RU2809246C2 - Recombinant viral vectors with modified tropism and ways of their use for targeted introduction of genetic material into human cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a composition containing a recombinant adeno-associated virus (AAV) vector containing an AAV capsid that encapsulates a nucleotide of interest, and a polyspecific binding molecule containing an antibody paratope that specifically binds a heterologous epitope, and a retargeting ligand, which specifically binds a cell surface protein or marker, as well as a method of delivering the nucleotide of interest to a target cell expressing the cell surface protein using it.

EFFECT: invention is effective for targeted introduction of genetic material into a cell.

43 cl, 15 dwg, 3 tbl, 4 ex

Description

LINK TO SEQUENCE LIST SUBMITTED AS A TEXT FILE VIA EFS WEB

[0001] The sequence listing recorded in file 10335WO01_ST25.txt is 88 kilobytes in size, was created on June 27, 2018, and is incorporated herein by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention herein generally relates to recombinant viral vectors with modified tropism and compositions containing them, useful for the targeted introduction of genetic material into cells and/or tissues.

BACKGROUND OF THE INVENTION

[0003] Gene delivery to specific target cells has become one of the most important technologies in modern medicine for the diagnosis and gene therapy of various chronic and genetic diseases. Until now, progress in the clinical application of gene therapy has been limited by the lack of ideal carrier vehicles for gene delivery. To achieve therapeutic success, gene delivery vehicles must be able to transduce target cells while avoiding transduction of non-target cells. In particular, when the native tropism of a virus does not meet current therapeutic needs, there is a need for recombinant viral vectors in which the natural tropism is eliminated or reduced and the desired tropism is successfully engineered. (Buchholz et al.)

[0004] In recent years, the greatest progress in vector development has been made using non-enveloped viruses (e.g., viruses containing a capsid formed by non-enveloped viral capsid proteins (e.g., lipid bilayer)), such as adeno-associated viruses (AAVs) and adenoviruses ( Ad), as well as enveloped viruses (e.g., viruses in which the capsid is surrounded by a lipid bilayer), such as retroviruses, lentiviruses, and herpes simplex virus. AAV-based vectors have been the subject of much research since AAVs are non-enveloped viruses that are only weakly immunogenic but are capable of transducing a wide range of species and tissues in vivo without evidence of toxicity.

[0005] AAVs are small, single-stranded, non-enveloped DNA viruses. The AAV genome is 4.7 kb. and is characterized by two inverted terminal repeats (ITRs) and two open reading frames that encode Rep proteins and Cap proteins, respectively. The two ITRs are the only cis elements required for AAV replication, packaging, and integration. The Rep reading frame encodes four proteins with molecular masses of 78 kDa, 68 kDa, 52 kDa and 40 kDa. These proteins function primarily in regulating AAV replication and AAV integration into host cell chromosomes. The Sar reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83-85 kDa (VP1), 72-73 kDa (VP2) and 61-62 kDa (VP3). More than 80% of all proteins in the AAV virion are VP3; in mature virions, VP1, VP2 and VP3 are found at a relative abundance of approximately 1:1:10. In vitro, the 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 in Kotin et al. (1994) Hum. Gene Ther. 5:793).

[0006] Among all known AAV serotypes, AAV2 is perhaps the best characterized serotype because its infectious clone was the first to be established. (Samulski et al. (1982) Proc. Natl. Acad. Sci. USA 79:2077-2081). Subsequently, complete sequences for 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). Typically, all AAVs share greater than 80% nucleotide sequence identity.

[0007] AAV is a promising vector for human gene therapy because, unlike other viral vectors, AAV has not been found 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). Moreover, AAV safely transdupes postmitotic tissues with relatively low immunogenicity and is able to integrate into host chromosomes in a site-specific manner and into cultured tissue cells on chromosome 19 if Rep proteins are provided by another vector. (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 Integrated AAV genomes have been shown to mediate long-term gene expression in a number of tissues, including muscle, liver, and brain (Fisher (1997) Nature Med. 3(3):306-312; Snyder et al. (1997) Nature Genetics 16: 270-276; Xiao et al. (1997) Experimental Neurology 144:113-124; Xiao et al. (1996) J. Virol. 70(11):8098-8108).

[0008] A number of viruses, including AAV, infect cells through virus/ligand:cell/receptor interactions, which ultimately result in endocytosis of the virus by the infected cell. This ligand:receptor interaction is the subject of much of the research on viral vectors, for example it can be manipulated to redirect the natural tropism of the virus from a cell naturally permissive to infection by the wild-type virus to a target cell, for example through a receptor that is expressed by the target cell .

[0009] Theoretically, retargeting a vector to any protein or cell surface marker should result in infection of the target cell, since most receptors or cell surface markers are involved in endocytosis pathways, either constitutive (e.g., for recycling) or ligand-induced (e.g., receptor-mediated). These receptors cluster in clathrin-coated cavities, enter the cell through clathrin-coated vesicles, pass through an acidic endosome in which the receptors are sorted, and then either return to the cell surface, remain intracellularly stored, or are degraded in lysosomes. Thus, viral vector retargeting platforms often seek to eliminate the natural tropism of the viral vector and redirect the viral vector to a receptor or marker expressed exclusively or predominantly by the target cell. Many of the advances in targeted gene therapy using viral vectors can be briefly described as non-recombinatorial (other than genetic) or recombinatorial (genetic) modification of a viral vector that results in pseudotyping, tropism expansion, and/or retargeting of the viral vector's natural tropism (reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[0010] Non-genetic approaches typically use an adapter that recognizes both the surface protein of the wild-type (unmodified) virus and the target cell. Soluble pseudoreceptors (for wild-type virus), polymers such as polyethylene glycol, and antibodies or parts thereof have been used as the virus-binding domain of the adapters, while natural ligands such as peptides or vitamins have been used for the cell-binding domain of the adapters described above , as well as antibodies and their parts. In this approach, retargeting of the viral vector to the target cell can be accomplished following binding of the vector:adapter complex to a protein expressed on the surface of the target cell, such as a cell surface protein.

[0011] This approach has been used for AAV (Bartlett et al. (1999) Nat. Biotechnol. 74:2777-2785), adenoviruses (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), herpesviruses (Nakano et al. ( 2005) Mol. Ther. 11:617-24) and paramyxoviruses (Bian et al. (2005) Cancer Gene Ther. 12:295-303; Bian et al. (2005) Int. J. Oncol. 29:1359-69 ), coronaviruses (Haijema et al. (2003) J. Virol. 77:4528-43]8; Wurdinger et al. (2005) Gene Therapy 12:1394-1404).

[0012] A more popular approach has been the recombinatorial genetic modification of viral capsid proteins and thus the surface of the viral capsid. In indirect recombinatorial approaches, the viral capsid is modified with a heterologous scaffold, which is then attached to an adapter. The adapter 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) Frameworks such as (1) Fc-binding molecules (e.g. Fc receptors, protein A, etc.) that bind to Fc adapter antibodies, (2) (strept)avidin, which binds to biotinylated adapters, (3) biotin, which binds to adapters fused to (strept)avidin, and (4) protein:protein binding pairs that form isometric peptide bonds, such as SpyCatcher, which binds the SpyTagged adapter, were included in 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).

[0013] In a direct recombinatorial targeting approach, a targeting ligand is directly inserted into or coupled to the viral capsid, that is, viral capsid proteins are modified to express a heterologous targeting ligand. The ligand then redirects, eg binds, a receptor or marker predominantly 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 recombinatorial approaches 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), retrovirus (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), poxvirus (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), paramyxovirus (Nakamura and Russell (2004) Expert Opinion on Biological Therapy 4:1685-1692; Hammond et al (2001) Journal of Virology 75:2087-2096;Galanis (2010) Clinical Pharmacology and Therapeutics 88:620-625; Blechacz and Russell (2008) Current Gene Therapy 8:162–175; Russell and Peng (2009) Current Topics in Microbiology and Immunology 330:213-241) and herpesvirus (Shah and Breakefield (2006) Current Gene Therapy 6:361-370; Campadelli-Fiume et al. (2011) Reviews in Medical Virology 21 :213-226).

[0014] Each of the three approaches has advantages and disadvantages. The main advantage of the direct recombinatorial approach is that the specificity of the viral vector is inherent in the viral genome and can be maintained during replication. However, for direct and indirect recombinatorial approaches, the ability to genetically modify a virus requires conservation of the capsid structure, and the targeting ligand or scaffold must be positioned in a position that will allow and properly display the targeting ligand or scaffold, thus limiting the repertoire of relevant ligands or scaffolds that can be used . As such, recombinatorial retargeting methods are limited to naturally occurring molecules useful as targeting ligands, resulting in the inclusion of other binding ligands such as antibodies or parts thereof. Both non-recombinatory and recombinatory adapter platforms have the advantage of flexibility in the adapter used. However, it is difficult to achieve optimal transduction efficiency with these two-component systems.

[0015] Provided herein is a viral retargeting strategy that overcomes the problems inherent in previous recombinatorial retargeting strategies by inserting a heterologous epitope into the viral capsid. The current recombinant viral capsid exhibits reduced natural tropism to the point of elimination, which is restored and redirected after combination with a polyspecific, optionally bispecific, binding molecule containing an antibody paratope, for example, Fv, which specifically binds a heterologous epitope, and a retargeting ligand that specifically binds target cell, especially at certain ratios of viral vector:polyspecific binding molecule.

BRIEF DESCRIPTION OF THE INVENTION

[0016] Disclosed herein are recombinant viral capsid proteins, viral capsids containing recombinant viral capsid proteins, viral vectors containing a nucleotide of interest embedded in a recombinant viral capsid; wherein said capsid proteins, capsids and viral vectors are genetically modified to include (display) a heterologous epitope, wherein the heterologous epitope (part thereof or in combination with a viral capsid protein) forms a binding pair with an antibody paratope, and wherein the recombinant viral capsid protein/capsid/vector may further comprise a mutation, insertion or deletion at an amino acid position that is involved in receptor binding, e.g., the (natural) tropism of the viral capsid protein/capsid/vector, such that the recombinant viral capsid protein or a viral capsid containing the recombinant viral capsid protein is characterized by reduced up to eliminated (natural) tropism (for example, has, in the absence of a polyspecific, not necessarily bispecific, binding molecule, a lower transduction efficiency than the transduction efficiency of a reference viral capsid protein/capsid/vector that lacks a heterologous epitope, or an undetectable transduction efficiency in the absence of a polyspecific, optionally bispecific, binding molecules). Such reduced to eliminated (natural) tropism of the recombinant viral capsid protein/capsid/vector can be enhanced or restored in the presence of a suitable multispecific, optionally bispecific, binding fragment. Accordingly, also described are compositions containing (1) recombinant viral vectors having a capsid containing the recombinant capsid protein described herein, and (2) a multispecific, optionally bispecific, binding molecule containing an antibody paratope and a targeting ligand, including compositions containing certain ratios of viral vector: polyspecific binding molecule; and their methods of use for directing and/or introducing genetic material into a target cell are also described herein. Methods for retargeting a recombinant viral vector, for example, to target delivery of a nucleotide of interest to a target cell, including contacting the recombinant viral vector with a polyspecific, optionally bispecific, binding molecule, and methods for producing the recombinant viral vector are also described.

[0017] Disclosed herein are recombinant viral capsid proteins containing an epitope that is heterologous to the capsid protein, wherein the epitope or portion thereof specifically binds an antibody paratope, and wherein the recombinant viral capsid protein or a viral capsid containing the recombinant viral capsid has natural tropism reduced to the point of being eliminated, for example, in the absence of a polyspecific, optionally bispecific, binding fragment.

[0018] 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 relative to a reference viral capsid that lacks the heterologous epitope, e.g. , the viral capsid contains a mutation involving the insertion of an epitope at an amino acid position and/or the replacement of an amino acid with an epitope at an amino acid position, where the mutation reduces or eliminates the (natural) tropism of the capsid protein, for example, in the absence of a polyspecific, optionally bispecific, binding moiety. In some embodiments, a heterologous epitope is inserted (displayed) by a viral capsid protein such that the insertion and/or display partially reduces the (natural) tropism of the recombinant viral capsid relative to a reference viral capsid that lacks the heterologous epitope, e.g., in the absence of a polyspecific, optionally a bispecific binding fragment, and the viral capsid further comprises an additional mutation (e.g., substitution, deletion, insertion other than a heterologous epitope insertion) that further reduces and/or eliminates the (natural) tropism of the recombinant viral capsid or recombinant viral vectors containing them , for example, in the absence of a polyspecific, optionally bispecific, binding fragment, compared to a reference viral capsid that lacks this mutation.

[0019] In general, the recombinant viral capsid proteins described herein can be produced using a capsid gene, for example, they are encoded by a capsid gene modified to express an epitope, and/or are a genetically modified non-enveloped virus that typically infects cells human, or serotypes of non-enveloped viruses that typically infect 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, e.g., is encoded by a capsid gene modified to express an epitope, and/or is a genetically modified adeno-associated virus (AAV) capsid protein of serotype AAV that infects primates, where optionally the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In some embodiments, the recombinant viral capsid protein is derived from an AAV2, AAV6, AAV8, or AAV9 capsid gene, such as a genetically modified AAV2 capsid protein, a genetically modified AAV6 capsid protein, a genetically modified AAV8 capsid protein, or a genetically modified AAV9 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, e.g., is 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, for which the amino acid sequence of the protein is Wild-type AAV2 VP1 is provided, respectively, as SEQ ID NO: 1. In some embodiments, the recombinant viral capsid protein is derived from an AAV6 capsid gene, e.g., is encoded by an AAV6 capsid gene modified to express an epitope, and/or is a genetically modified capsid an AAV6 VP1, VP2, and/or VP3 protein, for which the amino acid sequence of the wild-type AAV6 VP1 protein is provided by 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 for epitope expression, and/or is a genetically modified AAV8 VP1, VP2 and/or VP3 capsid protein, for which the amino acid sequence of the wild-type AAV VP1 protein is set forth, respectively, as SEQ ID NO: 21. In some embodiments, the recombinant viral capsid protein derived from an AAV9 capsid gene, for example, is encoded by an AAV9 capsid gene modified to express an epitope, and/or is a genetically modified AAV9 capsid protein VP1, VP2 or VP3, for which the amino acid sequence of the wild-type AAV9 VP1 protein is provided, respectively, as SEQ ID NO: 5. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, e.g., is 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. In some embodiments, the recombinant viral capsid protein is derived from an AAV6 capsid gene, e.g., is 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. In some embodiments, the recombinant viral capsid protein is derived from an AAV8 capsid gene, e.g., is 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. In some embodiments, the recombinant viral capsid protein is derived from an AAV9 capsid gene, e.g., is encoded by an AAV9 capsid gene modified to express an epitope, and/or is a genetically modified AAV9 capsid protein VP1, VP2, and/or VP3.

[0020] In some embodiments, the recombinant viral capsid protein is produced by, for example, encoded by a chimeric AAV capsid gene modified to express an epitope, wherein the chimeric AAV capsid protein 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.

[0021] Typically, a recombinant viral capsid protein, as described herein, contains a heterologous epitope inserted into and/or displayed on the recombinant capsid protein such that the heterologous epitope itself reduces and/or eliminates the natural tropism of the recombinant capsid protein or capsid containing it, compared to a reference capsid lacking the heterologous epitope, or a capsid including the reference capsid, respectively. In some embodiments, the heterologous epitope is inserted (displayed) into a region of the capsid protein involved in the natural tropism of the reference wild-type capsid protein, for example, into a region of the capsid protein involved in cell targeting. In some embodiments, the heterologous epitope is inserted into and/or displayed by the knob 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 on the heparin binding site of the AAV capsid protein. In some embodiments, the heterologous epitope is inserted into and/or displayed on the heparin binding site of the AAV2 capsid protein. In some embodiments, the heterologous epitope is inserted into and/or displayed on the heparin binding site of the AAV6 capsid protein. In some embodiments, the heterologous epitope is inserted into and/or displayed on the heparin binding site of the AAV8 capsid protein. In some embodiments, the heterologous epitope is inserted into and/or displayed on the heparin binding site of the AAV9 capsid protein. In some embodiments, (i) the viral capsid protein is derived from the AAV2 capsid gene and the epitope is inserted after and/or replaces amino acids at position I453 or I587 of the AAV2 VP1 capsid protein and/or amino acids at corresponding positions of the AAV2 VP2 and/or VP3 capsid proteins their; (ii) the viral capsid protein is derived from the AAV6 capsid gene and the epitope is inserted after and/or replaces the amino acid at position I585 of the AAV6 VP1 capsid protein and/or amino acids at the corresponding positions of the AAV6 VP2 and/or VP3 capsid protein; (iii) the viral capsid is derived from the AAV8 capsid gene and the epitope is inserted after and/or replaces the amino acid at position I590 of the AAV8 VP1 capsid protein and/or amino acids at the corresponding positions of the AAV8 VP2 and/or VP3 capsid proteins, or (iv) the viral capsid the protein is derived from the AAV9 capsid gene and the epitope is inserted after and/or replaces amino acids at position I453 or I589 of the AAV9 VP1 capsid protein and/or amino acids at the corresponding positions of the AAV9 VP2 and/or VP3 capsid proteins. In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) an amino acid selected from the group consisting of G453 of the AAV2 VP1 capsid protein (or corresponding positions of the VP2 and/or VP3 capsid proteins encoded by the same capsid gene, or corresponding amino acids of the capsid proteins VP1, VP2 and/or VP3 of another AAV that infects humans, e.g. AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9), N587 of the capsid protein VP1 of AAV2 (or the corresponding positions of the capsid proteins VP2 and /or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2 and/or VP3 of another AAV that infects humans, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9), Q585 capsid VP1 protein of AAV6 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2 and/or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8 and AAV9), N590 of the capsid protein VP1 of AAV8 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2 and/or VP3 of another AAV that infects humans , for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV9), G453 of the VP1 capsid protein of AAV9 (or the corresponding positions of the VP2 and/or VP3 capsid proteins encoded by the same capsid gene, or the corresponding amino acids of the VP1 capsid proteins, VP2 and/or VP3 of another AAV that infects humans, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and AAV8) or A589 of the VP1 capsid protein of AAV9 (or corresponding positions of the VP2 and/or VP3 capsid proteins encoded by those the same capsid genome, or the corresponding amino acids of the capsid proteins VP1, VP2 and/or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) G453 of the AAV2 capsid protein VP1 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2, and /or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) N587 of the AAV2 capsid protein VP1 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2, and /or VP3 of another AAV that infects humans, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) Q585 of the AAV6 capsid protein VP1 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2, and /or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, AAV8 and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) N590 of the AAV8 capsid protein VP1 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2, and /or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV9). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) G453 of the AAV9 capsid protein VP1 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2, and /or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8). In some embodiments, the heterologous epitope is inserted immediately after (e.g., fused to the C terminus of) A589 of the AAV9 capsid protein VP1 (or the corresponding positions of the capsid proteins VP2 and/or VP3 encoded by the same capsid gene, or the corresponding amino acids of the capsid proteins VP1, VP2, and /or VP3 of another AAV that infects humans, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 and AAV8). In some embodiments, a heterologous epitope is inserted and/or displayed between amino acids N587 and R588 of the AAV2 VP1 capsid protein (or the corresponding positions of VP2 and/or VP3 capsids encoded by the same capsid gene). In some embodiments, the recombinant viral capsid, a viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid contain the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant viral capsid, the viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid, contain the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the recombinant viral capsid, a viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid, contain an amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 25. In some embodiments, the recombinant viral capsid, a viral vector containing a recombinant viral capsid, and/or compositions containing a recombinant viral capsid contain an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 26. In some embodiments, the recombinant viral capsid, a viral vector comprising the recombinant viral capsid, and/or compositions comprising the recombinant viral capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 27.

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

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

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

[0025] In some embodiments, the recombinant viral capsid as described herein comprises the amino acid sequence EQKLISEEDL (represented by 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 the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked 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 EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I587 of the AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (represented by SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein, for example, contains the amino acid sequence presented by SEQ ID NO: 2.

[0026] In some embodiments, the recombinant viral capsid as described herein comprises the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the AAV6 VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I585 of the AAV6 VP1 capsid protein. In some embodiments, the recombinant viral capsid as described herein comprises EQKLISEEDL (represented by SEQ ID NO: 6) inserted between Q585 and S586 of the AAV6 VP1 capsid protein, for example, contains the amino acid sequence presented by SEQ ID NO: 4.

[0027] In some embodiments, the recombinant viral capsid as described herein comprises the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the AAV8 capsid VP1. In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I590 of the AAV8 VP1 capsid protein. In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented by SEQ ID NO: 6) inserted between N590 and T591 of the AAV8 VP1 capsid protein, for example, contains the amino acid sequence presented by SEQ ID NO: 25.

[0028] In some embodiments, the recombinant viral capsid as described herein comprises the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I453 of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented by SEQ ID NO: 6) inserted between G453 and S454 of the AAV9 VP1 capsid protein, for example, contains the amino acid sequence presented by SEQ ID NO: 26. In some embodiments, the recombinant viral capsid contains EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I589 of the AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid comprises EQKLISEEDL (represented by SEQ ID NO: 6) inserted between A589 and Q590 of the AAV9 VP1 capsid protein, for example, contains the amino acid sequence presented by SEQ ID NO: 27.

[0029] In some embodiments, the heterologous epitope comprises an affinity tag and one or more linkers. In some embodiments, the heterologous epitope comprises an affinity tag flanked by a linker, for example, the heterologous epitope comprises, from N-terminus to C-terminus, a first linker, an affinity tag, and a second linker. In some embodiments, the first and second linkers are each independently a polypeptide of at least 1 amino acid in length. In some embodiments, a heterologous epitope as described herein, for example, an affinity tag alone or in combination with one or more linkers, has a length of from about 5 amino acids to about 35 amino acids. In some embodiments, the first and second linkers are the same length and/or contain identical amino acid sequences.

[0030] Typically, in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid containing a recombinant viral capsid protein as described herein has a reduced or even eliminated natural tropism, e.g., a reduced ability or inability to target and bind a reference cell naturally permissive to transduction, compared with the ability of a reference viral capsid, for example a capsid containing a reference viral capsid protein, for example a viral capsid protein that would be identical to a recombinant viral capsid protein but without a heterologous epitope. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 10% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 20% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 30% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 40% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 50% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 60% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 70% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 75% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least an 80% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least an 85% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 90% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 95% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, a recombinant viral capsid comprising a recombinant viral capsid protein as described herein exhibits at least a 99% reduction in transduction efficiency compared to a reference viral capsid. In some embodiments, and in the absence of a suitable multispecific, optionally bispecific, binding molecule, transduction of a control cell by a recombinant viral capsid containing a recombinant viral capsid protein as described herein is eliminated, e.g., not detected.

[0031] In some embodiments, the viral capsid containing a recombinant viral capsid protein as described herein is a mosaic capsid, e.g., comprising a specified ratio of a recombinant viral capsid protein containing a heterologous epitope and a reference capsid protein that does not contain a heterologous epitope. epitope In some embodiments, the reference capsid protein is a wild-type reference capsid protein in that it comprises the amino acid sequence of a wild-type capsid protein having the same serotype as the recombinant viral capsid protein. In some embodiments, the reference capsid protein is a control reference capsid protein in that it contains the 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 in that it contains an amino acid sequence substantially identical to that of a wild-type capsid protein having the same serotype as the recombinant viral capsid protein, except for the mutation, ( e.g., insertion of an amino acid sequence, chimerization, etc.) that reduces the tropism of the wild-type capsid protein. In some embodiments, the composition described herein contains, or the method described herein combines, a recombinant viral capsid protein and a reference capsid protein in a ratio that ranges from 1:1 to 1:15. In some embodiments, the ratio is 1:2. In some embodiments, the ratio is 1:3. In some embodiments, the ratio is 1:4. In some embodiments, the ratio is 1:5. In some embodiments, the ratio is 1:6. In some embodiments, the ratio is 1:7. In some embodiments, the ratio is 1:8. In some embodiments, the ratio is 1:9. In some embodiments, the ratio is 1:10. In some embodiments, the ratio is 1:11. In some embodiments, the ratio is 1:12. In some embodiments, the ratio is 1:13. In some embodiments, the ratio is 1:14. In some embodiments, the ratio is 1:15.

[0032] Also disclosed herein are nucleic acids that encode the recombinant viral capsid protein described herein, compositions containing such nucleic acids (e.g., that can be used in methods for producing the recombinant viral capsid), and/or recombinant viral capsid proteins (e.g., compositions consisting essentially of a recombinant viral capsid protein, compositions containing only viral vectors enclosed in a capsid containing the viral capsid protein described herein, compositions containing such viral vectors and a multispecific, optionally bispecific, binding molecule ( for example, in certain ratios of a viral vector with a polyspecific, optionally bispecific, binding molecule (molecule: molecule), compositions containing such viral vectors, target modifying fragments and a pharmaceutically acceptable carrier, etc.). In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) and a nucleotide sequence that encodes at least 5 contiguous amino acids of an adenovirus or adeno-associated virus capsid protein.

[0033] In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the capsid protein VP1 AAV2. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented by SEQ ID NO: 6) inserted into I587 of the AAV2 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented by SEQ ID NO: 6) inserted between N587 and R588 of the AAV2 VP1 capsid protein, for example, in some embodiments, a nucleic acid such as described, encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 2.

[0034] In some embodiments, a nucleic acid such as

described herein, contains a nucleotide sequence that encodes the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the AAV6 VP1 capsid protein. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented by SEQ ID NO: 6) inserted into I585 of the AAV6 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented by SEQ ID NO: 6) inserted between Q585 and S586 of the AAV6 VP1 capsid protein, for example, in some embodiments, a nucleic acid such as described encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 4.

[0035] In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the capsid protein VP1 AAV8. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I590 of the AAV8 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented by SEQ ID NO: 6) inserted between N590 and T591 of the AAV8 VP1 capsid protein, for example, in some embodiments, a nucleic acid such as described encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25.

[0036] In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked by and/or operably linked to at least 5 contiguous amino acids of the capsid protein VP1 AAV9. In some embodiments, the nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented under SEQ ID NO: 6) inserted into I453 of the AAV9 VP1 capsid protein. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented under SEQ ID NO: 6) inserted between G453 and S454 of the AAV9 VP1 capsid protein, for example, in some embodiments, a nucleic acid such as described encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented in SEQ ID NO: 6) inserted into I589 of AAV9 capsid protein VP1. In some embodiments, a nucleic acid as described herein comprises a nucleotide sequence that encodes EQKLISEEDL (represented under SEQ ID NO: 6) inserted between A589 and Q590 of the AAV9 VP1 capsid protein, for example, in some embodiments, a nucleic acid such as described encodes an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0037] Typically, recombinant viral vectors, as described herein, contain a viral capsid containing a recombinant viral capsid protein, as described herein, wherein the viral capsid encapsulates the nucleotide of interest. In some embodiments, the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof. In some embodiments, the nucleotide of interest is under the control of a promoter other than a human promoter. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is an EF1α promoter.

[0038] Typically, the nucleotide of interest may be one or more genes that may encode a detectable marker, such as a reporter, or a therapeutic polypeptide. In some embodiments, the nucleotide of interest is a reporter gene. In some embodiments, the nucleotide of interest is a reporter gene that encodes a detectable marker selected from the group consisting of green fluorescent protein, luciferase, β-galactosidase, etc. In some embodiments, the detectable marker is a green fluorescent protein. In other embodiments, the nucleotide of interest is selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or fragment thereof, a nucleotide encoding the CRISPR/Cas system or part(s) thereof, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, secreted enzyme, etc. In one embodiment, the nucleotide of interest encodes a multidomain therapeutic agent, for example, a protein that contains at least two domains that provide two different functions.

[0039] The compositions described herein typically comprise a viral vector that contains a recombinant viral capsid protein as described herein, for example, comprising a capsid containing a recombinant viral capsid protein, wherein the capsid encapsulates a nucleotide of interest. In some embodiments, the composition described herein comprises (1) a viral vector having a capsid containing a recombinant viral capsid protein genetically modified to include a heterologous epitope, (2) a polyspecific, optionally bispecific, binding molecule comprising (i) a paratope an antibody that specifically binds the epitope, and (ii) a retargeting ligand that specifically binds the receptor, and optionally (3) a pharmaceutically acceptable carrier.

[0040] An antibody paratope as described herein typically contains at least a complementarity determining region (CDR) that specifically recognizes a heterologous epitope, for example, the CDR3 region of a heavy and/or light chain variable domain. In some embodiments, the multispecific, optionally bispecific, binding molecule comprises an antibody (or portion thereof) that contains an antibody paratope that specifically binds a heterologous epitope. For example, a 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 single domain light chain variable region comprises an antibody paratope that specifically binds a heterologous epitope. In some embodiments, the polyspecific, optionally bispecific, binding molecule may comprise an Fv region, for example, the polyspecific, optionally bispecific, binding molecule may comprise a scFv containing an antibody paratope that specifically binds a heterologous epitope. In some embodiments, the multispecific, optionally bispecific, binding molecule comprises an antibody (or portion thereof) comprising an antibody paratope that specifically binds a heterologous epitope, wherein the antibody (or portion thereof) further comprises one or more antibody constant domains (e.g., a heavy chains (eg, CH1, hinge, CH2, CH3, CH4, etc.) and/or a light chain constant domain (eg, CL), where one or more antibody constant domains do not bind a heterologous epitope.

[0041] The polyspecific, optionally bispecific, binding molecule as described herein further comprises a retargeting ligand in addition to a paratope (e.g., an antibody or a paratope-containing portion thereof) that specifically binds a heterologous epitope inserted into the recombinant viral capsid protein. displayed with it. In some embodiments, the retargeting ligand specifically binds a receptor on the surface of a bead (eg, to isolate and/or purify recombinant viral capsid protein, as described herein). In some embodiments, the retargeting ligand specifically binds a cell surface protein, such as a receptor, cell surface marker, etc., expressed on the surface of a eukaryotic mammalian (eg, human) cell, such as a target cell. In some embodiments, the retargeting ligand binds a (human) liver cell, a (human) brain cell, a (human) T cell, a (human) kidney cell, a (human) intestinal cell, a (human) pancreatic cell, a (human) cancer cell cell and/or (human) cell infected with a heterologous pathogen. In some embodiments, the retargeting ligand binds a (human) liver cell specific marker, a (human) brain cell specific marker, a (human) T cell specific marker, a (human) kidney cell specific marker, a (human) intestinal cell specific marker, a specific a (human) pancreatic cell marker, a (human) tumor cell specific marker, and/or a pathogenic epitope.

[0042] In some embodiments, the retargeting ligand binds a receptor expressed by a (human) liver cell, such as an asialoglycoprotein receptor, such as hASGR1. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) nerve cell, such as GABA, transferrin, etc. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) T cell, such as CD3, such as CD3ε. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) hematopoietic stem cell, such as CD34. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) kidney cell. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) muscle cell, such as an integrin. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) cancer cell, e.g., a tumor-associated antigen, e.g., adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B- RAF, BAGE-1, BCLX(L), BCR-ABL b3a2 fusion protein, beta-catenin, BING-4, CA-125, CALCA, oncoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, cyclin A1, dek-can fusion protein, DKK1, EFTUD2, elongation factor 2, ENAH (hMena ), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”), ETV6-AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2 ,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100/Pme117, GPNMB, HAUS3, hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA -A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1, also known as CCDC110, LAGE-1, LDLR-fucosyltransferase fusion protein AS, lengzin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE -C1, MAGE-C2, malicenzyme, 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, polypeptide P, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY -MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT fusion protein- SSX1 or -SSX2, TAG-1, TAG-2, telomerase, TGF-betaRII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (for lymphoma and other solid tumors), ErbB receptors, Melan A [MART1] , gp 100, tyrosinase, TRP-1/gp 75 and TRP-2 (for melanoma); MAGE-1 and MAGE-3 (for bladder, head and neck and non-small cell carcinoma); proteins EG and E7 HPV (for cervical cancer); mucin [MUC-1] (for breast, pancreatic, colon and prostate cancer); prostate specific antigen [PSA] (for prostate cancer); oncoembryonic antigen [CEA] (for colon, breast and gastrointestinal cancers) and such common tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3-7, 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 the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3).

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

[0044] In some embodiments, the polyspecific binding molecule is a bispecific binding molecule, such as an antibody comprising first and second antigen binding domains, wherein the first antigen binding domain comprises a paratope that specifically binds a heterologous epitope inserted into/displayed by the recombinant viral capsid protein , and a second antigen-binding domain that specifically binds a cell surface protein expressed by the target cell. In some embodiments, the bispecific binding molecule is a bispecific antibody comprising first and second antigen binding domains, wherein the first antigen binding domain comprises a paratope that specifically binds a heterologous epitope inserted into/displayed by a recombinant viral capsid protein, and the second antigen binding domain specifically binds a receptor expressed by a target cell, wherein a first antigen binding domain is operably linked to a first heavy chain region containing a first CH3 domain, wherein a second antigen binding domain is operably linked to a second heavy chain region containing a second CH3 domain, wherein the first and second C _H 3 -Ig domains 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 that lacks the amino acid difference. In one embodiment, the first Ig C _H 3 domain binds Protein A, and the second Ig C _H 3 domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (IMGT exon numbering; H435R EU numbering). The second C _H 3 domain may additionally contain the Y96F modification (by IMGT; Y436F by EU). Additional modifications that can be found within the second C _H 3 domain 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; EU N384S, K392N and V422I) for IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q and V82I (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU) for IgG4 antibodies.

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

[0046] Also described herein are methods for the preparation and use of recombinant viral capsid proteins, viral vectors containing them, compositions, etc. In some embodiments, methods of redirecting a virus, such as adenovirus, adeno-associated virus, etc.; delivery of diagnostic/therapeutic cargo to the target cell, etc. involve combining a recombinant viral vector containing a recombinant viral capsid protein as described herein, for example, a viral vector containing a capsid containing a recombinant viral capsid displaying a heterologous epitope with a bispecific binding molecule, wherein the bispecific binding molecule comprises (i) an antibody paratope , which specifically binds the epitope, and (ii) a retargeting ligand, which specifically binds the receptor. Such methods may include, as a first step, producing a recombinant viral vector, for example, culturing the packaging cell under conditions sufficient to produce viral vectors, wherein the packaging cell contains a plasmid encoding a capsid protein containing an epitope. When delivering a diagnostic/therapeutic payload to a target cell, the methods described herein may include contacting the target cell with a combination of a viral vector that contains a capsid, including a recombinant viral capsid displaying a heterologous epitope, and a polyspecific binding molecule, wherein the polyspecific binding molecule contains i) an antibody paratope that specifically binds an epitope, and (ii) a retargeting ligand that specifically binds a receptor expressed by the target cell. In some embodiments, the target cell is in an in vitro environment. In other embodiments, the target cell is present in vivo in a subject, such as a human.

[0047] In some embodiments, a composition described herein or a method described herein combines a recombinant viral vector containing a nucleotide of interest enclosed in a capsid containing a recombinant capsid protein as described herein, and a polyspecific binding molecule at a molecule:molecule ratio that restores the transduction efficiency of the viral vector to that of the reference viral vector. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is in the range of 1:0.5 to 1:100. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is in the range of 1:4 to 1:20. In some embodiments, the ratio of recombinant viral vector to bispecific binding molecule (molecule:molecule) is in the range of 1:8 to 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:4. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:8. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:20.

[0048] Also described herein are methods for inactivating a viral capsid and/or producing viral vectors, the methods typically 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, including a heterologous protein, and/or (b) culturing the packaging cell under conditions sufficient to produce viral vectors, wherein the packaging cell contains the nucleotide sequence. In some embodiments, the packaging cell further comprises a helper plasmid and/or a transfer plasmid containing the nucleotide of interest. In some embodiments, the methods further comprise isolating adeno-associated virus self-complementary vectors from the culture supernatant. In some embodiments, the methods further comprise lysing the packaging cell and isolating single-stranded adeno-associated virus vectors from the cell lysate. In some embodiments, the methods further comprise (a) removing cellular debris, (b) treating the supernatant containing the viral vectors with DNase I and MgCl2, (c) concentrating the viral vectors, (d) purifying the viral vectors, and (e) any combination of (a )-(d). Also provided herein is a viral vector produced in accordance with the method described herein and a packaging cell useful in producing the viral vector as described herein, for example, packaging cells containing a plasmid encoding the described recombinant capsid protein.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0049] The patent or application file contains at least one graphic material in color. Copies of this patent or patent application publication with color graphic(s) will be made available by the Office upon request and payment of the required fee.

[0050] Figure 1 shows immunofluorescence microscopy images (top panel) or fluorescence-activated cell sorting (FACS) histograms (bottom panel) to assess green fluorescent protein (GFP) expression by HepG2 cells incubated with (A) viral wild-type scAAV2-CMV-eGFP vectors alone; (B) scAAV2-N587Myc-CMV-hrGFP viral vectors alone; scAAV2-N587Myc viral vectors mixed with bispecific antibodies to Myc-ASGR1 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 viral vectors scAAV-N587Myc mixed with a monospecific antibody to Myc in a ratio of 1:8.

[0051] Figure 2A shows dot plots obtained by fluorescence-activated cell sorting (FACS) to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells incubated with wild-type scAAV2-based viral vectors alone (i), viral scAAV2-N587Myc-CMV-eGFP vectors alone (ii), scAAV2-N587Myc-CMV-eGFP viral vectors mixed with bispecific antibodies to Myc-ASGR1 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- viral vectors CMV-eGFP mixed with an irrelevant bispecific antibody to Myc-GCGR in a 1:8 ratio (xii). Expression of GFP by 29T3 cells incubated with scAAV-N587Myc viral vectors mixed with bispecific antibodies to Myc-ASGR1 in a 1:8 ratio is also shown (xi). For each experiment, 2×10 ⁵ cells and 5×10 ⁹ viral vectors were used.

[0052] Figure 2B shows fluorescence-activated cell sorting (FACS) histograms to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells incubated with unmodified AAV9-CAGG-GFP viral vectors alone (i) viral vectors AAV9-A589Myc-CAGG-eGFP alone (ii), AAV9-A589Myc-CAGG-eGFP viral vectors mixed with bispecific antibodies to Myc-ASGR1 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, 2 x 10 ⁵ cells and 1 x 10 ¹⁰ viral vectors (titrated by qPCR) were used.

[0053] Figure 3 shows fluorescence-activated cell sorting (FACS) dot plots to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells preincubated with 0 nM bivalent anti-ASGR1 antibody (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 then infected with viral vectors scAAV2-N587Myc-CMV-eGFP in a mixture with bispecific antibodies to Myc-ASGR1 in a ratio of 1:8 (L). 293T3-hASGR1 cells incubated with wild-type scAAV-based viral vectors alone (A) or with scAAV2-N587Myc-CMV-eGFP viral vectors alone (B) served as controls. For each experiment, 2x10 ⁵ cells and 5x10 ⁹ viral vectors (titrated by qPCR) were used.

[0054] Figure 4 shows immunofluorescence microscopy images to evaluate green fluorescent protein (GFP) expression by 293T-hASGR1 cells incubated with wild-type scAAV viral vectors alone (A), scAAV2-N587Myc-CMV-eGFP viral vectors alone (B ), or sequentially incubated with 1×10 ⁹ (C), 2×10 ⁹ (D), 4×10 ⁹ (E), 8×10 ⁹ (F), 2×10 ¹⁰ (G), 1×10 ¹¹ (H), 1x10 ¹² (I) bispecific antibodies to Myc-ASGR1 and then with 1x10 ⁹ scAAV2-N587Myc-CMV-eGFP viral vectors. Also shown are immunofluorescence microscopy images of 293T cells sequentially incubated with 1 × 10 ¹¹ anti-myc-ASGR1 antibody molecules and then with 1 × 10 ⁹ scAAV2-N587Myc-CMV-eGFP viral vectors (J) and 293T-hASGR1 cells sequentially incubated with 1 ×10 ¹¹ molecules of irrelevant bispecific antibody to Myc-GCGR and then with 1 × 10 ⁹ viral vectors scAAV2-N587Myc-CMV-eGFP (K).

[0055] Figure 5 shows dot plots obtained from fluorescence-activated cell sorting (FACS) to assess green fluorescent protein (GFP) expression by 29T3-hASGR1 cells incubated with wild-type ssAAV-based viral vectors alone (A), viral vectors ssAAV2-N587Myc-CMV-hrGFP alone (B), viral vectors ssAAV2-N587Myc-CMV-hrGFP mixed with bispecific antibodies to Myc-ASGR1 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 with ssAAV-N587Myc viral vectors mixed with an irrelevant bispecific antibody to Myc- GCGR in a ratio of 1:8 (K). Expression of GFP by 29T3 cells incubated with scAAV-N587Myc viral vectors mixed with bispecific antibodies to MycASGR1 in a 1:8 ratio is also shown (J). For each experiment, 2×10 ⁵ cells and 5×10 ⁹ viral vectors were used.

[0056] Figure 6 shows dot plots obtained by fluorescence-activated cell sorting (FACS) to assess green fluorescent protein (GFP) expression by 29T3-hGCGR cells incubated with wild-type scAAV-based viral vectors alone (A), viral scAAV2-N587Myc-CMV-eGFP vectors alone (B), scAAV2-N587Myc-CMV-eGFP viral vectors mixed with bispecific antibodies to Myc-GCGR 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 with viral vectors scAAV-2N587Myc-CMV-eGFP mixed with an irrelevant monospecific anti-Myc antibody (Regeneron Pharmaceuticals, Tarrytown, NY) in a 1:8 ratio (L). For each experiment, 2×10 ⁵ cells and 5×10 ⁹ viral vectors were used.

[0057] Figure 7 shows dot plots obtained by fluorescence-activated cell sorting (FACS) to assess green fluorescent protein (GFP) expression by Jurkat cells alone (A) or Jurkat cells incubated with scAAV6-EF1-eGFP viral vectors on wild-type basis alone (B), AAV6-Q585Myc-EF1a-eGFP viral vectors alone (C), AAV6-Q585Myc-EF1a-eGFP viral vectors mixed with bispecific antibodies to Myc-CD3 at the following ratios: 1:1 (D) , 1:5 (E), 1:10 (F), 1:100 or (G), 1:1000 (H). For each experiment, 2×10 ⁵ cells and 1×10 ⁹ viral vectors were used.

[0058] Figure 8A shows immunofluorescence microscopy images of the liver. Figure 8B shows immunofluorescence microscopy images of the spleen. Figure 8C shows immunofluorescence microscopy images of the kidney. All samples were from C57BL/6 mice transgenically modified to express human ASGR1 in liver cells (i-iv) or wild-type C57BL/6 mice (v-viii) ten days after intravenous injection of 1×10 ¹¹ scAAV2-CMV-eGFP wild type (i, v), saline (ii, vi), 1×10 ¹¹ scAAV2-N587myc-CMV-eGFP viral vectors alone (iii, vii) or scAAV2-N587myc-CMV-eGFP viral vectors with bispecific anti-myc antibody -ASGR1 (iv, viii).

[0059] Figure 9 shows immunofluorescence microscopy images of liver samples taken from C57BL/6 mice transgenically modified to express human ASGR1 on liver cells (DF, JL, P-R) or wild-type C57BL/6 mice (A-C , GI, M-O) four weeks after intravenous injection of 2.18 × 10 ¹¹ ssAAV2-CAGG-eGFP wild type (B, C, E, F), saline (A, D), 2.18 × 10 ¹¹ ssAAV2-N587myc-CAGG-eGFP viral vectors alone (GI, JL) or ssAAV2-N587myc-CAGG-eGFP viral vectors with a bispecific antibody to myc-ASGR1 (M-O, PR). Each image represents one mouse.

[0060] Figure 10 shows immunofluorescence microscopy images of liver samples taken from C57BL/6 mice transgenically modified to express human ASGR1 in liver cells, ten days after intravenous injection of (A) wild-type AAV9, (B) 250 nM NaCl (C ) AAV9-A589myc-CAGG-eGFP viral particles in combination with a bispecific antibody to myc-hCD3 or (D) AAV9-A589myc-CAGG-eGFP viral particles in combination with a bispecific antibody to myc-ASGR1.

[0061] Figure 11 provides illustrative, non-scale and non-limiting examples of multispecific binding molecule formats useful in some embodiments of the present invention.

[0062] Figure 12 shows dot plots obtained by fluorescence-activated cell sorting (FACS) to evaluate green fluorescent protein (GFP) expression by 29T3-hASGR1 cells incubated with (A)_ wild-type AAV8 vectors alone. (C) AA8-N590-myc viral vectors alone or pAAV RC8 N590myc viral vectors mixed with bispecific anti-hASGR1-IgG4-Fc/anti-myc antibody 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. Expression of GFP by mock-transfected 29T3-hASGR1 cells is also shown (B). For each experiment, 2×10 ⁵ cells and 1×10 ⁹ viral vectors were used.

[0063] Figure 13 shows immunofluorescence microscopy images of liver samples taken from C57BL/6 mice transgenically modified to express human ASGR1 in liver cells, ten days after intravenous injection of (A)-(C) wild-type AAV8, (D)- (F) AA8-N590-myc viral vectors and control bispecific binding molecule or (G)-(I) AAV8 N590myc-CAGG-eGFP viral particles in combination with anti-hASGR1-IgG4-Fc/anti-myc bispecific binding molecules.

[0064] Figure 14 shows dot plots obtained by fluorescence-activated cell sorting (FACS) to assess green fluorescent protein (GFP) expression by 29T3-h ENTPD3 cells incubated with (A) wild-type AAV2-based viral vectors alone, (B) AAV2-N587Myc-CAGG-eGFP viral vectors alone or AAV2-N587Myc-CAGG-eGFP viral vectors mixed with bispecific anti-hENTPD3-IgG4-Fc/anti-myc antibody 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, 2×10 ⁵ cells and 1×10 ⁹ viral vectors were used.

[0065] Figure 15A shows immunofluorescence microscopy images of liver samples, Figure 15B shows immunofluorescence microscopy images of intestinal samples, and Figure 15C shows immunofluorescence microscopy images of pancreas samples. All samples were collected from wild-type C57BL/6 mice ten days after intravenous injection of PBS (15A(i), 15B(i) and 15C(i)), 5×10 ¹¹ wild-type AAV9 (15A(ii), 15B(ii) ) and 15C(ii)), 5×10 ¹¹ AAV2-N587myc-CAGG-eGFP viral vectors with 1×10 ³ irrelevant IgG4-Fc/anti-myc bispecific binding proteins (15A(iii), 15B(iii), and 15C (iii)) or 5x10 ¹¹ AAV2-N587myc-CAGG-eGFP viral vectors with 1x10 ¹³ hENTPD3-IgG4-Fc bispecific binding proteins/anti-myc antibodies (15A(iv), 15B(iv) and 15C(iv) ).

DETAILED DESCRIPTION OF THE INVENTION

[0066] A common problem with adapter approaches using unmodified viral capsids or viral capsids with a modified scaffold has been the suboptimal transduction efficiency of the modified capsids. (Grifman et al. (2001) Mol. Ther. 3:964-75). For example, Curiel et al. describe the preparation and characterization of a recombinant adenoviral vector containing fibers with the RGD-4C sequence genetically included in the HI loop of the carboxy-terminal knob domain, and demonstrate the utility of the HI loop in the knob domain of the fiber protein as an optimal site for the incorporation of short peptide ligands. See, for example, US patent No. 7297542; see also Beany and Curiel (2012) Adv. Cancer. Res. 115:39-67. Likewise, insertion of ligand peptides into AAV capsid proteins resulted in the formation of capsids that were capable of displaying the ligand on the capsid surface and mediating transduction by interaction of the ligand with its receptor, thereby redirecting viral tropism through genetic modifications of the capsid (Girod et al. (1999) Nat Med 5(9): 1052-6, 1438 (errata included) (1999) Grifman et al (2001) Mol Ther 3(6):964-75 Nicklin et al (2001) Mol. Ther. 4(3): 174-81; Shi et al. (2001) Hum Gene Ther. 17(3):353-61 (2006); Wu et al. (2000) J. Virol. 74(18 ):8635-47). In particular, insertion of an integrin binding Arg-Gly-Asp (RGD) motif into the 1-587 insertion site of the AAV VP1 capsid protein has been demonstrated to allow AAV virus-based vectors to transduce cells with α _v β ₁ integrins (Girod et al. ( 1999) above). In contrast, although insertion of the 14 amino acid peptide L14 after amino acid R447 (1-447) resulted in the formation of capsids still recognized by the conformation-sensitive A20 antibody, such recombinant viral vectors were unable to transduce cells expressing the L-14 receptor (Girod et al., 1999; cf. Wu et al. (2000) (reported insertion of hemagglutinin (HA) peptide at position 1-447 and successful transduction of cells expressing the HA peptide). Insertion of the Myc epitope between T448 and N449 was recognized by anti-Myc antibody and was therefore present on the capsid surface but resulted in the formation of inactivated viral vectors (Grifman et al., 2001).In contrast, successful retargeting was again reported for the insertion of the NGR motif after N587, but not for the insertion c- myc after N587 (Grifman et al., 2001) US Patent No. 9,624,274 describes AAV capsid protein I-453 as a suitable insertion site for a heterologous epitope, although these studies demonstrate successful insertion and display of a heterologous peptide, e.g. AAV proteins, none of these studies suggest that a multispecific binding molecule, such as a bispecific binding molecule such as a bispecific antibody that specifically binds a heterologous peptide and a cell surface protein, can alter the targeting of modified viral vectors to cells expressing a cell surface protein. surfaces and restore their transduction efficiency.

[0067] Disclosed herein are recombinant viral capsid proteins that are modified with a heterologous epitope that can be used in connection with a polyspecific binding molecule containing a paratope, such as an Fv domain, that specifically binds the epitope, and a ligand that binds a surface expressed receptor target cells. As shown herein, contacting viral vectors having capsids formed by the capsid proteins described herein with a polyspecific binding molecule at certain ratios restores the transduction efficiency of the viral capsid to levels comparable to wild-type virus, see eg, Example 2. Typically, capsid proteins modified with heterologous epitopes, as described herein, can be obtained from a non-enveloped virus, such as, but not limited to, adenovirus (Ad) and adeno-associated virus (AAV).

[0068] Although the present invention has been specifically shown and described with reference to a number of embodiments, those skilled in the art will appreciate that changes in form and detail 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 limit the scope of the claims.

[0069] Although any methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, only certain preferred methods and materials are described herein. All publications referred to herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs.

Definitions

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention belongs.

[0071] Singular forms include references to plural forms unless the context clearly indicates otherwise. Thus, for example, reference to “method” includes one or more of the methods and/or steps described herein and/or that will become apparent to those skilled in the art upon reading the present disclosure.

[0072] The term "antibody" refers to immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain contains a heavy chain variable domain ( _VH ) and a heavy chain constant region ( _CH ). The heavy chain constant region contains at least three domains, CH ₁ , _CH 2, _CH 3 and optionally CH ₄ . Each light chain contains a light chain variable domain ( _CH ) and a light chain constant region ( _CL ). The heavy chain and light chain variable domains can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2 and LCDR3). Typical antibodies with a tetrameric structure contain two identical antigen-binding domains, each of which is formed by the association of V _H and V _L domains, and each of which, together with the corresponding C _H and C _L domains, forms the Fv region of the antibody. Single-domain antibodies contain one antigen-binding domain, for example, _VH or _VL . The term "antibody" includes monoclonal antibodies, multispecific (eg, bispecific) antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F(ab') fragments disulfide-linked Fv ( sdFv), intrabodies, minibodies, diabodies and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies 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 US Patent Application Publication No. 2007/0004909 and Ig-DARTS, such as those disclosed in US Patent Application Publication No. 2009/0060910. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e. molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0073] The antigen-binding domain of an antibody, eg, the portion of an antibody that recognizes and binds to an epitope of an antigen, is also called a “paratope.” This is a small region (5 to 10 amino acids) of the Fc region of an antibody, part of the antigen-binding fragment (Fab region), and may contain parts of the heavy and/or light chains of the antibody. A paratope specifically binds an epitope when the paratope binds an epitope with high affinity. The term “high affinity” antibody refers to an antibody that has a K _D for its target epitope of about 10 ^-9 M or less (e.g., about 1×10 ^-9 M, 1×10 ^-10 M, 1×10 ^{- 11} M or approximately 1×10 ^-12 M). In one embodiment, K _D is measured using surface plasmon resonance, for example, BIACORE™; in another embodiment, K _D is measured using ELISA.

[0074] The phrase "complementarity determining region" or the term "CDR" includes the amino acid sequence encoded by the nucleic acid sequence of the immunoglobulin genes of an organism, which normally (i.e., in a wild-type animal) is located between two backbone regions in the mild or severe variable region chains of an immunoglobulin molecule (such as an antibody or T-cell receptor). The CDR may be encoded by, for example, a germline sequence, or a rearranged or non-rearranged sequence, and, for example, an unprimed or mature B cell or T cell. The CDR may be somatically mutated (eg, different from the sequence encoded in the animal's germline), humanized, and/or modified by amino acid substitutions, additions, or deletions. In some cases (eg, in the case of CDR3), CDRs may be encoded by two or more sequences (eg, germline sequences) that are not contiguous (eg, in the case of a non-rearranged nucleic acid sequence), but they are contiguous in the nucleic acid sequence in B -cell, for example, as a result of splicing or joining of sequences (for example, V-D-J recombination to form CDR3 heavy chain).

[0075] An "epitope" is a portion of a macromolecule that is recognized by the immune system, particularly by antibodies, B cells, or cytotoxic T cells. Although epitopes are generally believed to be derived from foreign proteins, host-derived sequences that can be recognized are also classified as epitopes. Epitopes have at least 4 amino acids, preferably 4 to 30 amino acids, more preferably 5 to 20 amino acids, in particular 5 to 15 amino acids in length. Epitopes can be linear or three-dimensional, usually formed by amino acids that are distant from each other in the primary structure of the protein but become closely related in the secondary and/or tertiary structure. Epitopes that are specifically recognized by B cells are called B cell epitopes.

[0076] The phrase "inverted terminal repeat" or "ITR" includes symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs act as origins of replication for viral DNA synthesis and are important cis components for the production of AAV-based integrating vectors.

[0077] The phrase “light chain” includes the immunoglobulin light chain sequence from any organism, and unless otherwise noted, includes human κ and λ light chains and VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise noted. Typically, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. The light chain variable domain is encoded by a light chain variable region gene sequence that typically contains V _L and J _L segments derived from the repertoire of V and J segments present in the germ line. Sequences, locations, and nomenclature for light chain V and J segments for various organisms can be found in the IMGT database, www.imgt.org. Light chains include, for example, chains that do not selectively bind either a first or a second epitope selectively bound by the epitope-binding protein in which they occur. Light chains also include chains that bind and recognize, or assist a heavy chain or other light chain to bind and recognize one or more epitopes selectively bound by the epitope-binding protein in which they occur. Conventional or universal light chains include those derived from the human Vκ1-39Jκ gene or the human Vκ3-20Jκ gene, and include somatically mutated (eg, affinity matured) versions thereof. Exemplary human V _L segments include human Vκ1-39 gene segment, human Vκ3-20 gene segment, human Vλ1-40 gene segment, human Vλ1-44 gene segment, human Yλ2-8 gene segment, human Vλ2-14 gene segment, human Vλ3-21 gene, and include their somatically mutated (eg, affinity matured) versions. Light chains can be prepared that include a variable domain from one organism (e.g., human or rodent, e.g., rat or mouse; or bird, e.g., chicken) and a constant region from the same or another organism (e.g., human or rodent, e.g. rats or mice; or birds, such as chickens).

[0078] The term “about” or “about” includes being within a statistically significant range of value. Such a range may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of the given value or range. The tolerance covered by the terms "about" or "approximately" depends on the particular system under study and can be readily estimated by one skilled in the art.

[0079] The term "affinity tag" includes a polypeptide sequence that is a member of a specific binding pair, eg, that specifically binds to another polypeptide sequence, eg, an antibody paratope, with high affinity. Exemplary and non-limiting affinity tags include hexahistidine tag, FLAG tag, Strep II tag, streptavidin binding peptide (SBP) tag, calmodulin binding peptide (CBP), glutathione-S-transferase (GST), maltose binding protein (MBP), S-tag, label HA and label c-Myc. (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; incorporated herein by reference).

[0080] The term "capsid protein" includes a protein that is part of the capsid of a virus. For adeno-associated viruses, the capsid proteins are usually designated VP1, VP2, and/or VP3, and each is encoded by a single cap gene. For AAV, the three AAV capsid proteins are produced in an overlapping manner from the open reading frame (ORF) for cap through alternative mRNA splicing and/or alternative translation start codon usage, although all three proteins share a common stop codon. Warrington et al. (2004) J. Virol. 78:6595, which is incorporated herein by reference in its entirety. AAV2 VP1 is typically translated from the ATG (amino acid M1) start codon on a 2.4-kb mRNA, while AAV2 VP2 and VP3 arise from the smaller 2.3-kb mRNA. using the weaker ACG start codon to produce VP2 (amino acid T138) and with end-to-end translation to the next available ATG codon (amino acid M203) to produce the most abundant capsid protein, VP3. Warrington, above; 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, especially with respect to dependoparvoviruses. See Rutledge et al., supra. For example, Rutledge et al. (1998), supra, present in Figure 4B amino acid sequence alignments for the capsid proteins VP1, VP2 and VP3 of AAV2, AAV3, AAV4 and AAV6, where the start sites for each of the capsid proteins VP1, VP2 and VP3 are indicated by arrows and the variable domains are boxed. . Accordingly, while amino acid positions provided herein may be provided with respect to the AAV VP1 capsid protein, and amino acid positions provided herein that are not further specified are provided with respect to the AAV2 VP1 core protein sequence provided under SEQ ID NO: 1, a skilled person could respectively and easily determine the position of the same amino acid in the capsid protein of VP2 and/or VP3 of AAV and the corresponding amino acid position in different serotypes. Additionally, one of ordinary skill in the art will be able to swap domains between the capsid proteins of different AAV serotypes to form a “chimeric capsid protein.”

[0081] Domain exchange between two AAV capsid protein constructs to produce a “chimeric AAV capsid protein” is described, see, for example, Shen et al. (2007) Mol. Therapy 15(11):1955-1962, which is incorporated herein by reference in its entirety. A “chimeric AAV capsid protein” includes an AAV capsid protein that contains amino acid sequences, such as domains, from two or more different AAV serotypes and that is capable of and/or forms an AAV-like viral capsid/viral particle. The chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, for example, a nucleotide containing a plurality of, for example, at least two nucleic acid sequences, each of which is identical to a portion of the capsid gene encoding the capsid protein of different AAV serotypes, and the plurality together encodes a functional chimeric capsid AAV protein. Reference to a chimeric capsid protein in relation to a particular AAV serotype indicates that the capsid protein contains one or more domains from a capsid protein of that serotype and one or more domains from a capsid protein of another serotype. For example, a chimeric AAV2 capsid protein includes a capsid protein comprising one or more VP1, VP2 and/or VP3 capsid protein domains of AAV2 and one or more VP1, VP2 and/or VP3 capsid protein domains from another AAV.

[0082] A "mosaic capsid" contains at least two sets of proteins VP1, VP2 and/or VP3, where each set is encoded by a different cap gene.

[0083] In some embodiments, the mosaic capsid described herein comprises recombinant VP1, VP2, and/or VP3 proteins encoded by a cap gene genetically modified by insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprises VP1, VP2 and/or proteins VP3 encoded by a cap reference gene, e.g., a wild-type cap reference gene encoding wild-type VP1, VP2, and/or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and/or VP3 proteins, a cap reference reference gene encoding proteins VP1, VP2 and/or VP3, identical to the recombinant VP1, VP2 and VP3 proteins, but in the absence of a heterologous epitope, a mutant wild-type cap reference gene encoding essentially wild-type VP1, VP2 and/or VP3 proteins of the same AAV serotype as recombinant VP1, VP2 and/or VP3 proteins, but with a mutation (eg, insertion, substitution, deletion), wherein the mutation preferentially reduces the tropism of the wild-type VP1, VP2 and VP3 proteins. In some embodiments, the reference capsid protein is a chimeric reference protein comprising at least one domain of the VP1, VP2 and/or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2 and/or VP3 proteins. In some embodiments, the cap reference gene encodes a VP1, VP2, and/or VP3 chimeric protein.

[0084] The phrase “heavy chain” or “immunoglobulin heavy chain” includes an immunoglobulin heavy chain sequence, including an immunoglobulin heavy chain constant region sequence, from any organism. Heavy chain variable domains include three heavy chain CDRs and four FR regions unless otherwise noted. Heavy chain fragments include CDR, CDR and FR and combinations thereof. A typical heavy chain after the variable domain (from N-terminus to C-terminus) contains a _CH1 domain, a hinge, a _CH2 domain and a _CH3 domain. A functional heavy chain fragment includes a fragment that is capable of specifically recognizing an epitope (eg, recognizing an epitope with a K _D in the micromolar, nanomolar, or picomolar range), that is capable of expression and secretion from a cell, and that contains at least one CDR. Heavy chain variable domains are encoded by a variable region nucleotide sequence that typically contains _VH , _DH, and _JH segments derived from the repertoire of _VH , DH _, and _JH segments present in the germ line. Sequences, locations and nomenclature for heavy chain V, D and J segments for various organisms can be found in the IMGT database, which is accessible via the World Wide Web (www) at "imgt.org".

[0085] The term "heavy chain only antibody", "heavy chain only antigen binding protein", "single domain antigen binding protein", "single domain binding protein" or the like. refers to a monomeric or homodimeric immunoglobulin molecule containing an immunoglobulin-like chain containing a variable domain, operably linked to a heavy chain constant region, which cannot bind to a light chain because the heavy chain constant region usually lacks a functional _CH 1 domain. Accordingly, the term “heavy chain only antibody”, “heavy chain only antigen binding protein”, “single domain antigen binding protein”, “single domain binding protein” or the like. covers both (i) a monomeric single-domain antigen-binding protein containing one of the immunoglobulin-like chains containing a variable domain, operably linked to the constant region of the heavy chain in which there is no functional _CH 1 domain, and (ii) a homodimeric single-domain antigen-binding protein containing two immunoglobulin-like chains, each containing a variable domain, operably linked to a heavy chain constant region that lacks a functional _CH 1 domain. In various aspects, the homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each containing an identical variable domain, operably linked to an identical heavy chain constant region lacking a functional _CH 1 domain. In addition, each immunoglobulin-like chain of a single-domain antigen-binding protein contains a variable domain, which can be derived from heavy chain variable region gene segments (e.g., _VH , _DH , _JH ), light chain gene segments (e.g., _VL , _JL ) or a combination thereof, linked to a heavy chain constant region ( _CH ) gene sequence comprising a deletion or inactivating mutation in the _CH 1 (and optionally hinge region) coding sequence of a heavy chain constant region gene, e.g., IgG, IgA, IgE, IgD or combinations thereof. A single domain antigen binding protein containing a variable domain derived from heavy chain gene segments may be referred to as a “V _H single domain antibody” or “V _H single domain antigen binding protein”, see, for example, US Pat. No. 8,754,287; publication of US patent applications No. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556 and 20150197557, each of which is incorporated by reference in its entirety. A single domain antigen binding protein containing a variable domain derived from light chain gene segments may be referred to as a “V _L single domain antigen binding protein,” see, for example, US Patent Application Publication No. 20150289489, incorporated by reference in its entirety.

[0086] The phrase “light chain” includes the immunoglobulin light chain sequence from any organism, and unless otherwise noted, includes human kappa (κ) and lambda (λ) light chains and VpreB, as well as surrogate light chains. Light chain variable domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise noted. Typically, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain that includes FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. Light chain variable domains are encoded by a light chain variable region nucleotide sequence, which typically comprises light chain _VL and light chain _JL gene segments derived from the repertoire of light chain V and J gene segments present in the germline. Sequences, locations, and nomenclature for the light chain V and J gene segments for various organisms can be found in the IMGT database, which is accessible via the Internet on the World Wide Web (www) at “imgt.org.” Light chains include, for example, chains that do not selectively bind either a first or a second epitope selectively bound by the epitope-binding protein in which they occur. Light chains also include chains that bind and recognize, or assist the heavy chain to bind and recognize, one or more epitopes selectively bound by the epitope-binding protein in which they occur. Light chains also include chains that bind and recognize, or assist the heavy chain to bind and recognize, one or more epitopes selectively bound by the epitope-binding protein in which they occur. Conventional or universal light chains include those derived from the human Vκ1-39Jκ5 gene or the human Vκ3-20Jκ1 gene, and include somatically mutated (eg, affinity matured) versions thereof.

[0087] As used herein, the phrase "operably linked" includes the physical association (e.g., in three-dimensional space) of components or elements that directly or indirectly interact with each other or otherwise coordinate each other to participate in a biological event, and in such association such interaction and/or coordination is achieved or permitted. As just one example, a control sequence (e.g., an expression control sequence) in a nucleic acid is said to be “operably linked” to a coding sequence when it is located relative to the coding sequence such that its presence or absence affects expression and/or activity coding sequence. In many embodiments, a “functional linkage” includes covalently linking the respective components or elements to each other. Those skilled in the art will appreciate, however, that in some embodiments, a covalent bond is not required to achieve an effective functional connection. For example, in some embodiments, control nucleic acid sequences operably linked to the coding sequences they control are adjacent to the nucleotide of interest. Alternatively or additionally, in some embodiments, one or more such control sequences operate in trans or at a distance to control the coding sequence of interest. In some embodiments, the term “expression control sequence,” as used herein, refers to polynucleotide sequences that are necessary and/or sufficient to effect expression and processing of the coding sequences to which they are ligated. In some embodiments, the expression control sequences may be or contain appropriate transcription initiation, termination, promoter, and/or enhancer sequences; signals for efficient RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (eg, Kozak consensus sequence); sequences that increase 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. As an example, in prokaryotes, control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include promoters, enhancers, and/or transcription termination sequences. Those skilled in the art will appreciate from the context that in many embodiments, the term “control sequences” refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is beneficial for expression (including, for example, , leader sequences, targeting sequences and/or fusion partner sequences).

[0088] The term “recombinant capsid protein” includes a capsid protein that has at least one mutation compared to the corresponding capsid protein of a wild-type virus, which may be a reference and/or control virus for comparative study. A recombinant capsid protein includes a capsid protein containing a heterologous epitope that 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 acids may simply be inserted between two specified amino acids of the capsid protein. The insertion of amino acids may also be accompanied by a deletion of said capsid protein amino acids at the insertion site, for example, 1 or more amino acids of the capsid protein are replaced by 5 or more heterologous amino acids).

[0089] The terms “multispecific binding molecule” and “bispecific binding molecule” and the like generally and respectively refer to a binding molecule containing at least two and only two non-identical binding components, each binding component specifically binding a different epitope - either on two different molecules (for example, different epitopes on two different immunogens), or on the same molecule (for example, different epitopes on the same immunogen). Typically, one of the binding components of a bispecific binding molecule herein specifically binds a heterologous epitope displayed by a viral capsid protein, and the second binding component is specific for a protein, e.g., a cell surface marker, expressed predominantly and/or preferentially by the target cell, e.g. T cell marker (eg CD3, CD28, etc.). Bispecies binding molecules can be prepared, for example, by combining binding moieties that recognize different epitopes of the same immunogen. For example, nucleic acid sequences encoding binding components (e.g., light or heavy chain variable sequences) that recognize different epitopes can be fused to nucleic acid sequences encoding the same or different heavy chain constant regions, the same or different light chain constant regions, or , respectively, a heavy chain constant region and a light chain constant region, and such sequences can be expressed in a cell as a multispecific antigen binding protein in a format that is similar to the Fab structure, scFab structure, diabody structure, scFv structure, scFv-Fc structure, scFv- structure zipper, a tetramer structure like a typical antibody that includes a cognate universal light chain, a tetrameric structure containing a typical bivalent antibody that includes a cognate universal light chain and/or an additional binding moiety (e.g., scFv, scFv-zipper, scFab, etc. .) attached to one or both heavy chains (eg at the N- and/or C-terminus) and/or to one or both light chains (eg at the N- and/or C-terminus). Various formats of multispecific, especially bispecific, binding molecules are well known, see, for example, Brinkmann and Konterman (2017) mAbs 9:182-212, incorporated herein by reference in its entirety.

[0090] An exemplary polyspecific binding molecule has two heavy chains, each containing a heavy chain CDR followed (from N-terminus to C-terminus) by a _CH 1 domain, a hinge, a _CH 2 domain, and a _CH 3 domain. -domain, and an immunoglobulin light chain that either does not confer epitope-binding specificity but can bind to each heavy chain (eg, a common light chain), or that can bind to each heavy chain and can bind one or more epitopes bound by the epitope -binding regions of the heavy chain, or which can bind to each heavy chain and provide binding of one or both heavy chains to one or both 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 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 comprising a second amino acid sequence CH3 of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the first or second variable heavy chain domains (with or without a cognate light chain) bind a heterologous epitope as described herein, and another heavy chain variable domain (with or without a cognate light chain) binds a receptor on the target cell, and wherein the first heavy chain constant region chain binds to a second chain constant region in a manner that allows for easier isolation of the polyspecific binding protein, for example, where the first and second heavy chain constant regions form a knob-in-hollow (KIH) format, or where the second CH3 amino acid sequence contains a modification that reduces or eliminates the binding of the second amino acid sequence CH3 to protein A (see, for example, US 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 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 comprising a second amino acid sequence CH3 of a human IgG selected from IgG1, IgG2, IgG4, and a combination thereof, wherein the first and second variable domains are heavy chain domains (with or without a cognate light chain) bind the same or different antigens, wherein the first or second heavy chain constant regions are modified to further contain an additional binding domain (e.g., scFv or Fv that binds a heterologous epitope, as described herein, for example, wherein an additional binding domain is attached to the C-terminus or N-terminus of one or both of the heavy chains), and wherein the first heavy chain constant region is linked to the second chain constant region in a manner that allows for easier isolation of the multispecific binding protein for example, wherein the first and second heavy chain constant regions form a knob-in-hollow (KIH) format, or wherein the second CH3 amino acid sequence contains a modification that reduces or eliminates the binding of the second CH3 amino acid sequence to protein A. In some embodiments, in in which the second amino acid sequence CH3 has reduced or eliminated binding to protein A, the second amino acid sequence CH3 contains the modification H95R (exon numbering according to IMGT; H435R numbering according to EU). In one embodiment, the second CH3 amino acid sequence further comprises a Y96F modification (IMGT exon numbering; EU H436F). In another embodiment, the second amino acid sequence CH3 contains both the modification H95R (exon numbering according to IMGT; H435R numbering according to EU) and the modification Y96F (exon numbering according to IMGT; H436F according to EU). In some embodiments, the second CH3 amino acid sequence is derived from a modified human IgG1 and further comprises a mutation selected from the group consisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M, N384S, K392N, V397M, and V422I by EU). In some embodiments, the second amino acid sequence CH3 is derived from a modified human IgG2 and further comprises a mutation selected from the group consisting of N44S, K52N and V82I (IMGT: EU N384S, K392N and V422I). In some embodiments, the second CH3 amino acid sequence is derived from a modified human IgG4 and further comprises a mutation selected from the group consisting of 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 contains one or more modifications of any type described above.

[0091] In various embodiments, the Fc domains are modified to alter binding to the Fc receptor, which in turn affects effector function. In some embodiments, the engineered heavy chain constant region (CH) that includes the Fc domain is chimeric. Thus, the chimeric CH region combines CH domains derived from more than one immunoglobulin isotype. For example, a chimeric CH region comprises part or all of a CH2 domain derived from a human IgG1, human IgG2 or human IgG4 molecule in combination with part or all of a CH3 domain derived from a human IgG1, human IgG2 or human IgG4 molecule. In some embodiments, the chimeric CH region comprises a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216 to 227 according to EU numbering; amino acid residues 226 to 240 according to Kabat numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4, in combinations with a “lower hinge” sequence (amino acid residues 228 to 236 according to EU numbering; amino acid residues 241 to 249 according to Kabat numbering) derived from the hinge region of human IgG1, human IgG2 or human IgG4. In some embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge sequence and amino acid residues derived from a human IgG2 lower hinge sequence.

[0092] In some embodiments, the Fc domain can be designed to activate all, some, or none of the effector functions of a normal Fc without affecting the desired pharmacokinetic properties of the Fc-containing protein (eg, antibody). For examples of proteins containing chimeric CH regions and having altered effector functions, see WO 2014022540, which is incorporated herein in its entirety.

[0093] The term “target cells” includes any cells in which expression of the nucleotide of interest is desired. Preferably, target cells display a protein, such as a receptor, on their surface that allows the cell to be targeted by a retargeting ligand, as described below. Preferably, the target protein, eg, a receptor, is specific to the target cell, eg, a "cell-specific marker", "cell-specific antigen" or the like. The term “cell-specific marker,” “cell-specific antigen,” “organ-specific marker,” “tissue-specific marker,” or the like refers to and includes those proteins whose expression is enriched in a cell, tissue, and/or organ for which it is a specific marker. "Enriched" in the context of protein expression refers to and includes the expression or overexpression of a protein specific to a cell/tissue/organ, predominantly, preferentially or exclusively by the cell/tissue/organ for which the protein is a specific marker, although such marker may also be expressed by others cells/tissues/or organs at minimal levels. The Human Protein Atlas can be used to determine whether a protein is a cell/tissue/organ specific marker and also provides a repository of cell/tissue/organ specific proteins. See, www.proteinatlas.org; see also Uhlen et al. (2010) Nat. Biotech. 28:1248-50, which is incorporated herein by reference in its entirety.

[0094] The term "transduction" or "infection" or the like. refers to the introduction of a nucleic acid into a target cell using a viral vector. The term transduction efficiency or the like, eg, "transduction efficiency", refers to the proportion (eg, percentage) of cells expressing a nucleotide of interest after incubation with a specified amount of viral vectors containing the nucleotide of interest. Well-known methods for determining transduction efficiency include fluorescence activated cell sorting for cells transduced with a fluorescent reporter gene, PCR for expression of the nucleotide of interest, etc.

[0095] As used herein, the term “wild type” includes an entity having a structure and/or activity found in nature in a “normal” (as opposed to mutant, diseased, altered, etc.) state or environment. Those skilled in the art will appreciate that wild-type viral vectors, such as wild-type capsid proteins, can be used as a reference viral vector in comparative studies. Typically, the reference viral capsid protein/capsid/vector is identical to the test viral capsid protein/capsid/vector except for a change for which the effect needs to be tested. For example, to determine the effect, for example on transduction efficiency, of insertion of a heterologous epitope into a test viral vector, the transduction efficiency of the test viral vector (in the absence or presence of the corresponding multispecific binding molecule) can be compared with the transduction efficiency of a reference viral vector (in the absence or presence appropriate multispecific binding molecule, if necessary) that is identical to the test viral vector in each case (eg, additional mutations, nucleotide of interest, number of viral vectors and target cells, etc.), except for the presence of a heterologous epitope.

Recombinant viral capsid proteins and viral vectors and nucleic acids

[0096] In some embodiments, the recombinant viral capsid protein as described herein is an Ad capsid protein, e.g., a capsid protein of serotype Ad selected from the group consisting of Ad1, Ad2, Ad3, Ad4, Ad5, Ad6, and Ad7 . In some embodiments, the recombinant viral capsid protein is derived from the Ad2 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from the Ad5 capsid gene. In some embodiments, the recombinant viral capsid protein Ad, as described herein, contains a heterologous epitope in a fiber protein domain, e.g., at the carboxy terminus of the fiber protein, in the fiber protein knob domain, and/or in the HI loop of the fiber protein knob domain .

[0097] In some embodiments, the recombinant viral capsid protein described herein is derived from an adeno-associated virus (AAV) capsid gene, e.g., is a genetically modified capsid protein of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3 , AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, an AAV6 capsid gene, an AAV8 capsid gene, or an AAV9 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from the AAV2 capsid gene, e.g., is a genetically modified AAV2 VP1 capsid protein, the wild type amino acid sequence of which is set forth in SEQ ID NO: 1. In some embodiments, the recombinant viral capsid protein the protein is derived from the AAV8 capsid gene, for example, is a genetically modified AAV8 capsid protein VP1, the wild-type amino acid sequence of which is set forth in SEQ ID NO: 21. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, e.g. is a genetically modified AAV9 VP1 capsid protein, the wild-type amino acid sequence of which is set forth in SEQ ID NO: 5. In some embodiments, the recombinant viral capsid protein is derived from an AAV6 capsid gene, for example, is a genetically modified AAV6 VP1 capsid protein. In some embodiments, a heterologous epitope is inserted into I-453 of the AAV9 capsid protein.

[0098] Typically, a recombinant viral capsid protein, as described herein, contains a heterologous epitope inserted into and/or displayed by the capsid protein such that the heterologous epitope reduces and/or eliminates the natural tropism of the capsid protein or the capsid containing his. In some embodiments, the heterologous epitope is inserted into a region of the capsid protein that participates in the natural tropism of the reference wild-type capsid protein, for example, a region of the capsid protein associated with a cell receptor. In some embodiments, the heterologous epitope is inserted into and/or displayed by the knob 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 an amino acid position selected from the group consisting of G453 of AAV2 VP1 capsid protein, N587 of AAV2 VP1 capsid protein, Q585 of AAV6 VP1 capsid protein, G453 of AAV9 VP1 capsid protein, and A589 of AAV9 VP1 capsid protein. In some embodiments, a heterologous epitope is inserted and/or displayed between amino acid positions N587 and R588 of the AAV2 capsid VP1. In some embodiments, the recombinant viral capsid, a viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid contain the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant viral capsid, the viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid, contain the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the recombinant viral capsid, a viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid, contain an amino acid sequence encoded by the nucleic acid sequence represented by SEQ ID NO: 25. In some embodiments, the recombinant viral capsid, a viral vector containing a recombinant viral capsid, and/or compositions containing a recombinant viral capsid contain an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 26. In some embodiments, the recombinant viral capsid, a viral vector comprising the recombinant viral capsid, and/or compositions comprising the recombinant viral capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 27. Additional suitable insertion sites identified using AAV2 are well known in the art (Wu et al. (2000) J. Virol. 74:8635-8647) and include I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I- 459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713 and I-716. The recombinant viral capsid protein as described herein may be an AAV2 capsid protein containing a heterologous epitope inserted at a position selected from the group consisting of I-1, I-34, I-138, I-139, I- 161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, I-716 and combinations thereof. Additional suitable insertion sites identified 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 may be an AAV2 capsid protein containing a heterologous epitope inserted at a position selected from the group consisting of I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5) and combinations thereof.

[0099] The nomenclature I-### as used herein refers to an insertion site with ### indicating the amino acid number relative to the VP1 protein of the AAV capsid protein, however such an insertion may be located directly at the N- or C-terminus, preferably at C -end from one amino acid in a sequence of 5 amino acids from the N- or C-terminus of a given amino acid, preferably 3, more preferably 2, especially 1 amino acid from the N- or C-terminus of a given amino acid. In addition, the positions mentioned herein refer to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and their mutations) can be easily identified for the capsid proteins VP2 and VP3 encoded by the capsid gene by performing a sequence alignment of the VP1, VP2 proteins and VP3, encoded by the AAV capsid reference gene.

[00100] Thus, insertion into the appropriate nucleic acid coding position of one of these cap gene sites results in insertion into VP1, VP2 and/or VP3, since the capsid proteins are encoded by overlapping reading frames from the same gene with alternating start codons. Therefore, for AAV2, for example, according to this nomenclature, insertions between amino acids 1 and 138 are inserted in VP1 only, insertions between 138 and 203 are inserted in VP1 and VP2, and insertions between 203 and the C-terminus are inserted in VP1, VP2 and VP3, which, of course also applies to the I-587 insertion site. Therefore, the present invention covers the AAV structural genes with corresponding insertions into the VP1, VP2 and/or VP3 proteins.

[00101] In addition, due to the high conservation of at least large regions and the large number of closely related family members, relevant insertion sites for AAVs other than the listed AAV can be identified by performing amino acid alignments or comparing capsid structures. See, for example, Rutledge et al. (1998) J. Virol. 72:309-19 and US Pat. No. 9,624,274 for illustrative alignments of capsid proteins of various AAVs, each of which is incorporated herein by reference in its entirety.

[00102] In certain compositions disclosed herein consisting of a recombinant viral capsid (e.g., in the absence of a polyspecific binding molecule), the recombinant viral capsid protein is an AAV2 VP1 capsid protein with a heterologous epitope inserted at site 1587, where the heterologous epitope does not contain an Arg-Gly-Asp (RGD) motif, NGR motif, or c-myc. In certain compositions disclosed herein consisting of a recombinant viral capsid (e.g., in the absence of a polyspecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T448 and N449, where the heterologous epitope does not contain c-myc . In certain compositions disclosed herein consisting of a recombinant viral capsid (eg, in the absence of a polyspecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at site I-447, where the heterologous epitope does not contain L14 or ON THE.

[00103] In some compositions containing a recombinant viral capsid (eg, further comprising a polyspecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at site I587, where the heterologous epitope contains an Arg-Gly-Asp motif ( RGD), NGR motif or c-myc. In certain compositions disclosed herein containing a recombinant viral capsid (eg, further comprising a polyspecific binding molecule), the viral capsid is a VP1 capsid, the heterologous epitope contains c-myc, and the heterologous epitope is inserted between T448 and N449 or between N587 and R588. In certain compositions disclosed herein containing a recombinant viral capsid (eg, further comprising a polyspecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted at site I-447, where the heterologous epitope comprises L14 or HA. In certain compositions disclosed herein containing a recombinant viral capsid (eg, further comprising a polyspecific binding molecule), the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T448 and N449, where the heterologous epitope comprises c-myc. US Pat. No. 9,624,274 describes I-453 of the AAV capsid protein as a suitable insertion site for a heterologous epitope.

[00104] In some embodiments, insertion (display) of a heterologous epitope eliminates the natural tropism of the viral vector, e.g., transduction of a cell naturally permissive to infection by reference wild-type viral vectors and/or the target cell is not detected in the absence of a suitable multispecific binding molecule. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector, for example, compared to transduction of a cell naturally permissive to infection by reference wild-type viral vectors. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 10%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 20%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 30%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 40%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 50%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 60%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 70%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 80%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 90%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 95%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 90%. In these embodiments, where the insertion (display) of a heterologous epitope does not eliminate the natural tropism of the recombinant viral capsids, the natural tropism of such recombinant viral capsids can be eliminated by a second and different mutation. For example, in one embodiment, a recombinant viral capsid protein as described herein may be derived from the AAV9 capsid gene, contain a heterologous epitope, and may further contain a mutation, such as the W503A mutation.

[00105] This removal of targeted exposure of the virus to its natural host cell is important, especially if systemic administration is contemplated versus local or local-regional administration of viral vectors, since uptake of viral vectors by natural host cells limits the effective dose of viral vectors. In the case of AAV2 and AAV6, HSPG is reported to be the main receptor for viral uptake in a large number of cells, especially liver cells. For AAV2, HSPG binding activity depends on a group of 5 basic amino acids, R484, R487, R585, R588 and K532 (Kern et al., (2003) J Virol. 77(20): 11072-81). Recently, the amino acid substitution of lysine to glutamate K531E was reported to inhibit the ability of AAV6 to bind heparin or HSPG ((Wu et al., 2006) J. of Virology 80(22):11393–11397). Accordingly, preferred point mutations are those that reduce the natural receptor-mediated transduction activity of the viral vector for a given target cell by at least 50%, preferably by at least 80%, especially by at least 95% in the case of HSPGs such as the main receptor for binding viral vectors to HSPG.

[00106] Therefore, additional mutations preferred for HSPG-binding viral vectors are those that deplete or replace a basic amino acid, such as R, K or H, preferably R or K, which is involved in the binding of HSPG by the corresponding virus, to a non-basic amino acid such as A, D, G, Q, S and T, preferably A, or an amino acid that is present at the corresponding position of a different but highly conserved AAV serotype that lacks such a basic amino acid at that position. Therefore, 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. One particularly preferred embodiment of the present invention are those mutants of the AAV2 capsid protein that further contain two point mutations R585A and R588A, since these two point mutations are sufficient to eliminate the HSPG binding activity to a significant extent. These point mutations provide efficient targeting removal from HSPG-expressing cells, which for targeting increases the specificity of the corresponding mutant virus for its new target cell.

[00107] One embodiment of the present invention is a multimeric structure comprising a recombinant viral capsid protein of the present invention. The multimeric structure provides at least 5, preferably at least 10, more preferably at least 30, most preferably at least 60 recombinant viral capsid proteins containing a heterologous epitope as described herein. They can form regular viral capsids (empty viral particles) or viral vectors (capsids encapsulating the nucleotide of interest). The formation of viral vectors capable of packaging a viral genome is a highly advantageous feature for the use of recombinant viral capsids described herein as viral vectors.

[00108] One embodiment of the present invention is a nucleic acid encoding a capsid protein as described above. The nucleic acid is preferably a vector containing the claimed nucleic acid sequence. Nucleic acids, especially vectors, are required for the recombinant expression of the capsid proteins of the present invention.

[00109] Another embodiment of the present invention is the use of at least one recombinant viral capsid protein and/or nucleic acid encoding it, preferably at least one multimeric structure (for example, a viral vector), for manufacture and use as a transfer vector gene.

Heterologous epitopes

[00110] Typically, the recombinant viral capsid protein and/or a viral vector containing the recombinant viral capsid contains a heterologous epitope that is capable of retargeting the viral vector, for example, using a multispecific binding molecule. In some embodiments, the heterologous epitope is a B cell epitope, eg, is from about 1 amino acid to about 35 amino acids in length, and forms a binding pair with an antibody paratope, eg, an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises an affinity tag.

[00111] A large number of affinity 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, a polyhistidine tag (e.g., a His-6, His-8, or His-10 tag) that binds immobilized divalent cations (e.g., Ni ²⁺ ), a biotin moiety (e.g., on an in vivo biotinylated polypeptide sequence), that binds immobilized avidin, a GST (glutathione-8-transferase) sequence that binds immobilized glutathione, an S tag that binds immobilized protein S, an antigen that binds the immobilized antibody or domain or fragment thereof (including, for example, T7, myc tags , FLAG and B that bind the corresponding antibodies), a FLASH tag (a high-affinity tag that binds to specific arsenic-based moieties), a receptor or receptor domain that binds the immobilized ligand (or vice versa), protein A or its derivative (for example, Z ), which binds immobilized IgG, maltose-binding protein (MBP), which binds immobilized amylose, albumin-binding protein, which binds immobilized albumin, chitin-binding domain, which binds immobilized chitin, calmodulin-binding peptide, which binds immobilized calmodulin, and cellulose -binding domain that binds immobilized cellulose. Another exemplary tag is a SNAP tag commercially available from Covalys (www.covalys.com). In some embodiments, a heterologous epitope disclosed herein contains an affinity tag recognized only by a paratope of the antibody. In some embodiments, the heterologous epitope disclosed herein contains an affinity tag recognized by the antibody paratope and other specific binding pairs.

[00112] In some embodiments, the heterologous epitope and/or affinity tag does not form a binding pair with the immunoglobulin constant domain. In some embodiments, the heterologous epitope and/or affinity tag does not form a binding pair with a metal ion, for example, Ni ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , etc. In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A.

[00113] In some embodiments, the affinity tag is selected from the group consisting of FLAG (SEQ ID NO: 7), HA (SEQ ID NO: 8), and c-myc (EQKLISEEDL; SEQ ID NO: 6). In some embodiments, the heterologous epitope is c-myc.

[00114] In some embodiments, the recombinant viral capsid as described herein comprises the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked 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 the amino acid sequence EQKLISEEDL (represented by SEQ ID NO: 6) flanked 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 EQKLISEEDL (represented under 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 the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the recombinant viral capsid protein as described herein comprises the amino acid sequence set forth in SEQ ID NO: 2. NO: 4. In some embodiments, the recombinant viral capsid, a viral vector comprising the recombinant viral capsid, and/or compositions comprising the recombinant viral capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments embodiments, the recombinant viral capsid, viral vector containing the recombinant viral capsid, and/or compositions containing the recombinant viral capsid comprise an amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 26. In some embodiments, the recombinant viral capsid, viral vector containing a recombinant viral capsid, and/or compositions containing a recombinant viral capsid contain an amino acid sequence encoded by the nucleic acid sequence presented under SEQ ID NO: 27.

[00115] 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 a linker, for example, the heterologous epitope comprises, from N-terminus to C-terminus, a first linker, an affinity tag, and a second linker. In some embodiments, each of the first and second linkers is independently at least one amino acid long. In some embodiments, the first and second linkers are identical.

[00116] Typically, a heterologous epitope as described herein, for example, an affinity tag alone or in combination with one or more linkers, has a length of from about 5 amino acids to about 35 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is at least 5 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 6 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 7 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 8 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 9 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 10 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 11 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 12 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 13 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 14 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 15 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 16 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 17 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 18 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 19 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 20 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 21 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 22 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 23 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 24 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 25 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 26 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 27 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 28 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 29 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 30 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 31 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 32 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 33 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 34 amino acids. In some embodiments, the length of the heterologous epitope (by itself or in combination with one or more linkers) is 35 amino acids.

Retargeting fragments

[00117] The viral vector described herein is characterized by reduced or eliminated transduction ability in the absence of a multispecific binding molecule, in particular, a multispecific binding molecule containing (i) an antibody parasite that specifically binds the epitope, and (ii) a retargeting ligand , a specific binding receptor that can be conjugated to the bead surface (eg, for purification) or expressed by the target cell. 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" may include a scenario in which a wild-type viral vector is targeted to multiple cells in a tissue and/or multiple organs in the body, this broad tissue or organ targeting is reduced to the point of elimination by insertion of a heterologous epitope, and this retargeting to more specific cells in a tissue or a more specific organ in the body is achieved using a multispecific binding molecule. Such retargeting or redirection may also include a scenario in which a wild-type viral vector is targeted to a tissue, this tissue targeting is reduced to the point of elimination by insertion of a heterologous epitope, and this retargeting to an entirely different tissue is achieved using a multispecific binding molecule. An antibody paratope as described herein typically contains at least a complementarity determining region (CDR) that specifically recognizes a heterologous epitope, for example, the CDR3 region of a heavy and/or light chain variable domain. In some embodiments, the multispecific binding molecule comprises an antibody (or portion thereof) that contains an antibody paratope that specifically binds 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 single domain light chain variable region comprises an antibody paratope that specifically binds a heterologous epitope. In some embodiments, the polyspecific binding molecule may comprise an Fv region, for example, the polyspecific binding molecule may comprise a scFv containing an antibody paratope that specifically binds a heterologous epitope. In some embodiments, the multispecific binding molecule described herein comprises an antibody paratope that specifically binds c-myc.

[00118] In some embodiments, the multispecific binding molecule described herein comprises an antibody paratope that specifically binds c-myc, wherein the paratope comprises a scFv, scFv heavy and light chain variable domains, and/or an amino acid sequence of the set of HCDR1-HCDR2 -HCDR3-LCDR1-LCDR2-LCDR3 encoded by the nucleic acid sequence set forth in SEQ ID NO: 28, for example, a scFv comprising the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, a polyspecific binding molecule described herein , contains an Fv or scFv encoded by the nucleic acid sequence presented under SEQ ID NO: 28.

[00119] Accordingly, the present invention includes, as antibodies, antigen binding antibody fragments and polyspecific binding proteins that specifically bind c-myc, wherein the antibodies, antibody fragments and polyspecific binding proteins comprise a paratope that contains 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 antibody fragments and/or multispecific binding proteins that contain a paratope that specifically binds c-Myc, wherein the paratope contains heavy chain complementarity determining region 1 (HCDR1) containing SEQ ID NO: 31, HCDR2 containing SEQ ID NO: 32, HCDR3 containing SEQ ID NO: 33, light chain complementarity determining region 1 (LCDR1) containing SEQ ID NO : 34, LCDR2 containing SEQ ID NO: 35, and LCDR3 containing SEQ ID NO: 36.

[00120] The present invention provides antibodies, antigen-binding fragments thereof and/or polyspecific binding proteins containing a paratope that contains an HCVR containing the amino acid sequence represented by SEQ ID NO: 29, or a substantially similar sequence characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[00121] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains an LCVR containing the amino acid sequence presented under SEQ ID NO: 30, or a substantially similar sequence characterized by at least 90%, according to at least 95%, at least 98%, or at least 99% sequence identity with it.

[00122] The present invention provides antibodies, antigen-binding fragments thereof, and/or polyspecific binding proteins comprising a paratope that contains a pair of amino acid sequences HCVR and LCVR (HCVR/LCVR), containing the amino acid sequence HCVR represented by SEQ ID NO: 29, in a pair with the LCVR amino acid sequence set forth at SEQ ID NO: 30. In some embodiments, the HCVR/LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NO: 29/30.

[00123] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains a heavy chain CDR1 (HCDR1) containing the amino acid sequence represented by SEQ ID NO: 31, or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[00124] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains a heavy chain CDR2 (HCDR2) containing the amino acid sequence represented by SEQ ID NO: 32, or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[00125] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains a heavy chain CDR3 (HCDR3) containing the amino acid sequence represented by SEQ ID NO: 33, or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[00126] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains a light chain CDR1 (LCDR1) containing the amino acid sequence represented by SEQ ID NO: 34, or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[00127] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains a light chain CDR2 (LCDR2) containing the amino acid sequence represented by SEQ ID NO: 35, or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[00128] The present invention provides antibodies, antigen binding fragments and/or polyspecific binding proteins containing a paratope that contains a light chain CDR3 (LCDR3) containing the amino acid sequence represented by SEQ ID NO: 36, or a sequence substantially similar thereto, characterized by at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[00129] The present invention provides antibodies, antigen-binding fragments thereof, and/or polyspecific binding proteins comprising a paratope that contains a pair of amino acid sequences HCDR3 and LCDR3 (HCDR3/LCDR3), containing the amino acid sequence HCDR3 represented by SEQ ID NO: 33, in a pair with the LCDR3 amino acid sequence set forth at SEQ ID NO: 36. In some embodiments, the HCDR3/LCDR3 amino acid sequence pair is set forth at SEQ ID NO: 33/36.

[00130] The present invention provides antibodies, antigen binding fragments thereof and/or polyspecific binding proteins comprising a paratope that contains a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) encoded by a nucleotide sequence, presented under SEQ ID NO: 28. In certain embodiments, the set of amino acid sequences HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 is presented under SEQ ID NO: 31-32-33-34-35-36.

[00131] In a related embodiment, the present invention provides antibodies, antigen binding fragments thereof and/or polyspecific binding proteins comprising a paratope that contains a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3), contained in the pair of amino acid sequences HCVR/LCVR presented under SEQ ID NO: 29/30. Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in said HCVR and/or LCVR amino acid sequences disclosed herein. Exemplary approaches that can be used to determine the boundaries of the CDR include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the loop structural regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, MD. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 56:9268-9272 (1989). Public databases are also available to identify CDR sequences in an antibody.

[00132] The present invention also provides nucleic acid molecules encoding thuja antibodies or portions thereof.

[00133] A polyspecific binding molecule as described herein further comprises a retargeting ligand in addition to a paratope (e.g., an antibody or portion thereof) that specifically binds a heterologous epitope inserted into/displayed by the recombinant viral capsid protein. In some embodiments, the retargeting ligand binds 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, eg cell surface receptors, to which a retargeting ligand can be directed, and for which target altering ligands, eg antibodies or parts thereof, are already available. Such structures include, but are not limited to: class I and class II major histocompatibility complex antigens; receptors for various cytokines (for example, receptors for IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), cell type-specific growth hormones, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), colony-stimulating growth factors, endothelial growth factors, epidermal growth factors, fibroblast growth factors, glial neurotrophic factor, glial growth factors, gro-beta/mip 2, hepatocyte growth factors, insulin-like growth factor, interferons (α-IFN, β-IFN, γIFN, consensus IFN), interleukins (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14), keratinocyte growth factor, leukemia inhibitory factors, macrophage/monocyte chemotaxis activating factor, nerve growth factor activating neutrophil protein 2, platelet-derived growth factor, stem cell factor, transforming growth factor, tumor necrosis factors, vascular endothelial growth factor, lipoproteins including accessory or other type 1 transmembrane receptors such as PRLR, G protein-coupled receptors such as GCGR, ion channels such as Nav1.7, ASIC1 or ASIC2; cell adhesion molecules; transport molecules for metabolites such as amino acids; antigen receptors of B and T lymphocytes (for example, B cell receptors and associated proteins (for example, CD19, CD20, etc.) and T cell receptors and associated proteins (for example, CD3, CD4, CD8, etc. .); tetraspanin protein (e.g., CD63) The recombinant viral capsid described herein mediates cell type-specific infection by using a multispecific binding molecule containing a retargeting ligand that binds cell surface differentiation antigens as targets for the viral vector complex.

[00134] In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) liver cells, i.e. liver-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) brain cells, i.e. a brain cell-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (eg, exclusively) by (human) hematopoietic cells, i.e. marker specific for hematopoietic cells. In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) T cells, i.e. T-cell specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) B cells, i.e. B cell-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (eg, exclusively) by (human) dendritic cells, i.e. dendritic cell-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) macrophages, i.e. macrophage-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) NK cells, i.e. NK cell-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (eg, exclusively) by (human) kidney cells, i.e. kidney-specific marker. In some embodiments, the retargeting ligand binds a receptor expressed primarily (e.g., exclusively) by (human) pancreatic cells, i.e. pancreas-specific marker. In some embodiments, the retargeting ligand binds a receptor expressed primarily (e.g., exclusively) by intestinal (human) cells, i.e. gut-specific marker. In some embodiments, the retargeting ligand binds a protein expressed primarily (e.g., exclusively) by (human) cancer cells, i.e. tumor-associated antigen. In some embodiments, the retargeting ligand binds a protein expressed primarily (eg, exclusively) by (human) cells infected with a heterologous pathogen. Proteins that (1) are specifically expressed in, or enriched in, a cell/tissue/organ, and (2) are recognized by an antigen binding protein useful as a retargeting ligand as described herein are well known and can also be detected by 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 presents illustrative and non-limiting organ-specific markers for which antigen-binding proteins are available that may be useful as retargeting ligands, and the cells/tissue/organ expressing such markers.

[00135] In some embodiments, the retargeting ligand binds a receptor expressed by a (human) liver cell, such as an asialoglycoprotein receptor, such as hASGR1. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) brain cell. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) T cell, such as CD3, such as CD3ε. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) kidney cell. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) muscle cell, such as an integrin. In some embodiments, the retargeting ligand binds a receptor expressed by a (human) cancer cell, such as a tumor-associated antigen, such as E6 and E7. In some embodiments, the retargeting ligand binds the human glucagon receptor (hGCGR). In some embodiments, the retargeting ligand binds human ENTPD3.

[00136] In some embodiments, the retargeting ligand binds a tumor-associated antigen expressed by a tumor cell. Non-limiting examples of specific tumor-associated antigens include, for example, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR fusion protein -ABL b3a2, beta-catenin, BING-4, CA-125, CALCA, oncoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, SOA-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"), fusion protein ETV6-AML1, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7 , GAS7, glypican-3, GnTV, gp100/Pme117, GPNMB, HAUS3, hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3 , IL13Ralpha2, intestinal carboxylesterase, K-ras, kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1, also known as CCDC110, LAGE-1, LDLR-fucosyltransferase fusion protein AS, Lenzin, M -CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malik 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, polypeptide P, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGS5 , RhoC, RNF43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2 , telomerase, TGF-betaRII, TPBG, TRAG-3, triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (for lymphoma and other solid tumors), ErbB receptors, Melan A [MART1], gp 100, tyrosinase, TRP-1/gp 75 and TRP-2 (for melanoma); MAGE-1 and MAGE-3 (for bladder, head and neck and non-small cell carcinoma); proteins EG and E7 HPV (for cervical cancer); mucin [MUC-1] (for breast, pancreatic, colon and prostate cancer); prostate specific antigen [PSA] (for prostate cancer); oncoembryonic antigen [CEA] (for colon, breast and gastrointestinal cancers) and such common tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO-1/LAGE-2, NA-88, GnTV, TRP2-INT2. In some embodiments, the tumor-associated antigen is ErbB2/Her2. In some embodiments, the tumor-associated antigen is E6 and/or E7.

[00137] In some embodiments, the retargeting ligand binds to CD-based markers associated with the immune response, such as CD3, CD4, CD8, CD19, CD20, etc. In some embodiments, the CD-based marker is CD3.

[00138] In certain illustrative embodiments, the multispecific binding molecule is a bispecific antibody. Each antigen binding domain of a bispecific antibody contains a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen binding molecule comprising a first and a second antigen binding domain (eg, a bispecific antibody), the CDRs of the first antigen binding domain may be designated by the prefix "A1" and the CDRs of the second antigen binding domain may be designated by the prefix "A2". Thus, the CDRs of the first antigen binding domain may be referred to herein as A1-HCDR1, A1-HCDR2 and A1-HCDR3; and the second antigen binding domain CDRs may be referred to herein as A2-HCDR1, A2-HCDR2 and A2-HCDR3.

[00139] The first antigen binding domain and the second antigen binding domain can be directly or indirectly linked to each other to form a bispecific antigen binding molecule of the present invention. Alternatively, the first antigen binding domain and the second antigen binding domain may each be coupled to a separate multimerization domain. The association of one multimerization domain with another multimerization domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, the term “multimerization domain” means any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to bind to a second multimerization domain of the same or similar structure or composition. For example, the multimerization domain may be a polypeptide containing an immunoglobulin CH3 domain. A non-limiting example of a multimerization component is the Fc portion of an immunoglobulin (containing the CH2-CH3 domain), for example, the Fc domain of an IgG selected from the IgG1, IgG2, IgG3 and IgG4 isotypes, as well as any allotype within each isotype group.

[00140] The bispecific antigen binding molecules of the present invention typically contain two multimerization domains, for example two Fc domains, each of which is individually part of a distinct antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, such as, for example, IgG1/IgG1, IgG2/IgG2, IgG4/IgG4. Alternatively, the first and second multimerization domains may be of different IgG isotypes, such as, for example, IgG1/IgG2, IgG1/IgG4, IgG2/IgG4, etc.

[00141] In certain embodiments, the multimerization domain is an Fc fragment or amino acid sequence ranging from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerization domain is a cysteine residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides containing or consisting of a leucine zipper, a helix-loop motif, or a supercoil motif.

[00142] Any bispecific antibody format or technology can be used to produce the bispecific antigen binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen-binding specificity may be operably linked (e.g., by chemical binding, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity. specificity, yielding a bispecific antigen-binding molecule. Specific exemplary bispecific formats that may be used in the context of the present invention include, but are not limited to, scFv or diabody based bispecific formats, IgG-scFv hybrids, double variable domain (DVD)-Ig, quadra, peak-to-valley , conventional light chain (e.g., conventional peak-to-valley light chain, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, DuoBody, IgG1/IgG2, Dual Action Fab (DAF)-IgG, and bispecific Mab2 formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and sources cited herein for the above formats; see also Brinkmann and Konterman (2017) mAbs 9: 182 -212; each of which is incorporated by reference in its entirety).

[00143] The present invention also includes bispecific antigen binding molecules comprising a first CH3 domain and a second CH3 domain of Ig3, wherein the first and second CH3 domains of Ig3 differ from each other by at least one amino acid, and wherein there is at least one difference in amino acids reduces the binding of a bispecific antibody to protein A compared to a bispecific antibody in which there is no 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 the H95R modification (IMGT exon numbering; H435R EU numbering). The second CH3 may additionally contain the modification Y96F (according to IMGT; Y436F according to EU). Additional modifications that can 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-based antibodies; N44S, K52N and V82I (IMGT; EU N384S, K392N and V422I) in the case of IgG2-based 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-based antibodies; see for example WO 2010/151792.

[00144] In some embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain may comprise part or all of a CH2 sequence derived from the CH2 region of human IgG1, human IgG2, or human IgG4, and part or all of a CH3 sequence derived from human IgG1, human IgG2, or human IgG4. The chimeric Fc domain may also contain a chimeric hinge region. For example, a chimeric hinge may comprise a “upper hinge” sequence derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination 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 can be included in any of the antigen binding molecules provided herein contains, from N- to C-terminus: [CH1 IgG4] - [IgG4 upper hinge sequence] - [IgG2 lower hinge sequence] - [CH2 IgG4] - [CH3 IgG4]. Another example of a chimeric Fc domain that can be included in any of the antigen binding molecules provided herein contains, from N- to C-terminus: [CH1 IgG1] - [IgG1 upper hinge sequence] - [IgG2 lower hinge sequence] - [CH2 IgG4] - [CH3 IgG1]. These and other examples of chimeric Fc domains that can be included in any of the antigen binding molecules of the present invention are described in PCT application No. WO 2014/022540, incorporated by reference in its entirety. Chimeric Fc domains containing these common structural compositions and variants thereof may have altered binding to the Fc receptor, which in turn affects Fc effector function.

Methods of application and production

[00145] An additional embodiment of the recombinant viral capsid proteins described herein is their use to deliver a nucleotide of interest, such as a reporter gene or therapeutic gene, to a target cell. Typically, the nucleotide of interest may be a transfer plasmid, which may typically contain 5' and 3' inverted terminal repeat (ITR) sequences flanking the reporter gene(s) or therapeutic gene(s) ), which may be under the control of a viral or non-viral promoter when incorporated into an AAV-based vector. In one embodiment, the nucleotide of interest is a transfer plasmid containing 5' to 3': 5'-ITR, promoter, gene (eg, reporter and/or therapeutic gene), and 3'-ITR.

[00146] Non-limiting examples of useful promoters include, for example, the cytomegalovirus (CMV) promoter, spleen necrosis virus (SFFV) promoter, elongation factor 1 alpha (EF1a) promoter (1.2 kb EF1a promoter or EF1a promoter size 0.2 kb), chimeric EF1a/IF4 promoter and phosphoglycerate kinase (PGK) promoter. An internal enhancer may also be present in the viral construct to increase expression of the gene of interest. For example, a CMV enhancer can be used (Karasuyama et al. 1989. J. Exp. Med. 169:13, which is incorporated herein by reference in its entirety). In some embodiments, a CMV enhancer can be used in combination with a chicken 13-actin promoter.

[00147] A variety of reporter genes (or detectable fragments) can be contained in a multimeric structure containing the recombinant viral capsid proteins described herein. Exemplary reporter genes include, for example, β-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), cerulein, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof. The methods described herein demonstrate the construction of targeted vectors that use a reporter gene that encodes green fluorescent protein, however, those skilled in the art will understand upon reading this disclosure that the non-human animals described herein can be produced in the absence of a reporter gene or with any reporter gene known in the art.

[00148] A variety of therapeutic genes can also be contained in a multimeric structure containing recombinant viral capsid proteins described herein, for example, as part of a transfer vector. Non-limiting examples of a therapeutic gene include those encoding a toxin (eg, a suicide gene), a therapeutic antibody or fragment thereof, a CRISPR/Cas system or portion(s) thereof, antisense RNA, siRNA, shRNA, etc.

[00149] A further embodiment of the present invention is a method for producing a recombinant capsid protein, the method comprising the steps of:

a) expressing the nucleic acid encoding the recombinant capsid protein under suitable conditions, and

b) isolating the expressed capsid protein from step a).

[00150] Additional embodiments of the present invention provide a method for altering the tropism of a virus, comprising the steps of: (a) inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein, containing a heterologous epitope, and/or (b) culturing the packaging cell under conditions sufficient to produce viral vectors, where the packaging cell contains the nucleotide sequence. A further embodiment of the present invention is a method of displaying a heterologous epitope on the surface of a capsid protein, the method comprising the steps of: a) expressing a nucleic acid of the invention under suitable conditions and b) isolating the expressed capsid protein from step a).

[00151] In some embodiments, the packaging cell further comprises a helper plasmid and/or a transfer plasmid containing the nucleotide of interest. In some embodiments, the methods further comprise isolating adeno-associated virus self-complementary vectors from the culture supernatant. In some embodiments, the methods further comprise lysing the packaging cell and isolating single-stranded adeno-associated virus vectors from the cell lysate. In some embodiments, the methods further comprise (a) removing cellular debris, (b) treating the supernatant containing the viral vectors with DNase I and MgCl2, (c) concentrating the viral vectors, (d) purifying the viral vectors, and (e) any combination of (a )-(d).

[00152] Packaging cells useful for producing the viral vectors described herein include, for example, animal cells permissive for the virus, or cells modified to be permissive for the virus; or the design of the packaging cell, for example, using a transforming agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral vectors described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK- 293, which contain the SV40 large T antigen (HEK-293T or 293T), HEK293T/17 cells, the human sarcoma cell line HT-1080 (CCL-121), the Raji lymphoblast-like cell line (CCL-86), the epithelial-like glioblastoma cell line- U87-MG astrocytomas (HTV-14), T-lymphoma cell line HuT78 (TIB-161), NIH/3T3 cells, Chinese hamster ovary (CHO) cells (eg ATCC No. CRL9618, CCL61, CRL9096), HeLa cells (eg , 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.

[00153] L929 cells, the FLY virus cellular packaging system described in Cosset et al. (1995) J. Virol 69, 7430-7436, NSO cells (murine myeloma), human amniocyte cells (e.g. CAP, CAP-T), yeast cells (including but not limited to S. cerevisiae, Pichia pastoris), plant cells (including without limitation Tobacco NT1, BY-2), insect cells (including without limitation SF9, S2, SF21, Tni (e.g. High 5)) or bacterial cells (including without limitation E. coli).

[00154] For additional packaging cells and systems, packaging techniques and vectors for packaging a genome as a nucleic acid into a pseudotyped viral vector, see, for example, Polo et al., Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include the use of packaging cells that continuously express viral components, or by transiently transfecting cells with plasmids.

[00155] Additional embodiments include methods of redirecting a virus and/or delivering a reporter or therapeutic gene to a target cell, where the method includes a method of transducing cells in vitro or in vivo, the method comprising the steps of: contacting the target cell with a combination of viral a vector that contains a capsid, including a recombinant viral capsid displaying a heterologous epitope, and a polyspecific binding molecule, where the polyspecific binding molecule contains i) an antibody paratope that specifically binds the epitope, and (ii) a retargeting ligand that specifically binds a receptor expressed by the cell- target. In some embodiments, a composition described herein or a method described herein combines a recombinant viral vector and a polyspecific binding molecule in a molecule:molecule ratio that restores the transduction efficiency of the viral vector to similar efficiency to that of a control wild-type viral vector . In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is in the range of 1:0.5 to 1:100. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is in the range of 1:4 to 1:20. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is in the range of 1:8 to 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:4. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:8. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is 1:20. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:100. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:50. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:20. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:15. In some embodiments, the ratio of recombinant viral vector to polyspecific binding molecule (molecule:molecule) is less than 1:10.

[00156] In some embodiments, the target cell is in an in vitro environment. In other embodiments, the target cell is present in vivo in a subject, such as a human.

Target cells

[00157] A wide variety of cells can be used to deliver a nucleotide of interest using a recombinant viral vector as disclosed herein. Target cells are usually selected based on the nucleotide of interest and the desired effect.

[00158] In some embodiments, a nucleotide of interest may be delivered to allow a target cell to produce a protein that corrects a deficiency in the body, such as an enzymatic deficiency or an immunodeficiency such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that typically produce protein in the animal are targets. In other embodiments, cells in the area in which the protein would be most beneficial are targeted.

[00159] In other embodiments, a nucleotide of interest, such as a gene encoding an siRNA, can inhibit the expression of a particular gene in a target cell. The nucleotide of interest may, for example, inhibit the expression of a gene involved in the life cycle of the pathogen. Thus, cells susceptible to infection by a pathogen or infected by a pathogen can be targeted. In other embodiments, the nucleotide of interest may inhibit the expression of a gene that is responsible for the production of a toxin in a target cell.

[00160] In other embodiments, the nucleotide of interest may encode a toxic protein that causes the death of cells in which it is expressed. In this case, tumor cells or other unwanted cells may be targeted.

[00161] In other embodiments, a nucleotide of interest may be used that encodes a protein to be harvested, such as a therapeutic protein, and cells that are capable of producing and secreting the protein are targeted.

[00162] Once a specific population of target cells in which expression of the nucleotide of interest is desired has been identified, a target receptor that is specifically expressed in that population of target cells is selected. The target receptor may be expressed exclusively in this population of cells or to a greater extent in this population of cells than in other populations of cells. The more specific the expression, the more specifically the delivery can be directed to target cells. Depending on the context, the desired amount of marker specificity (and therefore gene delivery) may vary. For example, for introducing a toxic gene, high specificity is most preferable to avoid the death of non-target cells. For protein expression to harvest or express a secreted product when global exposure is required, less marker specificity may be required.

[00163] As discussed above, the target receptor can be any receptor for which a retargeting ligand can be identified or created. Preferably, the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target receptor may be a carbohydrate or other molecule that can be recognized by a binding partner. If the 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 obtained using standard procedures. These antibodies can then be used as a retargeting ligand as part of a multispecific binding molecule.

[00164] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the specific application (e.g., therapy, expression of the protein to be harvested, and conferring disease resistance) and (2) expression of a marker with the desired the value of specificity.

[00165] The target cells are not limited in any way and include both germline cells and cell lines and somatic cells and cell lines. The target cells can be stem cells of any origin. When the target cells are germline cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem (ES) cells.

Pharmaceutical compositions, dosage forms and administration

[00166] In a further embodiment, a medicinal product is provided comprising at least one recombinant viral capsid protein and a corresponding polyspecific binding molecule of the present invention and/or a nucleic acid of the present invention, preferably at least one multimeric structure of the present invention. Preferably, such a drug is useful as a vector for gene transfer.

[00167] Also disclosed herein are pharmaceutical compositions comprising the viral vectors described herein and a pharmaceutically acceptable carrier and/or excipient. In addition, this document discloses pharmaceutical dosage forms containing the viral vector described herein.

[00168] As discussed herein, the viral vectors described herein can be used for a variety of therapeutic applications (in vivo and ex vivo) and as research tools.

[00169] Pharmaceutical compositions based on the viral vectors disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and/or excipients. The viral vector can be formulated for administration, for example, by injection, inhalation or instillation (oral or nasal), or by oral, buccal, parenteral or rectal administration, or by administration directly into the tumor.

[00170] Pharmaceutical compositions can be formulated for various routes of administration, including systemic, local or localized administration. Methods and formulations can be found, for example, in Remrnington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pennsylvania. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal and subcutaneous. For injection, the pharmaceutical compositions can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hanks' solution or Ringer's solution. In addition, pharmaceutical compositions can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms of the pharmaceutical composition are also suitable.

[00171] For oral administration, the pharmaceutical compositions may be in the form of, for example, tablets or capsules prepared by conventional methods with pharmaceutically acceptable excipients such as binders (eg, pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (eg magnesium stearate, talc or silicon dioxide); disintegrants (eg potato starch or sodium starch glycolate) or wetting agents (eg sodium lauryl sulfate). Tablets can also be coated using methods well known in the art. Liquid preparations for oral administration may be in the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable carrier medium before use. Such liquid preparations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (for example, lecithin or acacia); non-aqueous carrier media (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) and preservatives (for example, methyl or propyl p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavorings, colors and sweeteners, if necessary.

[00172] Pharmaceutical compositions can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations may be presented in unit dosage form, such as ampoules or multi-dose containers, optionally with the addition of a preservative. Pharmaceutical compositions may further be formulated as suspensions, solutions or emulsions in oily or aqueous carrier vehicles and may contain other agents, including suspending, stabilizing and/or dispersing agents.

[00173] In addition, pharmaceutical compositions can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil) or ion exchange resins, or as sparingly soluble derivatives, eg, as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of local, non-invasive drug delivery over an extended period of time. This technology may include microspheres having a precapillary size that can be injected through a coronary catheter into any selected part of the organ without causing inflammation or ischemia. The administered therapeutic agent is slowly released from the microspheres and taken up by surrounding cells present in the selected tissue.

[00174] Systemic administration can also be accomplished by transmucosal or transdermal routes. For transmucosal or transdermal administration, the formulation uses penetrants suitable for penetration across the barrier. 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 facilitate penetration. Transmucosal administration can be accomplished using nasal sprays or suppositories. For topical administration, the viral vector described herein can be formulated into ointments, balms, gels or creams generally known in the art. The rinse solution can also be used topically to treat injury or inflammation to promote healing.

[00175] Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or an aqueous solution of propylene glycol; and sterile powders for the preparation of sterile injectable solutions or dispersions for immediate administration. In all cases, the form must be sterile and must be liquid. It must be stable under production conditions and under certain storage conditions (e.g. refrigeration and freezing) and must be protected from the contaminating action of microorganisms such as bacteria and fungi.

[00176] If the compositions disclosed herein are used as a therapeutic agent to enhance the immune response in a subject, the therapeutic agent may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and salts that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, etc. P. Salts formed with free carboxyl groups can also be prepared from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

[00177] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required size of the viral vector in the case of a dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved using various antibacterial and antifungal agents known in the art. In many cases it is preferable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using absorption retarding agents in the compositions, for example, aluminum monostearate and gelatin.

[00178] Sterile injectable solutions can be prepared by incorporating the active compounds or constructs in the required amount in a suitable solvent with various other ingredients listed above, if necessary, followed by sterilization by filtration.

[00179] Once formulated, the solutions can be administered in a manner appropriate to the dosage formulation and in an amount that is therapeutically effective. The compositions can be easily administered in various dosage forms, such as the type of injection solutions described above, but capsules or sustained release microparticles and microspheres and the like can also be used.

[00180] For example, for parenteral administration in an aqueous solution, the solution must be suitably buffered, if necessary, and the liquid diluent must first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous and intraperitoneal administration. In this context, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or administered at the proposed infusion site.

[00181] The person responsible for administration will in any case determine the appropriate dose for the individual subject. For example, a subject may be administered the viral vectors described herein daily or weekly for a period of time, or monthly, biannually, or annually, depending on the need or exposure to the pathogenic organism or condition in the subject (eg, cancer).

[00182] 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; delayed release capsules; biodegradable and any other forms currently in use.

[00183] Intranasal or inhalation solutions or sprays, aerosols or inhalers can also be used. Nasal solutions can be aqueous solutions intended to be administered into the nasal passages in the form of drops or sprays. Nasal solutions can be prepared so that they are similar in many ways to nasal secretions. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH between 5.5 and 7.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, and appropriate drug stabilizers, if necessary, can be included in the formulation. Various commercial nasal preparations are known, which may include, for example, antibiotics and antihistamines, and are used for the prevention of asthma.

[00184] Oral formulations may include excipients such as, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. pharmaceutical grade. These compositions are given in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. In certain embodiments, pharmaceutical compositions for oral administration will include an inert diluent or digestible edible carrier, or they may be enclosed in a hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be included directly in a food product. prescribed by the diet. For oral therapeutic administration, the active compounds can be included with excipients and administered in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers and the like.

[00185] Tablets, lozenges, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, gum acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a leavening agent such as corn starch, potato starch, alginic acid and the like; a glidant such as magnesium stearate; and a sweetener such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, oil of wintergreen or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or otherwise modify the physical form of the dosage form. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. The syrup or elixir may contain active sucrose compounds as a sweetener, methyl and propyl parabens as preservatives, colors and flavorings such as cherry or orange flavoring.

[00186] Other embodiments disclosed herein may provide kits for use in accordance with the methods and compositions. The kits may also include a suitable container, such as vials, tubes, mini or microcentrifuge tubes, test tube, flask, vessel, syringe or other container. If an additional component or component is provided, the kit may include one or more additional containers into which the component or component can be placed. The kits herein also generally include means for sealing the viral vectors and any other reagent containers for commercial sale. Such containers may include injection molded or blow molded plastic containers into which the required vials are placed. Optionally, the compositions described may require one or more additional active agents, such as, for example, anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antineoplastic agents.

[00187] Dosage ranges and frequency of administration may vary depending on the nature of the viral vectors and health conditions, as well as the characteristics of the individual patient and the route of administration used. In some embodiments, viral vector compositions can be administered to a subject at a dose ranging from about 1 x 105 plaque forming units (PFU) to about 1 x 10 PFU depending on the route of administration, the route of administration, the nature of the disease, and the condition of the subject. In some cases, viral vector compositions may be administered at a dose ranging from about 1 x 108 PFU to about 1 x 1015 PFU, or from about 1 x 1010 PFU to about 1 x 1015 PFU, or from about 1 x 108 PFU to about 1 ×1012 PFU. The more precise dose may also depend on the subject to whom it is administered. For example, a lower dose may be required if the subject is a minor, and a higher dose may be required if the subject is an adult human subject. In certain embodiments, the more precise dose may depend on the weight of the subject. In certain embodiments, for example, a juvenile human subject may receive a dose of from about 1×108 PFU to about 1×1010 PFU, while an adult human subject may receive a dose of from about 1×1010 PFU to about 1×1012 PFU.

[00188] The compositions disclosed herein can be administered by any method known in the art. For example, the compositions may be administered to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesional, intracranial, intraarticular, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, oral, topical, inhalation, injection, infusion, continuous infusion, local perfusion, catheter, rinse, cream or lipid composition.

[00189] Any method known to one skilled in the art can be used for large-scale production of the viral vectors, packaging cells, and vector constructs described herein. For example, master and working seeds can be obtained under GMP conditions in qualified primary CEFs or by other means. Packaging cells can be seeded into high surface area flasks, grown to near confluency, and viral vectors purified. Cells can be collected and viral vectors released into the culture medium, isolated and purified, or intracellular viral vectors released by mechanical disruption (cellular debris can be removed by large pore depth filtration and endonuclease digestion of host cell DNA). Viral viral vectors can subsequently be purified and concentrated by tangential flow filtration followed by diafiltration. The resulting concentrated mass can be formulated by diluting with a buffer containing stabilizers, filled into vials and lyophilized. The compositions and formulations can be stored for later use. Lyophilized viral vectors can be reconstituted for use by adding a diluent.

[00190] Certain additional agents used in combination therapy can be formulated and administered by any method known in the art.

[00191] The compositions disclosed herein may also include adjuvants such as aluminum salts and other mineral adjuvants, surfactants, bacterial components, carrier media and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants can stimulate Th1- or Th2-dependent immunity. The compositions and methods disclosed herein may also provide adjuvant therapy.

EXAMPLES

[00192] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1. Preparation of vectors based on an adeno-associated virus with a heterologous epitope

[00193] AAV capsid proteins are modified to contain one of several heterologous epitopes: FLAG, c-myc, hexahistidine, etc., using PCR to produce a plasmid encoding a recombinant capsid protein. Briefly, the sequence encoding FLAG, c-myc, or hexahistidine was inserted in frame after the codon encoding N587 of the AAV2 capsid protein, Q585 of the VP1 capsid protein of AAV6, N590 of the VP1 capsid protein of AAV8, A589 of the VP1 capsid protein of AAV9, or G453 of the VP1 capsid protein of AAV9.

[00194] Adeno-associated virus production was obtained using the triple transfection method of HEK293 cells (see, for example, Erik Arden and Joseph M. Metzger, J Biol Methods. 2016; 3(2)). Cells were seeded one day prior to PEFpro-mediated (Polyplus transfection, New York, NY) transfection with the appropriate vectors:

helper plasmid, pHelper (Agilent, catalog number 240074);

plasmid encoding wild type or modified AAV rep/cap gene (pAAV RC2 (Cell biolabs, cat. no. VPK-422), e.g. pAAV RC2/6/9 (Cell Biolabs, catalog no. VPK-426), pAAV RC8, pAAV RC2-N587myc, pAAV RC2/6-Q585myc, pAAVRC8-N590myc, pAAV RC9-A589myc, etc.; and

a plasmid encoding the AAV nucleotide sequence and ITR sequences of interest, for example pscAAV-CMV-eGFP, pAAV-CMVGFP (Agilent catalog number 240074), pAAV-EF1a-eGFP or pAAV-CAGG-eGFP, etc.

[00195] Seventy-two hours after transfection, the medium was collected and the cells were lysed in buffer [50 mM Tris-HCl, 150 mM NaCl, and 0.5% sodium deoxycholate (Sigma part number D6750-100G)]. Benzonase (Sigma, St. Louis, MO) was then added to both the medium and cell lysate to a final concentration of 0.5 U/μl before incubation at 37°C for 60 minutes. The cell lysate was centrifuged at 4000 rpm. within 30 minutes. Cell lysate and medium were combined and precipitated with PEG 8000 (Teknova, part number P4340) at a final concentration of 8%. The sediment was resuspended in 400 mM NaCl and centrifuged at 10,000 g for 10 minutes. Viruses in the supernatant were pelleted by ultracentrifugation at 149,000 g for 3 hours and titrated by qPCR.

[00196] For qPCR titration of AAV genomes, AAV samples were treated with DNase I (Thermofisher Scientific, part number EN0525) at 37°C for one hour and lysed using the DNA extract All Reagents kit (Thermofisher Scientific, part number 4403319) . Protein-enclosed viral genomes were quantified using a QuantStudio 3 real-time PCR system (Thermofisher Scientific) using AAV2 ITR primers. The primer sequences for the AAV2 ITR are 5'-GGAACCCCTAGTGATGGAGTT-3' (ITR forward primer; SEQ ID NO: 9) and 5'-CGGCCTCAGTGAGCGA-3' (ITR reverse primer; SEQ ID NO: 10) (Aurnhammer et al ., 2012), respectively cutting out the left internal inverted repeat (ITR) sequence from AAV and the right internal inverted repeat (ITR) sequence from AAV. The probe sequence for the AAV2 ITR is 5'-6-FAM-CACTCCCTCTCTGCGCGCTCG-TAMRA-3' (SEQ ID NO: 11) (Aurnhammer S., Haase M., Muether N., et al., 2012, Hum. Gene Ther Methods 23, 18-28). After the activation step at 95°C for 10 minutes, a two-step PCR cycle was performed at 95°C for 15 seconds and 60°C for 30 seconds for 40 cycles. TaqMan Universal PCR Master Mix (Thermofisher Scientific, part number 4304437) was used for qPCR. DNA plasmid (Agilent, catalog number 240074) was used as a standard for determining absolute titers.

[00197] Adeno-associated virus-based vectors containing a capsid with the c-myc epitope were produced. In this example, the c-myc epitope (EQKLISEEDL; SEQ ID NO: 6) was inserted between amino acids N587 and R588 of the AAV2 VP1 capsid protein or between amino acids A589 and Q590 of the AAV9 VP1 capsid protein, i.e. the nucleotide sequence encoding the c-myc epitope (GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG; SEQ ID NO: 12) was inserted into plasmid pAAV RC2 (Cell Biolabs, Inc., San Diego, Calif.) or plasmid pAAV RC2 /9 and each of the corresponding modified plasmids pAAV RC2-N587Myc, pAAV RC9-A589Myc, were used to encode a modified capsid protein for AAV-based viral vectors with reduced or eliminated tropism.

[00198] To generate pAAV RC2-N587Myc, a first polymerase chain reaction (PCR) product was obtained containing (5' to 3') the BsiWl restriction site, the nucleotide sequence between positions 3050 and 3773 of pAAV RC2, and the nucleotide sequence of the c-myc epitope with " sticky ends, and a second PCR product containing (5' to 3') the nucleotide sequence of the c-myc epitope with sticky ends, the nucleotide sequence between positions 3774 and 4370 of pAAV RC2 and the Pme1 restriction site, using the primers given in Table 2. Plasmid pAAV RC2-N587Myc (i.e., plasmid pAAV RC2 modified to encode the c-myc epitope between amino acids N587 and R588 of the VP1 capsid protein) was created by digesting pAAV RC2 with BsiW1 (New England Biolabs, R0553L) and Pmel (New England Biolabs, R0560L) and insertion of two PCR products using ligation-independent cloning described in (2012) Methods Mol. Biol. 52:51-9.

[00199] To create pAAV RC2/6-Q585Myc, a gblock DNA fragment containing positions 3700 and 3940 of pAAV RC2/6 with the c-myc epitope sequence inserted between 3757 and 3758 was ordered from Integrated DNA Technologies (Coralville, Iowa). Plasmid pAAV RC2/6-Q585Myc was generated by inserting a gblock fragment into pAAV RC2/6 digested with MscI (New England Biolabs, cat. no. R0534L) and Aflll (New England Biolabs, cat. no. R0520L) using ligation-independent cloning , described in (2012) Methods Mol. Biol. 52:51-9.

[00200] To generate pAAV RC9-A589Myc, a first polymerase chain reaction (PCR) product was obtained containing (5' to 3') the BsiW1 restriction site, the nucleotide sequence between positions 3052 and 3779 of pAAV RC9, and the nucleotide sequence of the c-myc epitope with " sticky ends, and a second PCR product containing (5' to 3') the nucleotide sequence of the c-myc epitope with sticky ends, the nucleotide sequence between positions 3779 and 4404 of pAAV RC9 and the Pme1 restriction site, using the primers given in table 2.

[00201] Plasmid pAAV RC9-A589Myc (i.e., plasmid pAAV RC2/9 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 insertion of the two PCR products using ligation-independent cloning described in (2012) Methods Mol. Biol. 52:51-9.

[00202] pscAAV-CMV-eGFP was produced by introducing a GFP fragment into the pscAAV MCS vector (Cell Biolabs, catalog number VPK-430) using restriction sites for BamHI and NotI. Plasmid pAAV-EF1a-eGFP and pAAV-CAGG-eGFP were synthesized de novo at Thermofisher Scientific (Waltham, MA).

Example 2: Antibody-Mediated Retargeting of AAV-Based Viral Vectors in Vitro

[00203] HepG2 is a human liver cancer cell line that expresses the liver-specific marker asialoglycoprotein receptor 1 (ASGR1). To test whether scAAV2-N587Myc-CMV-hrGFP viral vectors can infect HepG2 cells, for example, using a bispecific antibody that recognizes both c-myc and ASGR1 (anti-myc-ASGR1 antibody), mixtures containing different ratios of viral genomes (5e9 viral genomes) and number of antibody molecules (1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50 or 1:100) scAAV2 -N587Myc-CMV-eGFP and myc-ASGR1 bispecific antibody were incubated at room temperature for half an hour and then added to HepG2 cells. HepG2 cells were also incubated with scAAV2-CMV-eGFP wild-type viral vectors only, scAAV2-N587Myc-CMV-eGFP viral vectors only, or scAAV2-N587Myc-CMV-eGFP viral vectors with a monospecific anti-myc antibody (Regeneron Pharmaceuticals, Inc., Tarrytown , New York) at a ratio of 1:8 as controls. Three days postinfection, GFP expression by infected HepG2 cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 1). GFP expression was also detected in comparable percentages of HepG2 cells when incubated with wild-type scAAV2-CMV-eGFP and when incubated with viral vectors scAAV2-N587Myc-CMV-eGFP and bispecific antibody to myc-ASGR1 in a ratio of 1:8 (44.6% and 44.1%, respectively, Figures 1A and 1G, top and bottom panels). Lower GFP expression was detected by FACS in HepG2 cells incubated with a mixture of scAAV2-N587Myc-CMV-eGFP viral vectors and higher or lower amounts of myc-ASGR1 bispecific antibody (Regeneron Pharmaceuticals, Inc., Tarrytown, NY ) (Figure 1C, bottom panel). In addition, GFP was not detected in HepG2 cells infected with scAAV-N587Myc-CMV-eGFP in the absence of anti-myc-ASGR1 antibody or in HepG2 cells incubated with scAAV2-N587Myc-CMV-eGFP and monospecific anti-myc antibody.

[00204] Similarly, 293T-hASGR1 cells, which are 29T3 cells genetically modified to express human (h)ASGR1 on the cell surface, were incubated with mixtures containing varying ratios of viral genomes and amounts of antibody molecules (1:0.5, 1: 1, 1:2, 1:4, 1:8, 1:15, 1:20 or 1:100) scAAV2-N587Myc-CMV-eGFP and myc-ASGR1 bispecific antibody. Wild-type 293T cells (which do not express hASGR1) incubated with a 1:8 mixture of scAAV2-N587Myc-CMV-eGFP and anti-myc-ASGR1 bispecific antibody, and 293T-hASGR1 cells incubated with (a) wild-type scAAV viral vectors only type, (b) scAAV2-N587Myc-CMV-eGFP viral vectors alone or (c) scAAV2-N587Myc-CMV-eGFP viral vectors with an irrelevant myc-GCGR bispecific antibody that recognizes c-myc and the glucagon receptor (GCGR) (Regeneron Pharmaceuticals, Inc., Tarrytown, NY) not expressed by 293T cells at a ratio of 1:8 served as controls. Three days postinfection, 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, no. catalog 109-136-098, West Grove, PA) and GFP expression was analyzed by FACS. Expression of hASGR1 was detected on the surface of 293T-hASGR1 cells (Figures 2Ai-2Ax and 2Axii) but not 293T cells (Figure 2Axi). GFP-positive 293T-hASGR1 cells were detected after incubation with mixtures of scAAV2-N587Myc-CMV-eGFP and myc-ASGR1 bispecific antibody in ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20 and 1:50 at levels (56.9%-68.3%) comparable to 293T-hASGR1 cells incubated with wild-type scAAV (56.7%) (Figures 2Ai and 2Aiii-2Aix ). A ratio of 1:100 for scAAV2-N587Myc-CMV-eGFP and myc-ASGR1 bispecific antibody reduced infectivity (Figure 2Ax). Incubation with scAAV2-N587Myc-CMV-eGFP alone or scAAV-N587Myc with an irrelevant myc-GCGR bispecific antibody did not result in GFP expression by 293T-hASGR1 cells (Figures 2Aii and 2Axii).

[00205] In a similar experiment, 293T-hASGR1 cells were incubated with cells incubated with unmodified AAV9-CAGG-GFP viral vectors only, AAV9-A589Myc-CAGG-eGFP viral vectors only, or a mixture of AAV9-A589Myc-CAGG-eGFP viral vectors with bispecific antibodies to Myc-ASGR1 in different ratios. Three days postinfection, GFP expression was analyzed by FACS. GFP-positive 293T-hASGR1 cells were detected after incubation with mixtures of AAV9-A589Myc-CAGG-eGFP and myc-ASGR1 bispecific antibody in ratios of 1:1, 1:2, 1:4, 1:8, 1:20, 1: 50 and 1:100 at levels (41.1%-91%) comparable to 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)).

[00206] 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 various concentrations of bivalent anti-hASGR1 antibody for 1 hour at room temperature and then incubated with a mixture containing a 1:8 ratio of scAAV2-N587Myc-CMV-eGFP and bispecific anti-myc-ASGR1 antibody. Three days postinfection, cells were fixed and analyzed by FACS as described above. Figure 3 presents data showing that entry of scAAV2-N587Myc-CMV-eGFP can be blocked in a dose-dependent manner by a bivalent anti-hASGR1 antibody.

[00207] To determine whether sequential administration of a bispecific antibody and a modified AAV could result in an antibody-mediated change in the target of the modified AAV, varying amounts of anti-myc-ASGR1 bispecific antibody molecules (ranging from 1×10 ⁹ to 1×10 ¹² molecules) were added to 2×10 ⁵ 293T-hASGR1 cells for an hour, after which 1×10 ⁹ scAAV-N587Myc viral vectors were added. Controls included (1) 293T-hASGR1 cells incubated with wild-type scAAV only, i.e. in the absence of any antibody, (2) 293T-hASGR1 cells sequentially incubated with 1x10 ¹¹ molecules of an irrelevant bispecific antibody to myc-GCGR and 1x10 ⁹ scAAV2-N587Myc-CMV-eGFP viral vectors, and (3) cells 293T, which do not express hASGR1, sequentially incubated with 1×10 ¹¹ bispecific antibody molecules to myc-ASGR1 and 1×10 ⁹ scAAV-N587Myc viral vectors. Two days postinfection, GFP expression in infected 293T-hASGR1 cells was visualized 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 at all concentrations and scAAV2-N587Myc-CMV-eGFP (Figures 4A, 4C-4I). GFP was not detected in 29T3-hASGR1 cells after incubation with scAAV2-N587Myc-CMV-eGFP alone (Figure 4 B), nor in 293T cells, which do not express ASGR1, after sequential incubation with anti-myc-hASGR1 antibody and scAAV2-N587Myc- viral vectors. CMV-eGFP (Figure 4J) or in 29T3-hASGR1 cells after sequential incubation with an irrelevant myc-GCGR bispecific antibody and scAAV2-N587Myc-CMV-eGFP viral vectors (Figure 4K).

[00208] As described herein, the tropism of self-complementary AAV (scAAV) can be (1) inactivated, for example, by modifying the capsid protein, such as by insertion of a c-myc epitope, and optionally (2) redirected using bispecific antibodies, such as using a bispecific antibody that recognizes the c-myc epitope and the ligand expressed by the target cell. To determine whether single-chain AAV (ssAAV) tropism could similarly be (1) reduced or eliminated and optionally (2) redirected, 293T-hASGR1 cells were incubated with ssAAV2-N587Myc-CMV-hrGFP viral vectors prepared as described in Example 1, in the absence or presence of a bispecific antibody to myc-hASGR1 at various 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 vectors and an irrelevant myc-GCGR bispecific antibody at a viral vector:antibody ratio of 1:8, and cells 293T (which do not express hASGR1) incubated with viral vectors ssAAV2-N587Myc-CMV-hrGFP and a bispecific antibody to myc-hASGR1 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 and mixtures of ssAAV2-N587Myc-CMV-hrGFP viral vectors and myc-ASGR1 bispecific antibodies at all ratios (Figures 5A, 5C-5I). GFP expression was not detected on 29T3 cells incubated with the ssAAV2-N587Myc-CMV-hrGFP viral vectors and bisppeic anti-myc-ASGR1 antibodies (Figure 5J) or on 293T-hASGR1 cells incubated with the ssAAV2-N587Myc-CMV-hrGFP viral vectors only. or with ssAAV-N587Myc viral vectors and an irrelevant myc-GCGR bispecific antibody (Figures 5B, 5K).

[00209] Human (h)glucagon receptor (GCGR) is not normally expressed by 293T cells. However, 293T-hGCGR is a stable 293T cell line genetically modified to express hGCGR on the cell surface. To test whether retargeting of scAAV2-N587Myc-CMV-eGFP vectors could be mediated by anti-myc-GCGR bispecific antibody via the hGCGR receptor, 293T-hGCGR cells were incubated with different mixtures containing different ratios of scAAV2-N587Myc-CMV-eGFP:anti-myc-bispecific antibody. GCGR. Viral vectors scAAV2-N587Myc-CMV-eGFP were mixed with a bispecific antibody to myc-GCGR at ratios of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1 :50 or 1:100 at room temperature for half an hour before adding 293T-hGCGR cells. Three days postinfection, GFP expression by infected 293T-hCGCR cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 6). A significant percentage of GFP-positive cells were detected after incubation with wild-type scAAV (Figure 6A) or with scAAV2-N587Myc-CMV-eGFP/myc-GCGR bispecific antibody at a ratio of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:15, 1:20, 1:50 and 1:100 (figures 6C-6K). GFP was not detected in 293T-hGCGR cells incubated with scAAV2-N587myc-CMV-eGFP viral vectors alone or scAAV2-N587Myc-CMV-eGFP viral vectors with thuja monospecific antibody (Figures 6B and 6L, respectively).

[00210] Jurkat is a human acute T-cell leukemia cell line that expresses human (h)CD3. To test whether retargeting of AAV6-Q585Myc-EF1a-eGFP viral vectors could be mediated by anti-myc-CD3 bispecific antibody via the hCD3 receptor, Jurkat cells were incubated with different mixtures containing different ratios of AAV6-Q585Myc-EP1a-eGFP:anti-myc-CD3 bispecific antibody . Viral vectors AAV6-Q585Myc-EF1a-eGFP were mixed with bispecific antibody to myc-CD3 at ratios of 1:1, 1:5, 1:10, 1:100, 1:1000 at room temperature for half an hour before adding Jurkat cells. Three days postinfection, GFP expression by infected Jurkat cells was confirmed by fluorescence-activated cell sorting (FACS) (Figure 7). A significant percentage of GFP-positive cells was detected after incubation with wild-type AAV6-EF1a-eGFP (Figure 7B) or with AAV6-Q585Myc-EF1a-eGFP/anti-myc-CD3 bispecific antibody at a ratio of 1:1, 1:5, 1: 10 and 1:100 (figures 7D-7G). GFP was not detected on Jurkat cells incubated with AAV6-Q585Myc-EF1a-eGFP viral vectors (Figure 7C).

[00211] This example describes the inactivation of the natural tropism of several serotypes of self-complementary (sc) or single-chain (ss) AAVs, demonstrated by the failure 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 the c-myc epitope inserted between Q585 and S586, and AAV9 with infect cells normally infected with wild-type AAV (Figures 1A-1B, 2A-2B, 3A-3B, 4A-4B , 5A-5B, 6A-6B and 7). This example further demonstrates the ability of a bispecific antibody that recognizes a c-myc epitope and a second ligand expressed by a target cell (e.g., hASGR1, hGCGR, or hCD3) to alter the target tropism of a modified AAV and mediate infection of a target cell with a pseudotyped AAV-specific ligand manner (Figures 1-7). In addition, this example shows that the 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 genetically modified sc- or ss-AAV in vitro (Figure 4).

Example 3: Antibody-Mediated Redirection of Modified Viral Vectors in Vivo

Liver retargeting of viral vectors using different multispecific binding molecule formats

[00212] To determine whether a myc-ASGR1 bispecific antibody could alter the targeting of the scAAV2-N587myc-CMV-eGFP viral vectors to hASGR1-expressing liver cells in vivo, mice were genetically modified to have their liver cells express hASGR1 in a C57BL background. 6, and control wild-type C57BL/6 mice were injected with 1 x 10 ¹¹ (qPCR titrated) wild-type scAAV2-CMV-eGFP alone or scAAV2-N587myc-CMV-eGFP viral vectors in combination with a bispecific antibody to myc-ASGR1 in the ratio 1:8 ratio of viral genome and antibody molecules intravascularly. Controls included mice injected with saline [250 mM NaCl] or the scAAV2-N587myc-CMV-eGFP viral vector alone. Ten days after injection, mice were sacrificed and transcardially perfused with 4% PFA. Organs such as liver, kidney and heart were collected and dehydrated in 15% sucrose and then in 30% sucrose. The organs were then frozen sectioned on glass slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and Alexa-488-conjugated secondary anti-chicken antibody (Jackson ImmunoResearch Labs, Inc. , West Grove, Pennsylvania). (Figures 8A-8C). GFP-positive cells were detected in the livers of 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 (Figures 8A(i) and 8A(iv)), and in the livers of wild-type C57BL/6 mice injected with wild-type scAAV2-CMV-eGFP (Figure 8A(v)). GFP was not detected in spleen or kidney samples (Figures 8B and 8C), nor in liver, spleen or kidney samples from either saline-injected animal or the scAAV2-N587myc-CMV-eGFP viral vectors alone (Figures 8A(ii, iii, vi, vii)), nor in liver samples collected from wild-type C57BL/6 animals injected with scAAV2-N587myc-CMV-eGFP in combination with a bispecific antibody to myc-ASGR1 (Figure 8A(viii)). Thus, the combination of the scAAV2-N587myc-CMV-eGFP viral vectors and a bispecific antibody to myc-ASGR1 infected only those (liver) cells that express hASGR1, strongly suggesting that the scAAV2-CMV-eGFP viral vector is inactivated by modification of the capsid protein. for example, the natural tropism of a scAAV viral vector can be reduced to the point of elimination, for example by using a c-myc epitope, and such viral vectors can be specifically reactivated, for example specifically retargeted, for example in liver cells in vivo, for example with bispecific antibodies to myc-ASGR1.

[00213] Similarly, to determine whether bispecific antibodies to myc-ASGR1 can alter the targeting of hASGR1-expressing liver cells in vivo by the ssAAV2-N587myc-CAGG-eGFP viral vectors, mice genetically modified to have their liver cells express hASGR1 on a C57BL/6 background, and wild-type C57BL/6 control mice were injected with 2.18 x 10 ¹¹ (qPCR titrated) wild-type ssAAV2-CAGG-eGFP alone or ssAAV2-N587myc-CAGG-eGFP viral vectors in combination with a bispecific antibody to myc-ASGR1 in a 1:4 ratio of the viral genome and antibody molecules intravascularly. Controls included mice injected with PBS or the ssAAV2-N587myc-CAGG-eGFP viral vector alone. Four weeks after injection, mice were sacrificed and transcardially perfused with 4% PFA. Organs such as liver, kidney and heart were collected and dehydrated in 15% sucrose and then in 30% sucrose. The organs were then frozen sectioned on glass slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and Alexa-488-conjugated secondary anti-chicken antibody (Jackson ImmunoResearch Labs, Inc. , West Grove, Pennsylvania). GFP-positive cells were detected in the livers of 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 (Figures 9E-9F, 9P -9R), and in the livers of wild-type C57BL/6 mice injected with wild-type ssAAV2-CAGG-eGFP (Figure 9B-9C). Surprisingly, the infection efficiency of ssAAV2-N587myc-CAGG-eGFP in combination with the myc-ASGR1 bispecific antibody is much higher than that of WT ssAAV2-CAGG-GFP (Figures 9E-9F, 9P-9R). GFP was undetectable or virtually undetectable in liver samples from either saline-injected animal or the ssAAV2-N587myc-CAGG-eGFP viral vectors alone (Figures 9A, 9D, 9G-9L) or in liver samples collected from C57BL animals /6 wild type injected with ssAAV2-N587myc-CAGG-eGFP in combination with a bispecific antibody to myc-ASGR1 (Figure 9M-9O). Thus, the combination of the ssAAV2-N587myc-CAGG-eGFP viral vectors and a bispecific antibody to myc-ASGR1 infected only those (liver) cells that express hASGR1, strongly suggesting that the ssAAV2-N587myc-CAGG-eGFP viral vector is inactivated by modification of the capsid protein, for example, the natural tropism of a scAAV viral vector can be reduced to the point of elimination, for example by using a c-myc epitope, and such viral vectors can be specifically reactivated, for example specifically retargeted, for example in liver cells in vivo, for example using bispecific antibodies to myc-ASGR1.

[00214] Further experiments with the pseudotyped AAV9 viral vector were performed in mice genetically modified to have their liver cells express hASGR1 in a C57BL/6 background. These mice were injected with 1 × 10 ¹¹ (qPCR titrated) wild-type AAV9-CAGG-eGFP or AAV9-A589myc-CAGG-eGFP viral particles in combination with a bispecific antibody to myc-ASGR1 at a ratio of 1:100 viral genome to antibody molecules intravascular. Controls included mice injected with PBS or AAV9-N587myc-CAGG-eGFP viral particles in combination with an irrelevant myc-hCD3 bispecific antibody. Four weeks after injection, mice were sacrificed. The livers were fixed with 10% formaldehyde and sent to HistoWiz. Inc (New York, NY) to stain for GFP. GFP-positive cells were detected in the livers of animals injected with wild-type AAV9-CAGG-eGFP or AAV9-N587myc-CAGG-eGFP viral particles in combination with a bispecific antibody to myc-ASGR1 (Figures 10A and 10D). GFP was negligible or undetectable in liver samples from animals treated with saline or AAV9-N587myc-CAGG-eGFP virus particles in combination with anti-myc-hCD3 bispecific antibody, respectively (Figures 10B and 10C). Thus, similar to AAV2, the natural tropism of AAV9 can be reduced to the point of elimination by modifying the capsid protein, for example by insertion of a c-myc epitope, and such a modified viral vector, for example AAV9-A589myc, can be targeted to specific cell types using appropriate a bispecific binding protein to an epitope and a cell-specific marker, for example a bispecific antibody to myc-ASGR1 for liver cells.

Fc-firmate “protrusion-to-recess” based on IgG4 to hASGR1 with the addition of scFc to myc

[00215] To determine whether different formats of multispecific binding molecules could mediate AAV infection, an anti-myc scFv was fused to the C-terminus of a single chain of an anti-hASGR1 IgG4-based antibody in a knob-to-valley format (see Figure 11A). In particular, gblock encoding scFv to myc (SEQ ID NO: 28), flanked by homologous groups to 1459-1478 and 1479-1498 of pRG7078, encoding the heavy chain of the IgG4 stealth overhang to hASGR1, was provided by Integrated DNA technologies (Skokie, Illinois). The nucleotide (NA) and amino acid (AA) coding sequences of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 scFv (SEQ ID NO: 37) encoded by SEQ ID NO: 28 are presented in Table 3.

[00216] Gblock was cloned into pRG7078 encoding the hASGR1 IgG4 stealth overhang heavy chain to produce pRG7078 encoding an anti-hASGR1 IgG4 stealth overhang polypeptide fusion polypeptide and scFv anti-myc "hASGR1-IgG4-Fc/k bispecific antibody myc". Plasmid pRG7078 modified with c-myc, unmodified pRG7078 with an anti-hASGR1 IgG4 stealth hole, and a plasmid containing the antibody light chain were transfected into 293T cells cultured with OPTI-MEM (cat. no. 31985070, Thermo Fisher). Four days after transfection, the medium was collected and the hASGR1-IgG4-Fc/anti-myc bispecific binding molecule was purified using a protein A column (cat. no. 89948, Thermo Fisher).

[00217] To create a viral vector based on the AAV8 pseudotyped pAAV RC8 N590myc, gblock with the c-myc epitope (SEQ ID NO: 22) inserted between N590 and T591 of the AAV8 VP1 capsid protein was ordered from Integrated DNA technologies (Skokie, IL) . Plasmid pAAV RC8 (SEQ ID NO: 23) was digested with Mlu1 and Sbf1 enzymes and gblock was cloned into a vector using SLIC to produce pAAV RC8 N590myc (SEQ ID NO: 24).

[00218] 293T-hASGR1 cells, which are 293T cells genetically modified to express human (h)ASGR1 on the cell surface, were incubated with either mixtures containing various ratios of AAV8-N590Myc-CAGG-eGFP with hASGR1-IgG4 bispecific binding molecule molecules -Fc/k myc (1:0, 1:1, 1:2, 1:4, 1:8, 1:12, 1:15, 1:50 or 1:100), or with AAV8-CAGG viral genomes -eGFP. Three days postinfection, GFP expression was analyzed by FACS. GFP-positive 293T-hASGR1 cells were detected after incubation with mixtures of AAV8-N590Myc-CAGG-eGFP and bispecific molecules to hASGR1-IgG4-Fc/to myc at ratios of 1:1, 1:2, 1:4, 1:8, 1 :12, 1:15, 1:50, or 1:100 (2.12%-22.2%; Figures 12D-12K) comparable to 293T-hASGR1 cells incubated with wild-type AAV8-CAGG eGFP (46.1% ) (Figure 12A). Neither mock-infected cells (Figure 12B) nor incubation with AAV8-N590Myc-CAGG-eGFP alone resulted in GFP expression by 293T hASGR1 cells (Figure 12C).

[00219] To determine whether hASGR1-IgG4-Fc/k-myc bispecific binding proteins can alter the targeting of AAV8 N590myc-CAGG-eGFP viral particles to hASGR1-expressing liver cells in vivo, mice genetically modified so that their liver cells express hASGR1 in a C57BL/6 background, were injected intravascularly with 1 × 10 ¹¹ (qPCR titrated) wild-type AAV8-CAGG-eGFP or AAV8 N590myc-CAGG-eGFP viral particles in combination with bispecific binding molecules to hASGR1-IgG4-Fc/k myc in a 1:12 ratio of viral genome and binding protein molecules. Control animals were injected with AAV8 N590myc-CAGG-eGFP viral particles in combination with an irrelevant hCD3 x myc bispecific binding molecule having a similar format to the hASGR1-IgG4-Fc/myc bispecific binding molecule. Three weeks after injection, mice were sacrificed and transcardially perfused with 4% PFA. Organs such as liver, kidney and heart were collected and dehydrated in 15% sucrose and then in 30% sucrose. The organs were then frozen sectioned on glass slides and stained with chicken anti-EGFP antibody (Jackson ImmunoResearch Labs, Inc., West Grove, PA) and Alexa-488-conjugated secondary anti-chicken antibody (Jackson ImmunoResearch Labs, Inc. , West Grove, Pennsylvania). GFP-positive cells were detected in the livers of animals injected with wild-type AAV8-CAGG-eGFP viral particles (Figures 13A-13C) or AAV8 N590myc-CAGG-eGFP in combination with bispecific anti-hASGR1-IgG4-Fc/anti-myc binding molecules (Figures 13G-13I). Little or no GFP was detected in liver samples from animals injected with AAV8 N590myc-CAGG-eGFP viral particles in combination with an irrelevant hCD3/myc binding protein (Figures 13D-13F). Thus, epitope-tagged AAV8, such as AAV8-N590myc, can be targeted to a specific cell type using a bispecific binding molecule that specifically binds the epitope and the cell-specific marker expressed by the target cell type.

Retargeting of viral vectors towards the intestines and/or pancreas

[00220] 293T cells genetically modified to express human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3) on the cell surface (“293T-ENTPD3”) were incubated with mixtures containing varying ratios of AAV2-N587Myc-CAGG-eGFP or AAV2-CAGG- viral genomes GFP and a bispecific binding molecule formed by fusion of anti-myc scFv with the C-terminus of one chain of IgG4-based anti-hENTPD3 antibody in a knob-to-valve format (anti-hENTPD3-IgG4-Fc/anti-myc), in various ratios (1: 0, 1:1, 1:2, 1:4, 1:8, 1:20, 1:50, 1:100 or 1:200). Three days postinfection, GFP expression was analyzed by FACS. GFP-positive 293T-ENTPD3 cells were detected after incubation with mixtures of AAV2-N587Myc-CAGG-eGFP and anti-hENTPD3-IgG4-Fc/anti-myc molecules at levels (1.48%-16%; Figures 14C-14J) comparable to cells 293T-ENTPD3 incubated with wild-type AAV2-CAGG-eGFP (66%) (Figure 14A). Incubation with AAV2-N587Myc-CAGG-eGFP alone resulted in very low levels of GFP expression by 293T-ENTPD3 cells (Figure 14B).

[00221] ENTPD3 has been determined to be expressed in the intestinal mucosa (data not shown). To determine whether the hENTPD3-IgG4-Fc/k-myc binding protein could alter the targeting of AAV2-N587myc-CAGG-eGFP viral particles to ENTPD3-expressing intestinal cells in vivo, C57BL/6 mice were injected intravascularly with 5 x 10 ¹¹ (titrated in using qPCR) of AAV2-N587myc-CAGG-eGFP viral particles in combination with binding proteins to hENTPD3-IgG4-Fc/myc in a ratio of 1:20 of the viral genome and binding protein molecules. Control mice were treated with PBS, wild-type AAV9, or AAV2-N587myc-CAGG-eGFP viral particles in combination with an irrelevant binding protein molecule. Three weeks after injection, mice were sacrificed. The liver, intestines, and pancreas were fixed with 10% formaldehyde and sent to HistoWiz. Inc (New York, NY) to stain for GFP. GFP-positive cells were detected in mice injected with wild-type AAV9 (Figure 15B(ii)), but not in the livers of mice injected with PBS or AAV2-N587myc-CAGG-eGFP viral particles in combination with irrelevant binding molecules or hENTPD3-binding proteins. IgG4-Fc/k myc (Figure 15A(iii)-15A(iv), respectively), indicating that AAV2-N587myc viral vectors do not infect ENTPD3-negative liver cells even when co-administered with hENTPD3-IgG4-Fc binding proteins /to myc. GFP was detected in the intestine only in mice injected with wild-type AAV9 or AAV2-N587myc-CAGG-eGFP viral particles in combination with hENTPD3-IgG4-Fc/k myc binding proteins (Figures 15B(i)-15B(iv)). GFP was detected in the pancreatic islets of mice injected with AAV2-N587myc-CAGG-eGFP viral particles in combination with hENTPD3-IgG4-Fc/anti-myc binding proteins. (Figure 15C(iv)). WT AAV9 infected both islet and non-islet pancreatic cells (Figure 15C(ii)). GFP was not detected in pancreas samples from saline-injected mice or AAV2-N587myc-CAGG-eGFP viral particles in combination with irrelevant binding proteins. (Figures 7C(i) and 7C(iii)). Thus, the natural tropism of AAV can be reduced to the point of elimination by introducing a heterologous epitope and retargeting the same or different cells using a multispecific binding protein that binds the epitope tag and the marker expressed by the retargeted cells or tissues.

[00222] This example demonstrates that a vector based on several different adeno-associated virus serotypes can be genetically modified with a heterologous epitope (e.g., c-myc) as described herein to inactivate infectivity, and a bispecific antibody can be used (regardless of bispecific formats ), which simultaneously recognizes a heterologous epitope (eg, c-myc) and a marker expressed by the target cell to retarget the viral vector to deliver the nucleotide of interest.

Example 4. Use of genetically modified viral vectors AAV-N587Myc for delivery of therapeutic cargo to specific cells

[00223] This example demonstrates the ability of AAV-N587Myc viral vectors to specifically deliver a therapeutic payload, such as one or more suicide genes, a biological therapeutic (e.g., an antibody), a CRISPR/Cas gene editing system, shRNA, etc., in target cell type. Specifically, this example describes the delivery of a suicide gene, antibody coding sequence, or CRISPR/Cas gene editing system to cells expressing the target ligand.

[00224] Delivery of a suicide gene to cells expressing the target ligand

[00225] To test the ability of scAAV2-N587Myc viral vectors to deliver a suicide gene to a specific cell, a HER2 ⁺ breast cancer xenograft model in nude mice was used as described in Wang et al. ((2010) Cancer Gene Therapy 17:559-570).

[00226] Viral vectors. Viral vectors scAAV2-N587Myc or ssAAV2-N587Myc carrying the EGFP reporter gene (scAAV2-N587Myc-EGFP or ssAAV2-N587MycEGFP) were prepared as described in example 1. Viral vectors scAAV2-N587Myc or ssAAV2-N587Myc carrying the suicide gene (SG) , were obtained in a similar manner. Briefly, 293T17 cells were transfected with (1) pAd Helper and (2) pAAV RC2 (encoding a wild-type capsid) or pAAV RC2-N587Myc (encoding a c-myc epitope-modified capsid) as described above, and with (3) a pAAV vector carrying a suicide gene, for example a cytosine deaminase gene, a herpes simplex virus thymidine kinase gene, under the control of a promoter, for example CMV. Viral vectors ssAAV-N587MycSG or scAAV-N587SG were isolated and titrated as described in example 1.

[00227] Cell lines. Breast cancer cell lines BT474, breast cancer SK-BR-3, and lung cancer Calu-3 are HER2-positive human tumor cell lines (Bunn P. A. 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). Rhabdomyosarcoma A-673 and HeLa cervical cancer are HER2-negative human tumor cell lines, and BEAS-2b is an immortalized HER2-negative bronchial epithelial 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 ATCC-recommended media.

[00228] Mice. Female athymic mice aged 6 to 8 weeks were obtained and kept in special pathogen-free conditions. On day 0, mice were simultaneously (1) injected subcutaneously into the right flank with 10 ⁷ BT474, SK-BR-3, Calu-3, A-673, or HeLa tumor cells and (2) intravenously injected with a bispecific antibody to myc-HER2 and ss viral vectors - or scAAV-N587Myc carrying a reporter gene (for example, EGFP) or a suicide gene. Controls included animals without treatment (animals injected with tumor cells only), animals injected with viral vectors based on wild-type ss- or scAAV carrying a reporter gene or a suicide gene, animals injected with only a bispecific antibody to myc-HER2, or animals injected with viral vectors. ss- or scAAV-N587Myc vectors carrying only a reporter gene or a suicide gene. All animals were treated with the appropriate prodrug 1 day after injection and treatment. The size of each tumor was measured using a caliper 2 times a week, and the tumor volume was calculated as length × width ² × 0.52. When the tumor was painful, ulcerated, tumor diameter was 15 mm, or tumor volume was 1000 mm ³ , mice were sacrificed and the date of sacrifice was recorded as the date of death. The liver, spleen, kidneys and tumors of animals injected with viral vectors based on wild-type ss- or scAAV or ss- or scAAV-N587Myc viruses carrying a reporter gene are recorded and reporter gene expression is visualized.

[00229] 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 delivery of a suicide gene to a cell expressing the target HER2 ligand, using viral vectors described herein containing a heterologous epitope, such as c-myc, and a bispecific antibody that specifically binds the target ligand and the heterologous epitope. In additional experiments, the suicide gene is delivered into another cell type expressing one or more other target ligands using a viral vector containing a heterologous epitope as described herein and a bispecific antibody that specifically binds the heterologous epitope (eg, c-myc) and target receptor. Illustrative and non-limiting examples of receptors suitable for targeting include those receptors that mediate viral vector endocytosis, such as oncoembryonic antigen (CEA) (Qiu Y, et al. (2012) Cancer Lett. 316:31-38) and growth factor receptor vascular endothelium (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 may 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), mucins (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).

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LIST OF SEQUENCES

<110> Regeneron Pharmaceuticals, Inc.

Kuratsos, Christos

Murphy, Andrew J

Wang, Cheng

Sabin, Leah

<120> Recombinant viral vectors with modified tropism and pathways

their application for targeted introduction of genetic material into human cells

<130> 009108.335WO1/10335WO01

<150> 62/525,704

<151> 2017-06-27

<160> 40

<170>PatentIn version 3.5

<210> 1

<211> 735

<212> PROTEIN

<213> adeno-associated virus 2

<400> 1

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 2

<211> 745

<212> PROTEIN

<213> Artificial sequence

<220>

<223>AAV2 VP1 N587Myc

<400> 2

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Glu Gln Lys Leu Ile

580 585 590

Ser Glu Glu Asp Leu Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 3

<211> 736

<212> PROTEIN

<213> adeno-associated virus 6

<400> 3

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 4

<211> 746

<212> PROTEIN

<213> Artificial sequence

<220>

<223> AAV6 VP1 Q585Myc

<400> 4

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Glu Gln Lys Leu Ile Ser Glu

580 585 590

Glu Asp Leu Ser Ser Ser Thr Asp Pro Ala Thr Gly Asp Val His Val

595 600 605

Met Gly Ala Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu

610 615 620

Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His

625 630 635 640

Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Pro Gln

645 650 655

Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Pro Ala Glu Phe

660 665 670

Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln

675 680 685

Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg

690 695 700

Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr Ala Lys Ser Ala Asn

705 710 715 720

Val Asp Phe Thr Val Asp Asn Asn Gly Leu Tyr Thr Glu Pro Arg Pro

725 730 735

Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

740 745

<210> 5

<211> 736

<212> PROTEIN

<213> adeno-associated virus 9

<400> 5

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 6

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> c-myc

<400> 6

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 7

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FLAG

<400> 7

Asp Tyr Lys Asp Asp Asp Asp Lys

15

<210> 8

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223>HA

<400> 8

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

15

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> ITR forward primer

<400> 9

ggaaccccta gtgatggagt t 21

<210> 10

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<223> ITR reverse primer

<400> 10

cggcctcagt gagcga 16

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> AAV2 ITR probe

<400> 11

cactccctct ctgcgcgctc g 21

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> c-myc

<400> 12

gaacaaaaac tcatctcaga agaggatctg 30

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> pAAVRCBsiWF

<400> 13

ggagtaccag ctcccgtacg 20

<210> 14

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> N587mycR

<400> 14

ctcttctgag atgagttttt gttcgttgcc tctctggagg ttg 43

<210> 15

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> N587mycF

<400> 15

aaactcatct cagaagagga tctgagacaa gcagctaccg cag 43

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> pAAVRCPmeR

<400> 16

tccgcccgct gtttaaac 18

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> pAAVRC9BsiWF

<400> 17

actcagacta tcagctcccg tacg 24

<210> 18

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<223>A589mycR

<400> 18

ctcttctgag atgagttttt gttctgcttg ggcactctgg t 41

<210> 19

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<223> A589mycF

<400> 19

aaactcatct cagaggat ctgcaggcgc agaccgg 37

<210> 20

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> pAAVRC9PmeR

<400> 20

ctccgcccgc tgtttaaac 19

<210> 21

<211> 738

<212> PROTEIN

<213> adeno-associated virus 8

<400> 21

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly

450 455 460

Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile

530 535 540

Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala

580 585 590

Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 22

<211> 453

<212> DNA

<213> Artificial sequence

<220>

<223> AAV8 N590myc gblock

<400> 22

gaccctgtta ccgccaacaa cgcgtctcaa cgacaaccgg gcaaaacaac aatagcaact 60

ttgcctggac tgctgggacc aaataccatc tgaatggaag aaattcattg gctaatcctg 120

gcatcgctat ggcaacacac aaagacgacg aggagcgttt ttttcccagt aacgggatcc 180

tgatttttgg caaacaaaat gctgccagag acaatgcgga ttacagcgat gtcatgctca 240

ccagcgagga agaaatcaaa accactaacc ctgtggctac agaggaatac ggtatcgtgg 300

cagataactt gcagcagcaa aacgaacaaa aactcatctc agaagaggat ctgacggctc 360

ctcaaattgg aactgtcaac agccagggg ccttacccgg tatggtctgg cagaaccggg 420

acgtgtacct gcagggtccc atctgggcca aga 453

<210> 23

<211> 7336

<212> DNA

<213> Artificial sequence

<220>

<223> pAAV RC2/8

<400> 23

gcgcgccgat atcgttaacg ccccgcgccg gccgctctag aactagtgga tcccccggaa 60

gatcagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttctgatctg 120

cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga 180

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 240

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 300

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct 360

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac 420

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat 480

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac 540

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt 600

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag 660

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 720

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 780

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 840

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 900

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 960

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg 1020

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 1080

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1140

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1200

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1260

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1320

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat 1380

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1440

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1500

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1560

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1620

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac 1680

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1740

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 1800

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 1860

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 1920

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 1980

catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt 2040

ggctcgagga caacctctct gagggcattc gcgagtggtg ggcgctgaaa cctggagccc 2100

cgaagcccaa agccaaccag caaaagcagg acgacggccg gggtctggtg cttcctggct 2160

acaagtacct cggacccttc aacggactcg acaaggggga gcccgtcaac gcggcggacg 2220

cagcggccct cgagcacgac aaggcctacg accagcagct gcaggcgggt gacaatccgt 2280

acctgcggta taaccacgcc gacgccgagt ttcaggagcg tctgcaagaa gatacgtctt 2340

ttggggggcaa cctcgggcga gcagtcttcc aggccaagaa gcgggttctc gaacctctcg 2400

gtctggttga ggaaggcgct aagacggctc ctggaaagaa gagaccggta gagccatcac 2460

cccagcgttc tccagactcc tctacgggca tcggcaagaa aggccaacag cccgccagaa 2520

aaagactcaa ttttggtcag actggcgact cagagtcagt tccagaccct caacctctcg 2580

gagaacctcc agcagcgccc tctggtgtgg gacctaatac aatggctgca ggcggtggcg 2640

caccaatggc agacaataac gaaggcgccg acggagtggg tagttcctcg ggaaattggc 2700

attgcgattc cacatggctg ggcgacagag tcatcaccac cagcacccga acctgggccc 2760

tgcccaccta caacaaccac ctctacaagc aaatctccaa cgggacatcg ggaggagcca 2820

ccaacgacaa cacctacttc ggctacagca ccccctgggg gtattttgac tttaacagat 2880

tccactgcca cttttcacca cgtgactggc agcgactcat caacaacaac tggggattcc 2940

ggcccaagag actcagcttc aagctcttca acatccaggt caaggaggtc acgcagaatg 3000

aaggcaccaa gaccatcgcc aataacctca ccagcaccat ccaggtgttt acggactcgg 3060

agtaccagct gccgtacgtt ctcggctctg cccaccaggg ctgcctgcct ccgttcccgg 3120

cggacgtgtt catgattccc cagtacggct acctaacact caacaacggt agtcaggccg 3180

tgggacgctc ctccttctac tgcctggaat actttccttc gcagatgctg agaaccggca 3240

acaacttcca gtttacttac accttcgagg acgtgccttt ccacagcagc tacgcccaca 3300

gccagagctt ggaccggctg atgaatcctc tgattgacca gtacctgtac tacttgtctc 3360

ggactcaaac aacaggaggc acggcaaata cgcagactct gggcttcagc caaggtgggc 3420

ctaatacaat ggccaatcag gcaaagaact ggctgccagg accctgttac cgccaacaac 3480

gcgtctcaac gacaaccggg caaaacaaca atagcaactt tgcctggact gctgggacca 3540

aataccatct gaatggaaga aattcattgg ctaatcctgg catcgctatg gcaacacaca 3600

aagacgacga ggagcgtttt tttcccagta acgggatcct gatttttggc aaacaaaatg 3660

ctgccagaga caatgcggat tacagcgatg tcatgctcac cagcgaggaa gaaatcaaaa 3720

ccactaaccc tgtggctaca gaggaatacg gtatcgtggc agataacttg cagcagcaaa 3780

acacggctcc tcaaattgga actngtcaaca gccagggggc cttacccggt atggtctggc 3840

agaaccggga cgtgtacctg cagggtccca tctgggccaa gattcctcac acggacggca 3900

acttccaccc gtctccgctg atgggcggct ttggcctgaa acatcctccg cctcagatcc 3960

tgatcaagaa cacgcctgta cctgcggatc ctccgaccac cttcaaccag tcaaagctga 4020

actctttcat cacgcaatac agcaccggac aggtcagcgt ggaaattgaa tgggagctgc 4080

agaaggaaaa cagcaagcgc tggaaccccg agatccagta cacctccaac tactacaaat 4140

ctacaagtgt ggactttgct gttaatacag aaggcgtgta ctctgaaccc cgccccattg 4200

gcacccgtta cctcacccgt aatctgtaat tgcctgttaa tcaataaacc gtttaattcg 4260

tttcagttga actttggtct ctgcgtattt ctttcttatc tagtttccat ggctacgtag 4320

ataagtagca tggcgggtta atcattaact acagcccggg cgtttaaaca gcgggcggag 4380

gggtggagtc gtgacgtgaa ttacgtcata gggttaggga ggtcctgtat tagaggtcac 4440

gtgagtgttt tgcgacattt tgcgacacca tgtggtctcg ctgggggggg gggcccgagt 4500

gagcacgcag ggtctccatt ttgaagcggg aggtttgaac gagcgctggc gcgctcactg 4560

gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt 4620

gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 4680

tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa 4740

aattcgcgtt aaatttttgt taaatcagct cattttttta accaataggc cgaaatcggc 4800

aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg 4860

aacaagagtc cactattaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 4920

agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 4980

gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 5040

cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 5100

caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 5160

agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 5220

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 5280

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 5340

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 5400

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 5460

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 5520

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 5580

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 5640

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 5700

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 5760

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 5820

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 5880

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 5940

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 6000

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 6060

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 6120

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 6180

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 6240

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 6300

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 6360

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 6420

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 6480

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 6540

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 6600

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcggggctga acggggggtt 6660

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac cctacagcgtg 6720

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 6780

gcagggtcgg aacaggag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 6840

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 6900

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 6960

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 7020

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 7080

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 7140

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 7200

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 7260

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 7320

accatgatta cgccaa 7336

<210> 24

<211> 7366

<212> DNA

<213> Artificial sequence

<220>

<223> Plasmid paav RC2/8 myc

<400> 24

gcgcgccgat atcgttaacg ccccgcgccg gccgctctag aactagtgga tcccccggaa 60

gatcagaagt tcctattccg aagttcctat tctctagaaa gtataggaac ttctgatctg 120

cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga 180

gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 240

gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 300

gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggccct 360

tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac 420

caccggggtg aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat 480

tcagagaatt taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac 540

cagaaatggc gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt 600

gctccccaaa acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag 660

cgcctgtttg aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc 720

gcagacgcag gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag 780

atcaaaaact tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac 840

ctcggagaag cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc 900

caactcgcgg tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac 960

taaaaccgcc cccgactacc tggtgggcca gcagcccgtg gaggacatt ccagcaatcg 1020

gatttataaa attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct 1080

gggatgggcc acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac 1140

taccgggaag accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt 1200

aaactggacc aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg 1260

ggaggagggg aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag 1320

caaggtgcgc gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat 1380

cgtcacctcc aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca 1440

ccagcagccg ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga 1500

ctttgggaag gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt 1560

ggttgaggtg gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc 1620

cagtgacgca gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac 1680

gtcagacgcg gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca 1740

cgtgggcatg aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc 1800

aaatatctgc ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc 1860

tcaacccgtt tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat 1920

gggaaaggtg cgacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg 1980

catctttgaa caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt 2040

ggctcgagga caacctctct gagggcattc gcgagtggtg ggcgctgaaa cctggagccc 2100

cgaagcccaa agccaaccag caaaagcagg acgacggccg gggtctggtg cttcctggct 2160

acaagtacct cggacccttc aacggactcg acaaggggga gcccgtcaac gcggcggacg 2220

cagcggccct cgagcacgac aaggcctacg accagcagct gcaggcgggt gacaatccgt 2280

acctgcggta taaccacgcc gacgccgagt ttcaggagcg tctgcaagaa gatacgtctt 2340

ttggggggcaa cctcgggcga gcagtcttcc aggccaagaa gcgggttctc gaacctctcg 2400

gtctggttga ggaaggcgct aagacggctc ctggaaagaa gagaccggta gagccatcac 2460

cccagcgttc tccagactcc tctacgggca tcggcaagaa aggccaacag cccgccagaa 2520

aaagactcaa ttttggtcag actggcgact cagagtcagt tccagaccct caacctctcg 2580

gagaacctcc agcagcgccc tctggtgtgg gacctaatac aatggctgca ggcggtggcg 2640

caccaatggc agacaataac gaaggcgccg acggagtggg tagttcctcg ggaaattggc 2700

attgcgattc cacatggctg ggcgacagag tcatcaccac cagcacccga acctgggccc 2760

tgcccaccta caacaaccac ctctacaagc aaatctccaa cgggacatcg ggaggagcca 2820

ccaacgacaa cacctacttc ggctacagca ccccctgggg gtattttgac tttaacagat 2880

tccactgcca cttttcacca cgtgactggc agcgactcat caacaacaac tggggattcc 2940

ggcccaagag actcagcttc aagctcttca acatccaggt caaggaggtc acgcagaatg 3000

aaggcaccaa gaccatcgcc aataacctca ccagcaccat ccaggtgttt acggactcgg 3060

agtaccagct gccgtacgtt ctcggctctg cccaccaggg ctgcctgcct ccgttcccgg 3120

cggacgtgtt catgattccc cagtacggct acctaacact caacaacggt agtcaggccg 3180

tgggacgctc ctccttctac tgcctggaat actttccttc gcagatgctg agaaccggca 3240

acaacttcca gtttacttac accttcgagg acgtgccttt ccacagcagc tacgcccaca 3300

gccagagctt ggaccggctg atgaatcctc tgattgacca gtacctgtac tacttgtctc 3360

ggactcaaac aacaggaggc acggcaaata cgcagactct gggcttcagc caaggtgggc 3420

ctaatacaat ggccaatcag gcaaagaact ggctgccagg accctgttac cgccaacaac 3480

gcgtctcaac gacaaccggg caaaacaaca atagcaactt tgcctggact gctgggacca 3540

aataccatct gaatggaaga aattcattgg ctaatcctgg catcgctatg gcaacacaca 3600

aagacgacga ggagcgtttt tttcccagta acgggatcct gatttttggc aaacaaaatg 3660

ctgccagaga caatgcggat tacagcgatg tcatgctcac cagcgaggaa gaaatcaaaa 3720

ccactaaccc tgtggctaca gaggaatacg gtatcgtggc agataacttg cagcagcaaa 3780

acgaacaaaa actcatctca gaagaggatc tgacggctcc tcaaattgga actgtcaaca 3840

gccaggggggc cttacccggt atggtctggc agaaccggga cgtgtacctg cagggtccca 3900

tctgggccaa gattcctcac acggacggca acttccaccc gtctccgctg atgggcggct 3960

ttggcctgaa acatcctccg cctcagatcc tgatcaagaa cacgcctgta cctgcggatc 4020

ctccgaccac cttcaaccag tcaaagctga actctttcat cacgcaatac agcaccggac 4080

aggtcagcgt ggaaattgaa tgggagctgc agaaggaaaa cagcaagcgc tggaaccccg 4140

agatccagta cacctccaac tactacaaat ctacaagtgt ggactttgct gttaatacag 4200

aaggcgtgta ctctgaaccc cgccccattg gcacccgtta cctcacccgt aatctgtaat 4260

tgcctgttaa tcaataaacc gtttaattcg tttcagttga actttggtct ctgcgtattt 4320

ctttcttatc tagtttccat ggctacgtag ataagtagca tggcgggtta atcattaact 4380

acagcccggg cgtttaaaca gcgggcggag gggtggagtc gtgacgtgaa ttacgtcata 4440

gggttaggga ggtcctgtat tagaggtcac gtgagtgttt tgcgacattt tgcgacacca 4500

tgtggtctcg ctgggggggg gggcccgagt gagcacgcag ggtctccatt ttgaagcggg 4560

aggtttgaac gagcgctggc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa 4620

aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 4680

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 4740

tggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 4800

cattttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc 4860

gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaag aacgtggact 4920

ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 4980

cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 5040

gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 5100

aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 5160

ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa 5220

atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 5280

tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 5340

aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 5400

acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 5460

acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 5520

ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 5580

ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 5640

caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 5700

ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 5760

aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 5820

aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 5880

tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 5940

aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 6000

cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 6060

ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 6120

gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 6180

agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 6240

atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 6300

cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6360

cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6420

cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6480

tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 6540

tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6600

ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6660

aggcgcagcg gtcggggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6720

cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6780

ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggag cgcacgaggg 6840

agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6900

ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6960

acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 7020

cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 7080

gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa 7140

tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt 7200

ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt 7260

aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg 7320

gataacaatt tcacacagga aacagctatg accatgatta cgccaa 7366

<210> 25

<211> 748

<212> PROTEIN

<213> Artificial sequence

<220>

<223> pAAV8N590myc

<400> 25

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly

450 455 460

Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile

530 535 540

Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Glu Gln

580 585 590

Lys Leu Ile Ser Glu Glu Asp Leu Thr Ala Pro Gln Ile Gly Thr Val

595 600 605

Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp Gln Asn Arg Asp Val

610 615 620

Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn

625 630 635 640

Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro

645 650 655

Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr

660 665 670

Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr

675 680 685

Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser

690 695 700

Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser

705 710 715 720

Thr Ser Val Asp Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro

725 730 735

Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 26

<211> 746

<212> PROTEIN

<213> Artificial sequence

<220>

<223> pAAV9A589myc

<400> 26

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Glu Gln Lys

580 585 590

Leu Ile Ser Glu Glu Asp Leu Gln Ala Gln Thr Gly Trp Val Gln Asn

595 600 605

Gln Gly Ile Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu

610 615 620

Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His

625 630 635 640

Pro Ser Pro Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln

645 650 655

Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe

660 665 670

Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln

675 680 685

Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg

690 695 700

Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn

705 710 715 720

Val Glu Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro

725 730 735

Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 27

<211> 746

<212> PROTEIN

<213> Artificial sequence

<220>

<223> pAAV9G453myc

<400> 27

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Ser

450 455 460

Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser Val Ala Gly Pro Ser Asn

465 470 475 480

Met Ala Val Gln Gly Arg Asn Tyr Ile Pro Gly Pro Ser Tyr Arg Gln

485 490 495

Gln Arg Val Ser Thr Thr Val Thr Gln Asn Asn Asn Ser Glu Phe Ala

500 505 510

Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn Gly Arg Asn Ser Leu Met

515 520 525

Asn Pro Gly Pro Ala Met Ala Ser His Lys Glu Gly Glu Asp Arg Phe

530 535 540

Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly Lys Gln Gly Thr Gly Arg

545 550 555 560

Asp Asn Val Asp Ala Asp Lys Val Met Ile Thr Asn Glu Glu Glu Ile

565 570 575

Lys Thr Thr Asn Pro Val Ala Thr Glu Ser Tyr Gly Gln Val Ala Thr

580 585 590

Asn His Gln Ser Ala Gln Ala Gln Ala Gln Thr Gly Trp Val Gln Asn

595 600 605

Gln Gly Ile Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu

610 615 620

Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly Asn Phe His

625 630 635 640

Pro Ser Pro Leu Met Gly Gly Phe Gly Met Lys His Pro Pro Pro Gln

645 650 655

Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asp Pro Pro Thr Ala Phe

660 665 670

Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln

675 680 685

Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg

690 695 700

Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Tyr Lys Ser Asn Asn

705 710 715 720

Val Glu Phe Ala Val Asn Thr Glu Gly Val Tyr Ser Glu Pro Arg Pro

725 730 735

Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 28

<211> 814

<212> DNA

<213> Artificial sequence

<220>

<223> cmyc-scfv-gblock

<400> 28

tctccctgtc tctgggtaaa ggcggaggtg gatccggagg gggcggatct gacatccaga 60

tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc 120

gggcaagtca gagcattagc agctatttaa attggtatca acagaaacca gggaaagccc 180

ctaagctcct gatctatgct gtttccatt tgcaaagtgg ggtcccatca aggttcagtg 240

gcagtggatc tgggacagat ttcactctca ccatcaactc tctgcaacct gaagattttg 300

caacttactc ctgtcaacag acttacagta cccctccgat caccttcggc caagggacac 360

gactggagat taaaggaggg ggcggctcag gaggaggggg tagtggggga ggcggctccg 420

aggtgcagct ggtggagtct gggggaggct tggtacagcc tggagggtcc ctgagactct 480

cctgtgtcac ctctggattt cgttttagta gttatgaaat gaactgggtc cgccaggctc 540

cagggaaggg gctggaatgg atttcataca ttaacagaag tggttatacc atatactacg 600

cagcctctct gaagggccga ttcaccatct ccagagacaa cgccaagaac tcactgtatc 660

ttcaaatgaa cagcctgaga gacgaggaca cggctatcta ttactgtgcg agagggagta 720

tcgcccctcg gggggagatt cgacacatct ttgactactg gggccaggga accctggtca 780

ccgtctcctc atgatgagcg gccgctaatc agcc 814

<210> 29

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc HCVR AA

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Thr Ser Gly Phe Arg Phe Ser Ser Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Ser Tyr Ile Asn Arg Ser Gly Tyr Thr Ile Tyr Tyr Ala Ala Ser Leu

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Ile Ala Pro Arg Gly Glu Ile Arg His Ile Phe Asp

100 105 110

Tyr Trp Gly Gln Gly Thr Leu Val

115 120

<210> 30

<211> 108

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc LCVR AA

<400> 30

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 31

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc HCDR1

<400> 31

Gly Phe Arg Phe Ser Ser Tyr Glu

15

<210> 32

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc HCDR2 AA

<400> 32

Ile Asn Arg Ser Gly Tyr Thr Ile

15

<210> 33

<211> 2

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc HCDR3

<400> 33

Ala Arg

1

<210> 34

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc LCDR1 AA

<400> 34

Gln Ser Ile Ser Ser Tyr

15

<210> 35

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc LCDR2

<400> 35

Ala Ala Ser

1

<210> 36

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> myc LCDR3 AA

<400> 36

Gln Gln Ser Tyr Ser Thr Pro

15

<210> 37

<211> 247

<212> PROTEIN

<213> Artificial sequence

<220>

<223> anti-myc scFv

<400> 37

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Val Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Ser Cys Gln Gln Thr Tyr Ser Thr Pro Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Gly Gly Gly Gly

100 105 110

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val

115 120 125

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

130 135 140

Cys Val Thr Ser Gly Phe Arg Phe Ser Ser Tyr Glu Met Asn Trp Val

145 150 155 160

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser Tyr Ile Asn Arg

165 170 175

Ser Gly Tyr Thr Ile Tyr Tyr Ala Ala Ser Leu Lys Gly Arg Phe Thr

180 185 190

Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser

195 200 205

Leu Arg Asp Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Gly Ser Ile

210 215 220

Ala Pro Arg Gly Glu Ile Arg His Ile Phe Asp Tyr Trp Gly Gln Gly

225 230 235 240

Thr Leu Val Thr Val Ser Ser

245

<210> 38

<211> 774

<212> DNA

<213> Artificial sequence

<220>

<223> scFv to myc NA

<400> 38

ggcggaggtg gatccggagg gggcggatct gacatccaga tgacccagtc tccatcctcc 60

ctgtctgcat ctgtaggaga cagagtcacc atcacttgcc gggcaagtca gagcattagc 120

agctatttaa attggtatca acagaaacca gggaaagccc ctaagctcct gatctatgct 180

gtttccatt tgcaaagtgg ggtcccatca aggttcagtg gcagtggatc tgggacagat 240

ttcactctca ccatcaactc tctgcaacct gaagattttg caacttactc ctgtcaacag 300

acttacagta cccctccgat caccttcggc caagggacac gactggagat taaaggaggg 360

ggcggctcag gaggaggggg tagtggggga ggcggctccg aggtgcagct ggtggagtct 420

gggggaggct tggtacagcc tggagggtcc ctgagactct cctgtgtcac ctctggattt 480

cgttttagta gttatgaaat gaactgggtc cgccaggctc cagggaaggg gctggaatgg 540

atttcataca ttaacagaag tggttatacc atatactacg cagcctctct gaagggccga 600

ttcaccatct cccagagacaa cgccaagaac tcactgtatc ttcaaatgaa cagcctgaga 660

gacgaggaca cggctatcta ttactgtgcg agagggagta tcgcccctcg gggggagatt 720

cgacacatct ttgactactg gggccaggga accctggtca ccgtctcctc atga 774

<210> 39

<211> 372

<212> DNA

<213> Artificial sequence

<220>

<223> myc HCVR NA

<400> 39

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60

tcctgtgtca cctctggatt tcgttttagt agttatgaaa tgaactgggt ccgccaggct 120

ccagggaagg ggctggaatg gatttcatac attaacagaa gtggttatac catatactac 180

gcagcctctc tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat 240

cttcaaatga acagcctgag agacgaggac acggctatct attactgtgc gagagggagt 300

atcgcccctc ggggggagat tcgacacatc tttgactact ggggccaggg aaccctggtc 360

accgtctcct ca 372

<210> 40

<211> 324

<212> DNA

<213> Artificial sequence

<220>

<223> myc LCVR NA

<400> 40

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccgtca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgat caccttcggc 300

caagggacac gactggagat taaa 324

<---

Claims (64)

1. Composition for targeted introduction of genetic material into a cell, containing (a) a recombinant adeno-associated virus (AAV) vector containing an AAV capsid that encapsulates a nucleotide of interest, wherein the AAV capsid contains a recombinant AAV capsid protein modified to include a heterologous epitope, wherein the recombinant AAV capsid protein is derived from an AAV capsid gene modified to express a heterologous epitope, and (b) a multispecific binding molecule comprising an antibody paratope that specifically binds a heterologous epitope, and a retargeting ligand that specifically binds a protein or cell surface marker, wherein the ratio of the recombinant AAV vector to the multispecific binding molecule (molecule:molecule) is from 1:0, 5 to 1:100, and where the AAV capsid exhibits, compared to the wild-type AAV capsid of the same serotype: reduced or eliminated natural tropism in the absence of a polyspecific binding molecule, and restored and/or redirected tropism in combination with a polyspecific binding molecule. 2. The composition of claim 1, wherein the recombinant AAV capsid protein contains an additional mutation in addition to the heterologous epitope. 3. The composition of claim 1 or 2, wherein the heterologous epitope is inserted such that the insertion partially reduces the tropism of the AAV capsid relative to a wild-type AAV capsid of the same serotype, and the recombinant AAV capsid protein optionally includes an additional mutation, wherein the additional mutation includes a substitution , a deletion or insertion other than the insertion of a heterologous epitope, in addition to the insertion of a heterologous epitope, wherein the mutation further reduces and/or eliminates the tropism of the recombinant AAV capsid protein compared to a wild-type AAV capsid of the same serotype. 4. The composition according to claim 2 or 3, wherein the additional mutation is selected from the group consisting of R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A or R588T. 5. The composition according to any of the previous paragraphs, wherein the recombinant AAV vector and the polyspecific binding molecule are present in a ratio of 1:4. 6. The composition according to any of the previous paragraphs, wherein the paratope of the antibody contains an Fv domain. 7. The composition according to claim 6, wherein the Fv domain is directly fused to the heavy chain constant domain. 8. The composition according to any of the previous claims, wherein the retargeting ligand comprises an antibody or an antigen-binding portion thereof. 9. The composition according to any of the previous claims, wherein the polyspecific binding molecule comprises a bispecific antibody, and wherein each of the antibody paratope and the retargeting ligand contains a separate Fv domain fused to a first and second heavy chain constant domain. 10. Composition according to any one of paragraphs. 1-8, wherein the multispecific binding molecule comprises a bispecific antibody, and wherein the retargeting ligand comprises an antibody with a tetrameric structure comprising two identical immunoglobulin heavy chains and two identical light chains, and wherein the antibody paratope that binds the heterologous epitope is attached to the C-terminus or N-terminus of one or both heavy chains. 11. The composition of claim 10, wherein the paratope is a scFv, wherein the scFv optionally contains the amino acid sequence set forth in SEQ ID NO: 37. 12. Composition according to any of the previous paragraphs, where (i) the heterologous epitope contains an affinity tag and optionally a linker and/or (ii) the recombinant AAV capsid protein contains the amino acid sequence EQKLISEEDL (SEQ IDNO: 6) or a portion thereof flanked by and/or operably linked to at least 5 contiguous amino acids of the AAV capsid protein, and wherein the antibody paratope specifically binds the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) or a portion thereof. 13. The composition of any of the preceding claims, wherein the heterologous epitope comprises the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) or a portion thereof, and wherein the antibody paratope specifically binds the amino acid sequence EQKLISEEDL (SEQ ID NO: 6) or a portion thereof. 14. The composition according to any of the previous claims, wherein the nucleotide of interest is: (i) is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter or an insect promoter, (ii) flanked by AAV ITR sequences; (iii) is a reporter gene, or selected from the group consisting of a suicide gene, a nucleotide encoding an antibody or a binding portion thereof, a nucleotide encoding the CRISPR/Cas system or portion(s) thereof, a nucleotide encoding antisense RNA, or a nucleotide encoding siRNA; or (iv) any combination of (i) - (iii). 15. The composition according to any of the previous claims, wherein the nucleotide of interest is under the control of a non-human promoter. 16. The composition according to claim 14 or 15, where the reporter gene encodes a green fluorescent protein. 17. The composition according to any of the previous claims, wherein AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. 18. The composition according to any of the previous claims, wherein a heterologous epitope is inserted after and/or replaces a position selected from the group consisting of I-453 AAV2, I-587 AAV2, I-585 AAV6, I-590 AAV8, I-453 AAV9 , I-589 AAV9 and any corresponding amino acids of the AAV serotype infecting primates. 19. The composition according to any of the preceding claims, wherein the AAV capsid is a mosaic capsid that contains a reference AAV capsid protein, wherein the reference AAV capsid protein is the same serotype as the recombinant AAV capsid protein and does not include a heterologous epitope, and wherein the AAV capsid comprises a reference AAV capsid protein and a recombinant AAV capsid protein in a ratio of 1:1 to 15:1. 20. The composition according to any of the preceding claims, wherein the recombinant AAV capsid protein is produced using a chimeric AAV capsid gene modified to express a 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 part of the capsid protein of a different AAV serotype, and wherein the plurality of nucleic acid sequences together encode a chimeric AAV capsid protein. 21. The composition according to any of the previous claims, wherein the protein or cell surface marker comprises asialoglycoprotein 1 (ASGR1). 22. Composition according to any one of paragraphs. 1-20, wherein the protein or cell surface marker comprises CD3. 23. Composition according to any one of paragraphs. 1-20, wherein the protein or cell surface marker comprises ENTPD3. 24. Composition according to any one of paragraphs. 1-20, wherein the protein or cell surface marker comprises CD71. 25. The composition according to any of the previous claims, wherein the protein or cell surface marker is a human protein. 26. The composition according to any of the previous claims, further containing a pharmaceutically acceptable carrier. 27. A method of delivering a nucleotide of interest to a target cell expressing a cell surface protein, comprising contacting the target cell with a composition according to any of the preceding claims, wherein the target cell expresses a cell surface protein or marker on its surface. 28. The method according to claim 27, where the target cell is under in vitro conditions. 29. The method of claim 27, wherein the target cell is located in vivo in a subject. 30. The method according to claim 29, where the subject is a person. 31. Method according to any one of paragraphs. 27-30, where the target cell is a human cell. 32. Method according to any one of paragraphs. 27-31, wherein the target cell is selected from the group consisting of a liver cell, a brain cell, a T cell, a kidney cell, an intestinal cell, a pancreatic cell, a cancer cell, a muscle cell, a nerve cell, and a cell infected with a heterologous pathogen. 33. Method according to any one of paragraphs. 27-32, where the target cell is a human liver cell. 34. Method according to any one of paragraphs. 27-33, wherein the protein or cell surface marker is human asialoglycoprotein receptor 1 (hASGR1). 35. Method according to any one of paragraphs. 27-34, where the target cell is a human T cell. 36. Method according to any one of paragraphs. 27-32 and 35, wherein the protein or cell surface marker is CD3. 37. Method according to any one of paragraphs. 27-32, wherein the protein or cell surface marker is the human glucagon receptor (hGCGR). 38. Method according to any one of paragraphs. 27-32, where the target cell is an intestinal cell. 39. Method according to any one of paragraphs. 27-32, where the target cell is a pancreatic cell. 40. Method according to any one of paragraphs. 27-32 and 38, 39, wherein the protein or cell surface marker is ENTPD3. 41. Method according to any one of paragraphs. 27-32, where the target cell is a muscle cell. 42. Method according to any one of paragraphs. 27-32, where the target cell is a nerve cell. 43. Method according to any one of paragraphs. 27-32 and 42, wherein the protein or cell surface marker is CD71.