cells are human neuronal cells.

27. The use according to claim 25, wherein the target cell is a human muscle cell.

28. The use according to claim 25, wherein the target cell is selected from the group consisting of liver cells, brain cells, T cells, kidney cells, intestinal cells, pancreatic cells, cancer cells and cells infected by pathogens.

29. The use according to claim 25, wherein: (a) the target cell is a human hepatocyte, wherein the retargeting ligand binds to human asialoglycoprotein receptor 1 (hASGR1) expressed by the human hepatocyte; or (b) the retargeting ligand binds to a GABA receptor expressed by a human neuronal cell; or (c) the retargeting ligand binds to a transferrin receptor expressed by human neuronal cells; (d) the target cell is a human T cell, wherein the retargeting ligand binds to CD3 expressed by the human T cell; or (e) the target cell is a human T cell, wherein the retargeting ligand binds to CD3ε expressed by the human T cell; or (f) the target cell is a human hematopoietic cell, wherein the retargeting ligand binds CD34; or (g) the target cell is a human cancer cell, wherein the retargeting ligand binds to a tumor-associated antigen; or (h) the target cell is a human cancer cell, wherein the retargeting ligand binds to a tumor-associated antigen, wherein the tumor-associated antigen is E6, E7 or Her2; or (i) the cell surface protein is human glucagon receptor (hGCGR); or (j) The target cell is an intestinal cell or a pancreatic cell, wherein the receptor is ENTPD3.

30. The use according to claim 25, wherein: (i) the retargeting ligand binds to CD20; or (ii) the retargeting ligand binds to the human glucagon receptor; or (iii) the retargeting ligand specifically binds to CD63 or human exonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

31. An in vitro or ex vivo method for delivering a target nucleotide to a target cell expressing a cell surface protein, wherein the method comprises contacting the target cell with a composition according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23, wherein the multispecific binding molecule comprises a retargeting ligand that binds to the cell surface protein.

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