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1. (WO2019006043) TROPISM-MODIFIED RECOMBINANT VIRAL VECTORS AND USES THEREOF FOR THE TARGETED INTRODUCTION OF GENETIC MATERIAL INTO HUMAN CELLS
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What is claimed is:

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

wherein the heterologous epitope or portion thereof specifically binds an antibody paratope, and

wherein the viral capsid protein forms recombinant viral capsid with reduced or abolished natural tropism.

2. The recombinant viral capsid protein of claim 1, wherein the epitope is at least 1 amino acid in length.

3. The recombinant viral capsid protein of claim 1 or claim 2, comprising a substitution, insertion, or deletion at an amino acid position involved with the natural tropism of the viral capsid such that the recombinant viral capsid protein forms viral capsid with reduced to abolished natural tropism.

4. The recombinant viral capsid protein of claims 1-3, wherein the viral capsid protein is derived from adeno-associated virus (AAV) capsid gene, optionally wherein the heterologous epitope is inserted into a position selected from the group consisting of 1587 of AAV2, 1585 of AAV6, 1590 of AAV8, 1453 of AAV9, 1589 of AAV9, and any corresponding amino acids of an AAV serotype that infects primates, optionally wherein the AAV serotype that infects primates is selected from the group consisting of AAVl, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

5. The recombinant viral capsid protein of claim 4, wherein the adeno-associated virus is AAV2.

6. The recombinant viral capsid protein of claim 4, wherein the adeno-associated virus is AAV6.

7. The recombinant viral capsid protein of claim 4, wherein the adeno-associated virus is AAV8.

8. The recombinant viral capsid protein of claim 4, wherein the adeno-associated virus is AAV9.

9. The recombinant viral capsid protein of any one of claims 1-8, wherein

(i) the viral capsid protein is a genetically modified AAV2 VP1 capsid protein and the epitope is inserted after and/or replaces an amino acid at position 1453 or 1587;

(ii) the viral capsid protein is a genetically modified AAV6 VP 1 capsid protein and the epitope is inserted after and/or replaces an amino acid at position 1585;

(iii) the viral capsid is a genetically modified AAV8 VP1 capsid protein and the epitope is inserted after and/or replaces an amino acid at position 1590.

(iv) the viral capsid protein is a genetically modified AAV9 VP1 capsid protein and the epitope is inserted after and/or replaces an amino acid at position 1453 or 1589.

10. The recombinant viral capsid protein of any one of claims 1-9, wherein the viral capsid protein is encoded by an AAV2 capsid gene modified to express the epitope is flanked between amino acids N587 and R588 of the AAV2 VP1 capsid protein.

11. The recombinant viral capsid protein of any one of claims 1-9, wherein the viral capsid protein is derived from an AAV6 capsid gene, and the epitope is inserted immediately after amino acid Q585.

12. The recombinant viral capsid protein of any one of claims 1-9, wherein the viral capsid protein is derived from an AAV8 capsid gene, and wherein the epitope is inserted immediately after amino acid N590 of the AAV8 VP1 capsid protein.

13. The recombinant viral capsid protein of any one of claims 1-9, wherein the viral capsid protein is derived from an AAV9 capsid gene, the epitope is inserted into the capsid protein after and/or replaces the amino acid at position G453 or A589 of the AAV9 VPl capsid protein, and wherein the capsid protein further comprises an additional mutation.

14. The recombinant viral capsid protein of claim 13, wherein the additional mutation is W503A.

15. The recombinant viral capsid protein of any one of claims 1-14, wherein the epitope comprises the amino acid sequence EQKLISEEDL, which amino acid sequence or a portion thereof specifically binds the antibody paratope, and optionally.

16. The recombinant viral capsid protein of any of claims 1-15, wherein the epitope comprises the amino acid sequence EQKLISEEDL, which amino acid sequence or a portion thereof specifically binds the antibody paratope, and

wherein the amino acid sequence EQKLISEEDL flanked by and operably linked to at least 5 contiguous amino acids of an AAV capsid protein.

17. The recombinant viral capsid protein of claim 16, wherein the viral capsid protein comprises an amino acid sequence set forth as SEQ ID NO:2, an amino acid sequence set forth as SEQ ID NO:4, an amino acid sequence set forth as SEQ ID NO:25, an amino acid sequence set forth as SEQ ID NO:26, or an amino acid sequence set forth as SEQ ID NO:27.

18. The recombinant viral capsid protein of any one of claims 1-17, wherein the recombinant viral capsid protein or a viral capsid comprising the recombinant viral capsid protein is unable to infect a target cell in the absence of a multispecific binding molecule comprising the antibody paratope, optionally wherein in the absence of a multispecific binding molecule comprising the antibody paratope, the transduction efficient of the viral capsid protein or a viral capsid comprising the recombinant viral capsid protein is:

(i) reduced by at least 10%,

(ii) reduced by at least 20%,

(iii) reduced by at least 30%,

(iv) reduced by at least 40%,

(v) reduced by at least 50%,

(vi) reduced by at least 60%,

(vii) reduced by at least 70%,

(viii) reduced by at least 80%,

(ix) reduced by at least 90%, or

(x) abolished.

19. An isolated nucleic acid comprising a nucleotide sequence encoding the recombinant viral capsid protein of any one of claims 1-18.

20. The isolated nucleic acid of claim 19, wherein the nucleotide sequence encodes recombinant viral capsid protein that comprises an amino acid sequence set forth as SEQ ID NO:2, an amino acid sequence set forth as SEQ ID NO:4, an amino acid sequence set forth as SEQ ID NO:25, an amino acid sequence set forth as SEQ ID NO:26, or an amino acid sequence set forth as SEQ ID NO:27.

21. A recombinant viral vector comprising a nucleotide of interest encapsulated by the recombinant viral capsid of claim 20, optionally wherein the recombinant viral capsid is a mosaic capsid.

22. The recombinant viral vector of claim 21, wherein the nucleotide of interest is under the control of a promoter selected from the group consisting of a viral promoter, a bacterial promoter, a mammalian promoter, an avian promoter, a fish promoter, an insect promoter, and any combination thereof.

23. The recombinant viral vector of claim 22, wherein the nucleotide of interest is under the control of a non-human promoter.

24. The recombinant viral vector of claim 23, wherein the nucleotide of interest is flanked by AAV ITR sequences.

25. The recombinant viral vector of any one of claims 21-24, wherein the nucleotide of interest is a reporter gene.

26. The recombinant viral vector of claim 25, wherein the reporter gene encodes green fluorescent protein.

27. The recombinant viral vector of any one of claims 21-24, wherein 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 a CRISPR/Cas system or portion(s) thereof, a nucleotide encoding antisense RNA, a nucleotide encoding siRNA, and a combination thereof.

28. A composition comprising (a) the recombinant viral vector of any one of claims 21-27 and (b) a multispecific binding molecule comprising an antibody paratope that specifically binds the epitope, optionally wherein multispecific binding molecule further comprises a retargeting ligand that specifically binds a receptor expressed on a target cell, wherein the multspecific binding molecule is optionally a bispecific binding molecule.

29. The composition of claim 28, wherein the viral vector and the multispecific binding molecule are present at a ratio of 1 :4.

30. The composition of claim 28 or 29, wherein the antibody paratope is an Fv domain.

31. The composition of claim 30, wherein the Fv domain is directly fused to a heavy chain constant domain.

32. The composition of any one of claims 28-31, wherein the retargeting ligand is an antibody, or portion thereof.

33. The composition of any one of claims 28-32, wherein the multispecific binding molecule is a bispecific antibody, and

wherein the paratope and the retargeting ligand each comprise a distinct Fv domain fused to a first and second heavy chain constant domain.

34. The composition of claim 33, wherein the first and second heavy chains bind to Protein A with differential binding affinities.

35. The composition of any one of claims 28-32, wherein the multispecifc binding molecule is a bispecific antibody, and

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

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

36. The composition of claim 35, wherein the paratope is an scFv, optionally wherein the scFv comprises an amino acid sequence set forth as SEQ ID NO:37.

37. The composition of any one of claims 28-36, wherein the heterologous epitope comprises an amino acid sequence EQKLISEEDL or a portion thereof, and wherein the antibody paratope specifically binds the amino acid sequence EQKLISEEDL or a portion thereof.

38. The composition of any one of claims 28-37, wherein the retargeting ligand specifically binds a cell surface protein is a cell surface marker.

39. The composition of claim 38, wherein the cell surface marker is asialoglycoprotein 1 (ASGR1).

40. The composition of claim 38, wherein the cell surface marker is CD3.

41. The composition of claim 38, wherein the cell surface marker is ENTPD3.

42. The composition of any one of claims 28-41, further comprising a pharmaceutically acceptable carrier.

43. A method of directing the recombinant viral vector of any one of claims 21-27 to a target cell comprising

contacting the recombinant viral vector with a multispecific binding molecule, wherein the multispecific binding molecule comprises (i) an antibody paratope that specifically binds the epitope and (ii) a retargeting ligand that specifically binds a protein expressed on the surface of the target cell.

44. The method of claim 43, wherein the target cell is in vivo.

45. The method of claim 43 or claim 44, wherein said contacting is performed ex vivo.

46. A method of delivering a nucleotide of interest to a target cell expressing a cell surface protein comprising contacting the target cell with the composition of any one of claims 28-42, wherein the multispecific binding molecule comprises a retargeting ligand that binds the cell surface protein.

47. The method of claim 46, wherein the target cell is in vitro.

48. The method of claim 46, wherein the target cell is in vivo in a subject.

49. The method of claim 48, wherein the subject is a human.

50. The method of any one of claims 46-49, wherein the target cell is a human cell.

51. The method of any one of claims 46-50, 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 pancreas cell, a cancerous cell, and a cell infected with heterologous pathogen.

52. The method of any one of claims 46-51, wherein the target cell is a human liver cell.

53. The method of any one of claims 46-52, wherein the cell surface protein is human asialoglycoprotein receptor 1 (hASGRl).

54. The method of any one of claims 46-51, wherein the target cell is a human T cell.

55. The method of claim 54, wherein the cell surface protein is CD3.

56. The method of any one of claims 46-51, wherein the cell surface protein is human glucagon receptor (hGCGR).

57. The method of any one of claims 46-50, wherein the target cell is an intestinal cell.

58. The method of any one of claims 46-50, wherein the target cell is a pancreas cell.

59. The method of claim 57 and 58, wherein the cell surface protein is ENTPD3.

60. A method of inactivating a viral capsid protein comprising

(a) inserting a nucleic acid encoding a heterologous epitope into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the heterologous epitope, and

(b) culturing a packaging cell in conditions sufficient for the production of viral vectors, wherein the packaging cell comprises the nucleotide sequence.

61. A method of producing a viral vector comprising culturing a packaging cell in conditions sufficient for the production of viral vectors, wherein the packaging cell comprises a plasmid encoding a recombinant capsid protein according to any one of claim 1-18.

62. The method of claim 60 or claim 61, wherein the packaging cell further comprises a helper plasmid and/or a transfer plasmid comprising a nucleotide of interest.

63. The method of any one of claims 60-62, further comprising isolating self-complementary adeno-associated viral vectors from culture supernatant.

64. The method of any one of claims 60-63, further comprising lysing the packaging cell and isolating single-stranded adeno-associated viral vectors from the cell lysate.

65. The method of any one of claims 60-64, further comprising

a. clearing cell debris,

b. treating the supernatant containing viral vectors with DNase I and MgC12, c. concentrating viral vectors,

d. purifying the viral vectors, and

e. any combination of a.-d.

66. A viral vector made according to the method of any one of claims 60-65.

67. A packaging cell for producing a viral vector comprising a plasmid encoding capsid protein according to any one of claims 1-18.

68. A binding molecule comprising a paratope that binds SEQ ID NO:6, wherein the paratope comprises an HCVR, LCVR, HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and/or LCDR3 sequences of the scFv encoded by the nucleic acid sequence set forth as SEQ ID NO:28.

69. The binding molecule of claim 68, wherein the antibody paratope is an Fv domain.

70. The binding molecule of claim 69, wherein the Fv domain is directly fused to a heavy chain constant domain.

71. The binding molecule of any one of claims 68-70, wherein the binding molecule is a bispecific antibody and further comprises a retargeting ligand, and

wherein the paratope and the retargeting ligand each comprise a distinct Fv domain fused to a first and second heavy chain constant domain.

72. The binding protein of claim 71, wherein the first and second heavy chains bind to Protein A with differential binding affinities.

73. The binding protein of any one of claims 68-69, wherein the binding molecule is a bispecific antibody and further comprises a retargeting ligand, and

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

wherein the paratope is appended to the C-terminus or N-terminus of one or both heavy chains and/or to the C-terminus or N-terminus of one or both heavy chains.

74. The binding protein of claim 73, wherein the paratope is an scFv, optionally wherein the scFv comprises an amino acid sequence set forth as SEQ ID NO:37.

75. The binding protein of any one of claims 69-72, further comprising a retargeting ligand that is an antibody, or portion thereof.

76. The binding protein of claim 75, wherein the retargeting ligand specifically binds a cell surface protein is a cell surface marker.

77. The binding protein of claim 76, wherein the cell surface marker is asialoglycoprotein 1 (ASGR1).

78. The binding protein of claim 76, wherein the cell surface marker is CD3.

79. The binding protein of claim 76, wherein the cell surface marker is GCGR.

80. The binding protein of claim 76, wherein the cell surface marker is ENTPD3.

81. A composition comprising (a) the recombinant viral vector of any one of claims 21-27 and (b) the binding protein of any one of claims 68-81.

82. The composition of claim 81, wherein the viral vector and the multispecific binding molecule are present at a ratio of 1 :4.

83. The composition of claim 81 or claim 82, further comprising a pharmaceutically acceptable carrier.