Parvovirus compositions and related methods for gene therapy

Abstract

The present disclosure provides technologies comprising parvovirus compositions, preparations, constructs, and methods for gene therapy.

Description

This disclosure provides techniques for gene therapy, including parvovirus compositions, formulations, constructs, and related methods. Abstract

Claims

Docket No.: 2017359-0072 (CAR-002.WO) CLAIMS We claim:

1. A construct comprising a VP1 capsid coding sequence encoding a parvovirus VP1 capsid polypeptide (e.g., a VP1 capsid polypeptide, e.g., a variant VP1 capsid polypeptide) (e.g., wild type or otherwise functional, e.g., codon optimized); and one or more of the following: (i) an expression control sequence (e.g., operably linked to the VP1 capsid coding sequence), (ii) a 5’ untranslated region (UTR) sequence, (iii) an alternative translation initiation codon sequence (e.g., wherein the VP1 capsid coding sequence comprises the alternative translation initiation codon), (iv) wherein the VP1 capsid coding sequence comprises fewer ATG sequence(s) across the length of the VP1 capsid coding sequence, relative to a parvovirus reference VP1 capsid coding sequence selected from the group consisting of those in Table 3A, or (v) any combination thereof.

2. The construct of claim 1, wherein the one or more of (i)-(v) direct transcription and / or translation of the parvovirus VP1 capsid.

3. The construct of claim 1, wherein the expression control sequence comprises a promoter, and wherein the promoter is a polyhedrin, P10, or OpiE1 promoter.

4. The construct of claim 1, wherein the 5’ UTR sequence further comprises either a (i) nucleotide spacer sequence or (ii) a Kozak consensus sequence or both.Docket No.: 2017359-0072 (CAR-002.WO) 5. The construct of claim 4, wherein the nucleotide spacer sequence comprises a nucleotide sequence according to SEQ ID NO:

138.

6. The construct of claim 4, wherein the nucleotide spacer sequence comprises a nucleotide sequence according to SEQ ID NO:

139.

7. The construct of claim 4, wherein the Kozak consensus sequence is a eukaryotic conventional Kozak consensus sequence (GCCGCC-GG), Viral-derived Kozak consensus sequence (CCTGTTAAG), or alternative Kozak consensus sequence (AAA).

8. The construct of any one of the preceding claims, wherein the alternative translation initiation codon sequence comprises a sequence according to CTG, TTG, ACG, or ATC.

9. The construct of any one of the preceding claims, wherein the one or more of (i)-(v) directs translation start at the alternative translation initiation codon sequence.

10. The construct of any one of the preceding claims, further comprising a sequence that encodes a parvovirus VP2 capsid polypeptide (e.g., a reference VP2 capsid polypeptide, e.g., a variant VP2 capsid polypeptide) (e.g., wherein the sequence that encodes a parvovirus VP2 capsid polypeptide is or comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 195).

11. The construct of claim 10, wherein the one or more of (i)-(v) directs transcription and / or translation start such that the parvovirus VP2 capsid polypeptide is present in excess of theDocket No.: 2017359-0072 (CAR-002.WO) parvovirus VP1 capsid polypeptide (e.g., wherein the ratio of parvovirus VP2 capsid polypeptide to VP1 capsid polypeptide is 25:1, 20:1, 15:1, 10:1, 5:1).

12. The construct of any one of the preceding claims, further comprising a parvovirus VP3 capsid polypeptide.

13. The construct of any one of the preceding claims, further comprising a heterologous peptide tag.

14. The construct of claim 13, wherein the heterologous peptide tag allows affinity purification using an antibody, an antigen-binding fragment of an antibody, or a nanobody.

15. The construct of claim 13 or 14, wherein the heterologous peptide tag comprises an epitope / tag selected from hemagglutinin, His (e.g., 6X-His), FLAG, E-tag, TK15, Strep-tag II, AU1, AU5, Myc, Glu-Glu, KT3, and IRS.

16. The construct of any one of the preceding claims, wherein the construct further comprises a nucleic acid sequence that encodes one or more heterologous peptides having a length from about 10 amino acids to 20 amino acids (e.g., according to SEQ ID NOs: 8-87) (e.g., wherein the one or more heterologous peptides comprises or is a heterologous targeting peptide).

17. The construct of claim 16, wherein the one or more heterologous peptides are inserted into one or more residues of a parvovirus VP1 capsid polypeptide corresponding to one or more residues within a variable region of a parvovirus (e.g., AAV) capsid (e.g., wherein the one or more residues of a parvovirus VP1 capsid polypeptide map(s) onto a structural overlay of one or more residues within a variable region of a parvovirus VP1 capsid (e.g., AAV capsid)).Docket No.: 2017359-0072 (CAR-002.WO) 18. The construct of claim 16, wherein the one or more heterologous targeting peptides are inserted into one or more residues along a 3-fold axis of symmetry of a common VP3 region of the parvovirus VP1 capsid polypeptide.

19. The construct of claim 16, wherein the one or more heterologous targeting peptides are inserted into one or more residues along a 3-fold axis of symmetry of a common VP2 region of the parvovirus VP1 capsid polypeptide.

20. The construct of claim 16, wherein the one or more heterologous peptides targets a cell e.g., a PymT tumor cell, a cervix cancer cell (e.g., a HeLa cell), a K562 cell, a Raji cell, a SKOV- 3 cell, a breast cancer cell (e.g., a MCF-7 cell), a M07e cell, a human saphenous vascular endothelial cell (HSaVEC), a MT1-MMP cell, a primary hepatocyte cell (e.g., a Huh7 cell), an immune cell (e.g., a human T cell, e.g., a CD4+ T cell, e.g., a Th2 cell, e.g., a CAR T cell, e.g., a NK cell), a neuron cell (e.g., a LX-2 cell, e.g., a stellate cell, e.g., a primary neuron cell, e.g., a neuroblastoma cell (e.g., a SH-SY5Y cell)), a lung cell (e.g., a lung fibroblast cell), a myoblast cell, a myotube cell, a primary cardiomyocyte, a skeletal muscle cell (e.g., a differentiated skeletal muscle cell), a human vein endothelial cell, a T84 cell, a ileum cell (intestinal), a primary human airway epithelia cell), a kidney cell (e.g., a human renal proximal tubule (HRCE) cell, e.g., a bile duct cell, e.g., an outer medullary cell, e.g., a mixed medullary cell, e.g., renal cortical epithelial cells, e.g., renal epithelial cells), a bone marrow MSC cell, a blood cell (e.g., a PBMC cell), a small intestine cell, a muscle cell, a heart cell, a spleen cell, a liver cell, a brain cell (e.g., a brain-striatum cell), an ocular cell, a testes cell, an oocyte, a medulla cell, a striatum cell, a spinal cord (or chord) cell, or a duodenum cell (e.g., wherein the one or more heterologous peptides comprises or is a heterologous targeting peptide).

21. The construct of claim 16, wherein the one or more heterologous peptides increases cell specificity and / or viral transduction efficiency and / or increases virion performance.Docket No.: 2017359-0072 (CAR-002.WO) 22. The construct of any one of the preceding claims, wherein the parvovirus VP1 capsid polypeptide comprises a nuclear localization signal sequence (NLS).

23. The construct of any one of the preceding claims, wherein the parvovirus VP1 capsid polypeptide is a protoparvovirus VP1 capsid polypeptide, a bocaparvovirus VP1 capsid polypeptide, an erythroparvovirus VP1 capsid polypeptide, a tetraparvovirus VP1 capsid polypeptide, or a copiparvovirus VP1 capsid polypeptide.

24. The construct of any one of the preceding claims, wherein the construct reduces toxicity in a host cell, relative to a reference construct lacking (i)-(v).

25. The construct of any one of the preceding claims, wherein the construct increases virion production in a host cell, relative to a reference construct lacking (i)-(v).

26. The construct of any one of the preceding claims, wherein the construct increases capsid polypeptide yield in a host cell, relative to a reference construct lacking (i)-(v).

27. The construct of claim 26, wherein the host cell is an insect cell.

28. The construct of claim 26, wherein the host cell is a mammalian cell.

29. The construct of any one of the preceding claims, wherein the parvovirus VP1 capsid polypeptide comprises an amino acid sequence with at least 60% identity to an amino acid sequence in Table 3B.Docket No.: 2017359-0072 (CAR-002.WO) 30. The construct of any of the preceding claims, wherein the parvovirus VP1 capsid polypeptide diminishes human humoral immune response against a virion, and / or reduces neutralization of a virion by human antibodies.

31. The construct of any one of claims 1-30, wherein the VP1 capsid coding sequence comprises or is single-stranded deoxyribonucleic acid (ssDNA).

32. The construct of any one of claims 1-31, wherein the VP1 capsid coding sequence comprises or is double stranded DNA (dsDNA).

33. The construct of any one of claims 1-32, wherein the VP1 capsid coding sequence comprises or is RNA (e.g., an mRNA).

34. The construct of any one of claims 1-33, wherein the VP1 capsid coding sequence comprises or is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

171.

35. The construct of any one of claims 1-34, wherein the VP1 capsid coding sequence comprises or is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO:

178.

36. The construct of claim 34 or 35, wherein the sequence that encodes a parvovirus VP2 capsid polypeptide is or comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 195.Docket No.: 2017359-0072 (CAR-002.WO) 37. A construct comprising a sequence having at least 70% identity (e.g., 80%, 85%, 90%, 95%, 100% identity) to a sequence in Table 4 (e.g., wherein the sequence comprises or is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 171) (e.g., wherein the sequence comprises or is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 178) (e.g., wherein sequence comprises or is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 194) (e.g., wherein sequence comprises or is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 195).

38. The construct of claim 37, further comprising a sequence that encodes a parvovirus VP2 capsid polypeptide (e.g., a reference VP2 capsid polypeptide, e.g., a variant VP2 capsid polypeptide) (e.g., wherein sequence comprises or is least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identical to SEQ ID NO: 194) (e.g., wherein the sequence that encodes a parvovirus VP2 capsid polypeptide is or comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 195).

39. A parvovirus VP1 capsid polypeptide according to any one of claims 1-38.

40. A virion comprising the parvovirus VP1 capsid polypeptide of claim 39.

41. The virion of claim 40, wherein the parvovirus VP1 capsid polypeptide diminishes human humoral immune response against the virion, and / or reduces neutralization of the virion by human antibodies.Docket No.: 2017359-0072 (CAR-002.WO) 42. The virion of claim 40 or 41, wherein the parvovirus VP1 capsid polypeptide increases affinity and / or specificity of the virion to at least one cellular receptor involved in internalization of the virion.

43. The virion of any one of claims 40-42, wherein the parvovirus VP1 capsid polypeptide further comprises an insertion of one or more heterologous targeting peptides having a length of from 10 amino acids to 20 amino acids into one or more residues of a parvovirus VP1 capsid polypeptide corresponding to one or more residues within a variable region of a parvovirus (e.g., AAV) capsid (e.g., wherein the one or more residues of a parvovirus VP1 capsid polypeptide map(s) onto a structural overlay of one or more residues within a variable region of a parvovirus VP1 capsid (e.g., AAV capsid)).

44. The virion of claim 42 or 43, wherein the parvovirus VP1 capsid polypeptide targets a cell, e.g., a PymT tumor cell, a cervix cancer cell (e.g., a HeLa cell), a K562 cell, a Raji cell, a SKOV-3 cell, a breast cancer cell (e.g., a MCF-7 cell), a M07e cell, a human saphenous vascular endothelial cell (HSaVEC), a MT1-MMP cell, a primary hepatocyte cell (e.g., a Huh7 cell), an immune cell (e.g., a human T cell, e.g., a CD4+ T cell, e.g., a Th2 cell, e.g., a CAR T cell, e.g., a NK cell), a neuron cell (e.g., a LX-2 cell, e.g., a stellate cell e.g., a primary neuron cell, e.g., a neuroblastoma cell (e.g., a SH-SY5Y cell)), a lung cell (e.g., a lung fibroblast cell), a myoblast cell, a myotube cell, a primary cardiomyocyte, a skeletal muscle cell (e.g., a differentiated skeletal muscle cell), a human vein endothelial cell, a T84 cell, a ileum cell (intestinal), a primary human airway epithelia cell), a kidney cell (e.g., a human renal proximal tubule (HRCE) cell, e.g., a bile duct cell, e.g., an outer medullary cell, e.g., a mixed medullary cell, e.g., renal cortical epithelial cells, e.g., renal epithelial cells), a bone marrow MSC cell, a blood cell (e.g., a PBMC cell), a small intestine cell, a muscle cell, a heart cell, a spleen cell, a liver cell, a brain cell (e.g., a brain-striatum cell), an ocular cell, a testes cell, an oocyte, a medulla cell, a striatumDocket No.: 2017359-0072 (CAR-002.WO) cell, a spinal cord (or chord) cell, or a duodenum cell (e.g., wherein the one or more heterologous peptides comprises or is a heterologous targeting peptide).

45. The virion of any one of claims 40-44, wherein the parvovirus VP1 capsid polypeptide confers increased infectivity compared to the infectivity by a reference virion comprising the corresponding parvovirus reference VP1 capsid polypeptide.

46. The virion of any one of claims 40-45, wherein the one or more heterologous peptides comprises the amino acid sequence according to any one of SEQ ID NOs: 8-87.

47. The virion of any one of claims 40-46, wherein the one or more heterologous peptides increases cell specificity and / or viral transduction efficiency and / or increases virion performance.

48. The virion of any one of claims 40-46, further comprising a polynucleotide comprising a heterologous nucleic acid sequence.

49. The virion of claim 48, wherein the heterologous nucleic acid comprises a nucleic acid sequence that is at least about 60% identical to a nucleic acid sequence of a target cell.

50. The virion of claim 48 or 49, wherein the heterologous nucleic acid is at least about 60% identical to a nucleic acid of a mammal, preferably wherein the mammal is a human.

51. The virion of any one of claims 48-50, wherein the polynucleotide comprises at least one inverted terminal repeat (ITR).Docket No.: 2017359-0072 (CAR-002.WO) 52. The virion of claim 51, wherein the at least one ITR comprises one or more of the following: (a) a dependoparvovirus ITR, (b) a bocaparvovirus ITR (c) a protoparvovirus ITR, (d) a tetraparvovirus ITR, (e) an erthythroparvovirus ITR, or (f) a copiparvovirus ITR.

53. The virion of any one of claims 48-52, wherein the polynucleotide is deoxyribonucleic acid (DNA).

54. The virion of claim 53, wherein the DNA is single-stranded or self-complementary duplex.

55. The virion of any one of claims 48-54, wherein the polynucleotide comprises a Rep protein-dependent origin of replication (ori).

56. The virion of any one of claims 48-55, wherein the heterologous nucleic acid sequence is operably linked to a transgene promoter, optionally placed between two ITRs.

57. The virion of any one of claims 48-56, wherein the heterologous nucleic acid sequence comprises a transgene coding sequence.Docket No.: 2017359-0072 (CAR-002.WO) 58. The virion of claim 57, wherein the transgene coding sequences comprises one or more of: (a) a gene encoding a protein or a fragment thereof, preferably a human protein or a fragment thereof; (b) a nucleic acid encoding a nuclease, optionally a Transcription Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, a megaTAL, or a CRISPR endonuclease, (e.g., a Cas9 endonuclease or a variant thereof); (c) a nucleic acid encoding a reporter, e.g., luciferase or GFP; or (d) a nucleic acid encoding a drug resistance protein, e.g., neomycin resistance.

59. The virion of claim 57 or 58, wherein the transgene coding sequence is codon-optimized for expression in a target cell.

60. The virion of claim 59, wherein the target cell is or comprises a PymT tumor cell, a cervix cancer cell (e.g., a HeLa cell), a K562 cell, a Raji cell, a SKOV-3 cell, a breast cancer cell (e.g., a MCF-7 cell), a M07e cell, a human saphenous vascular endothelial cell (HSaVEC), a MT1-MMP cell, a primary hepatocyte cell (e.g., a Huh7 cell), an immune cell (e.g., a human T cell, e.g., a CD4+ T cell, e.g., a Th2 cell, e.g., a CAR T cell, e.g., a NK cell), a neuron cell (e.g., a LX-2 cell, e.g., a stellate cell, e.g., a primary neuron cell, e.g., a neuroblastoma cell (e.g., a SH- SY5Y cell)), a lung cell (e.g., a lung fibroblast cell), a myoblast cell, a myotube cell, a primary cardiomyocyte, a skeletal muscle cell (e.g., a differentiated skeletal muscle cell), a human vein endothelial cell, a T84 cell, a ileum cell (intestinal), a primary human airway epithelia cell), a kidney cell (e.g., a human renal proximal tubule (HRCE) cell, e.g., a bile duct cell, e.g., an outer medullary cell, e.g., a mixed medullary cell, e.g., renal cortical epithelial cells, e.g., renal epithelial cells), a bone marrow MSC cell, a blood cell (e.g., hematopoietic stem cell (HSC), e.g., a PBMC cell), a small intestine cell, a muscle cell, a heart cell, a spleen cell, a liver cell, a brainDocket No.: 2017359-0072 (CAR-002.WO) cell (e.g., a brain-striatum cell), an ocular cell, a testes cell, an oocyte, a medulla cell, a striatum cell, a spinal cord (or chord) cell, or a duodenum cell.

61. The virion of any one of claims 57-60, wherein the transgene coding sequence comprises a hemoglobin gene (HBA1, HBA2, HBB, HBG1, HBG2, HBD, HBE1, and / or HBZ), a gene encoding an alpha-hemoglobin stabilizing protein (AHSP), coagulation factor VIII, coagulation factor IX, von Willebrand factor, dystrophin or truncated dystrophin, micro-dystrophin, utrophin or truncated utrophin, micro-utrophin, usherin (USH2A), CEP290, glial cell line-derived neurotrophic factor (GDNF), neuturin (NTN), HTT, neuronal apoptosis inhibitory protein (NAIP), cystic fibrosis transmembrane conductance regulator (CFTR), F8 or a fragment thereof (e.g., fragment encoding B-domain deleted polypeptide (e.g., VIII SQ, p-VIII)), T cell receptor (e.g., TCR alpha or TCR beta), a gene associated with lysosomal storage diseases, a gene associated with Alport syndrome (e.g., Col4a3, Col4a4, Col4a5), a gene associated with Fabry disease (e.g., GLA), a gene associated with autosomal dominant polycystic kidney disease (PKD) (e.g., PKD, PKD1, PKD2), a gene associated with congenital nephrotic syndrome (e.g., NPHS1 (Nephrin), NPHS2 (Podocin), a gene associated with hypertrophic cardiomyopathy (e.g., MYBPC3, JPH2, ALPK3), a gene associated with dilated cardiomyopathy (e.g., RBM20), or a gene associated with dilated cardiomyopathy (e.g., ALPK3, LMNA, BAG3).

62. The virion of any one of claims 48-61, wherein the heterologous nucleic acid sequence comprises a non-coding sequence.

63. The virion of claim 62, wherein the non-coding sequence comprises or is RNA.

64. The virion of claim 63, wherein the RNA comprises or is lncRNA, miRNA, shRNA, siRNA, antisense RNA, and / or guide RNA.Docket No.: 2017359-0072 (CAR-002.WO) 65. The virion of claim 62, wherein the non-coding sequence comprises or is DNA.

66. The virion of claim 65, wherein the DNA comprises or is: (a) a transcription regulatory element (e.g., an enhancer, a transcription termination sequence, an untranslated region (5’ or 3’ UTR), a proximal promoter element, a locus control region, a polyadenylation signal sequence), and / or (b) a translation regulatory element (e.g., Kozak sequence, woodchuck hepatitis virus post-transcriptional regulatory element).

67. The virion of claim 66, wherein the DNA comprises or is a transcription regulatory element, and wherein the transcription regulatory element is a locus control region, optionally a β-globin LCR or a DNase hypersensitive site (HS) of β-globin LCR.

68. The virion of any one of claims 57-67, wherein the transgene coding sequence (or the protein translated therefrom) or the non-coding sequence increases or restores the expression of an endogenous gene of the target cell.

69. The virion of any one of claims 57-68, wherein the transgene coding sequence (or the protein translated therefrom) or the non-coding sequence decreases or eliminates the expression of an endogenous gene of the target cell.

70. The virion of any one of claims 57-69, wherein the transgene promoter is selected from: (a) a promoter heterologous to a nucleic acid; (b) a promoter that facilitates the tissue-specific expression of a nucleic acid, preferably wherein the transgene promoter facilitates hematopoietic cell-specific expression or erythroid lineage-specific expression;Docket No.: 2017359-0072 (CAR-002.WO) (c) a promoter that facilitates the constitutive expression of a nucleic acid; and (d) a promoter that is inducibly expressed, optionally in response to a metabolite or small molecule or chemical entity.

71. The virion of any one of claims 57-70, wherein the transgene promoter is selected from the CMV promoter, β-globin promoter, CAG promoter, AHSP promoter, MND promoter, Wiskott-Aldrich promoter, cardiac troponin T promoter, and PKLR promoter.

72. The virion of any one of claims 40-71, wherein the virion is icosahedral.

73. The virion of any one of claims 68-72, wherein the parvovirus VP1 capsid polypeptide is phosphorylated.

74. The virion of any one of claims 68-73, wherein the virion detargets the liver relative to a virion comprising an AAV capsid (e.g., detargets the liver about 50 times more than a virion comprising an AAV capsid, e.g., 100 times more than a virion comprising an AAV capsid, e.g., 150 times more than a virion comprising an AAV capsid. e.g., 200 times more than a virion comprising an AAV capsid, e.g., 150 times more than a virion comprising an AAV capsid, e.g., 200 times more than a virion comprising an AAV capsid, e.g., 250 times more than a virion comprising an AAV capsid, e.g., 300 times more than a virion comprising an AAV capsid).

75. A population of virions according to any one of claims 40-74, wherein the population is characterized as having reduced toxicity in a host cell, improved virion production in a host cell, increased capsid polypeptide yield, or any combination thereof, relative to a population of virions comprising a parvovirus reference VP1 capsid polypeptide encoded by the reference construct.Docket No.: 2017359-0072 (CAR-002.WO) 76. A system comprising a construct of any one of claims 1-38 and / or a second construct comprising a sequence that encodes a parvovirus VP2 capsid polypeptide, wherein the parvovirus VP2 capsid polypeptide is present in excess of the parvovirus VP1 capsid polypeptide (e.g., wherein the ratio of parvovirus VP2 capsid polypeptide to VP1 capsid polypeptide is 25:1, 20:1, 15:1, 10:1, 5:1).

77. A system comprising a parvovirus VP1 capsid polypeptide of claim 40 and a parvovirus VP2 capsid polypeptide, wherein the parvovirus VP2 capsid polypeptide is present in excess of the parvovirus VP1 capsid polypeptide (e.g., wherein the ratio of parvovirus VP2 capsid polypeptide to VP1 capsid polypeptide is 25:1, 20:1, 15:1, 10:1, 5:1).

78. A composition comprising a construct of any one of claims 1-38.

79. A composition comprising a virion of any one of claims 40-74.

80. A composition comprising a population of virions of claim 75.

81. A composition comprising a parvovirus VP1 capsid polypeptide of claim 39.

82. The composition of any one of claims 78-81, wherein the composition is a pharmaceutical composition.

83. The composition of claim 82, further comprising a pharmaceutically acceptable carrier.Docket No.: 2017359-0072 (CAR-002.WO) 84. A kit comprising a construct of any one of claims 1-38 and a construct comprising a second coding sequence encoding a least one capsid replication protein (e.g., NS1 protein) of a parvovirus operably linked to at least one expression control sequence for expression in a host cell.

85. A host cell comprising a construct of any one of claims 1-38.

86. A host cell comprising a parvovirus VP1 capsid polypeptide of claim 39.

87. A host cell comprising a virion of any one of claims 40-74.

88. A host cell comprising a population of virions of claim 75.

89. A host cell comprising a composition of any one of claims 78-83.

90. The host cell of claim 89, comprising a second construct comprising a polynucleotide comprising at least one ITR nucleotide sequence.

91. The host cell of claim 90, wherein the at least one ITR comprises a parvovirus ITR.

92. The host cell of claim 90, wherein the at least one ITR comprises one or more of the following: (a) a dependoparvovirus ITR, (b) a bocaparvovirus ITRDocket No.: 2017359-0072 (CAR-002.WO) (c) a protoparvovirus ITR, (d) a tetraparvovirus ITR, (e) an erthythroparvovirus ITR, or (f) a copiparvovirus ITR.

93. The host cell of claim 92, wherein the at least one ITR comprises a dependoparvovirus ITR, wherein the at least one dependoparvovirus ITR comprises an AAV ITR, optionally an AAV2 ITR.

94. The host cell of any one of claims 85-93, further comprising a third construct comprising a polynucleotide comprising: (1) at least one capsid replication protein (e.g., NS1) of a parvovirus operably linked to at least one expression control sequence for expression in a host cell, (2) (i) at least one ITR replication protein of a protoparvovirus, bocaparvovirus, dependoparvovirus, tetraparvovirus, copiparvovirus or erythroparvovirus, or (ii) at least one ITR replication protein of an AAV, optionally wherein the at least one ITR replication protein of an AAV comprises (a) a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in a host cell, and / or (b) a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in a host cell, or (3) a combination of (1) and (2i) or (1) and (2ii).

95. The host cell of any one of claims 85-94, wherein at least the first construct, the second construct, or the third construct is stably integrated in the host cell genome.Docket No.: 2017359-0072 (CAR-002.WO) 96. The host cell of any one of claims 85-95, wherein the at least one capsid replication protein of a parvovirus is an NS1 protein (e.g., having at least 30% identity to SEQ ID NO: 7).

97. The host cell of any one of claims 85-96, wherein the host cell is an insect cell.

98. The host cell of any one of claims 85-96, wherein the host cell is a mammalian cell.

99. The host cell of any one of claims 85 or 97, wherein the host cell is derived from a species of lepidoptera.

100. The host cell of claim 99, wherein the species of lepidoptera is Spodoptera frugiperda, Spodoptera littoralis, Spodoptera exigua, or Trichoplusia ni.

101. The host cell of claim 97, wherein the insect cell is Sf9.

102. The host cell of any one of claims 85-101, wherein the construct is a baculoviral construct, a viral construct, or a plasmid.

103. The host cell of any one of claims 85-102, wherein the construct is a baculoviral construct.

104. The host cell of any one of claims 85-103, wherein the expression control sequence for expression in a host cell comprises: (a) a promoter, and / or (b) a Kozak consensus sequence.Docket No.: 2017359-0072 (CAR-002.WO) 105. The host cell of any one of claims 85-104, wherein the promoter is a polyhedrin, P10, or OpiE1 promoter.

106. The host cell of any one of claims 94-105, wherein the polynucleotide comprising at least one ITR replication protein of an AAV comprises a nucleotide sequence encoding Rep52 and / or Rep78.

107. The host cell of claim 106, wherein the AAV is AAV2.

108. A method of producing a virion according to any one of claims 40-74 or a population of virions according to claim 75, comprising: (1) providing one or more of the following: (i) a first construct comprising at least one ITR nucleotide sequence, optionally further comprising a heterologous nucleic acid operably linked to a promoter for expression in a target cell, (ii) a second construct comprising a construct according to any one of claims 1-38 and / or a construct comprising a second coding sequence linked to an expression control sequence, wherein the second coding sequence encodes a parvovirus VP1 capsid polypeptide, wherein the expression control sequence comprises or is an expression control sequence for expression in a host cell, and (2) introducing the first construct, the second construct, and / or the third construct into a host cell, and (3) maintaining said host cell under conditions such that a virion according to any one of claims 40-74 or a population of virions according to claim 75 is produced.Docket No.: 2017359-0072 (CAR-002.WO) 109. The method of claim 108, further comprising (4) providing a third construct comprising: (A) at least one capsid replication protein (e.g., NS1 protein) of parvovirus operably linked to at least one expression control sequence for expression in a host cell (e.g., wherein the at least one capsid replication protein of a parvovirus enhances encapsidation, relative to encapsidation without the at least one capsid replication protein of a protoparvovirus), (B) at least one ITR replication protein of an AAV, optionally wherein the at least one ITR replication protein of an AAV comprises (a) a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in a host cell, and / or (b) a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in a host cell, or (C) a combination of (A) and (B).

110. The method of claim 109, wherein the host cell achieves a cell viability of greater than 50% (e.g., of greater than 60%, 70%, or 80%).

111. A method of producing a virion according to any one of claims 40-74 or a population of virions according to claim 75 in a host cell, the method comprising: (1) providing a host cell comprising (i) a first construct comprising at least one ITR nucleotide sequence, optionally further comprising a heterologous nucleic acid operably linked to a promoter for expression in a target cell, (ii) a second construct comprising a construct according to any one of claims 1-38 and / or a construct comprising a VP1 capsid coding sequence linked to an expression control sequence, wherein the VP1 capsid coding sequence encodes a parvovirus VP1 capsid polypeptide, wherein the expression control sequence comprises or is an expression control sequence for expression in a host cell, andDocket No.: 2017359-0072 (CAR-002.WO) (iii) a third construct comprising (A) at least one capsid replication protein (e.g., NS1) of parvovirus operably linked to at least one expression control sequence for expression in a host cell (e.g., wherein the at least one capsid replication protein of a parvovirus enhances encapsidation, relative to encapsidation without the at least one capsid replication protein of a protoparvovirus), (B) at least one ITR replication protein of an AAV, optionally wherein the at least one ITR replication protein of an AAV comprises (a) a Rep52 or a Rep40 coding sequence operably linked to at least one expression control sequence for expression in a host cell, and / or (b) a Rep78 or a Rep68 coding sequence operably linked to at least one expression control sequence for expression in a host cell, or (C) a combination of (A) and (B), optionally, a fourth construct or more, wherein at least one of (i), (ii), (iii)(A), (iii)(B), and (iii)(C) is / are stably integrated in the host cell genome, and the fourth construct or more, when present, comprises the remainder of the (i), (ii), (iii)(A), (iii)(B), and (iii)(C) nucleotide sequences which is / are not stably integrated in the host cell genome, and (2) maintaining the host cell under conditions such that a virion according to any one of claims 40-74 or a population of virions according to claim 75 is produced.

112. The method of claim 111, wherein the host cell achieves a cell viability of greater than 50% (e.g., of greater than 60%, 70%, or 80%).

113. The method of any one of claims 108-112, wherein the host cell is an insect cell.Docket No.: 2017359-0072 (CAR-002.WO) 114. The method of claim 113, wherein the insect cell is derived from a species of lepidoptera (e.g., Spodoptera frugiperda, Spodoptera littoralis, Spodoptera exigua, or Trichoplusia ni).

115. The method of any one of claims 113 or 114, wherein the insect cell is Sf9.

116. The method of any one of claims 108-112, wherein the host cell is a mammalian cell.

117. The method of any one of claims 108-116, wherein the at least one construct is a baculoviral construct, a viral construct, or a plasmid.

118. The method of any one of claims 108-117, wherein the at least one construct is a baculoviral construct.

119. The method of any one of claims 111-118, wherein the at least one ITR comprises one or more of the following: (a) a dependoparvovirus ITR, (b) a bocaparvovirus ITR (c) a protoparvovirus ITR, (d) a tetraparvovirus ITR, (e) an erthythroparvovirus ITR, or (f) a copiparvovirus ITR.

120. The method of any one of claims 111-118, wherein the at least one expression control sequence for expression in a host cell comprises:Docket No.: 2017359-0072 (CAR-002.WO) (a) a promoter, and / or (b) a Kozak consensus sequence.

121. The method of claim 120, wherein the promoter is a polyhedrin, P10, or OpiE1 promoter.

122. The method of any one of claims 108-121, wherein the polynucleotide comprising at least one ITR replication protein of an AAV comprises a nucleotide sequence encoding Rep52 and / or Rep78.

123. The method of claim 122, wherein the AAV is AAV2.

124. A method of purifying a virion according to any one of claims 40-70 or a population of virions according to claim 75, wherein the virion or the population of virions is purified using an antibody, an antigen-binding fragment of an antibody, or a nanobody that binds the virion.

125. The method of claim 124, wherein the antibody, an antigen-binding fragment of an antibody, or a nanobody binds the heterologous peptide tag in the capsid of the virion.

126. The method of claim 125, wherein the heterologous peptide tag comprises an epitope / tag selected from hemagglutinin, His (e.g., 6X-His), FLAG, E-tag, TK15, Strep-tag II, AU1, AU5, Myc, Glu-Glu, KT3, and IRS.

127. A method of preventing or treating a disease (e.g., a kidney disease, e.g., a cardiac (or heart) disease), comprising:Docket No.: 2017359-0072 (CAR-002.WO) administering (e.g., systemically) to a subject in need thereof an effective amount of virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82.

128. A method of preventing or treating a disease (e.g., a kidney disease, e.g., a cardiac (or heart) disease), comprising: (a) obtaining a plurality of cells; (b) transducing the cells with a virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82, optionally further selecting or screening for the transduced cells; and (c) administering an effective amount of the transduced cells to a subject in need thereof.

129. The method of any one of claims 127 or 128, further comprising co-administering an immune suppressant and / or a prophylactic to mitigate an immune response.

130. The method of any one of claims 127-129, wherein the subject has not been prescreened for antibodies against a virion according to any one of claims 36-70 or a population of virions according to claim 71 or a pharmaceutical composition of claim 78.

131. The method of any one of claims 127-130, wherein a virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82 selectively target a cell (e.g., a PymT tumor cell, a cervix cancer cell (e.g., a HeLa cell), a K562 cell, a Raji cell, a SKOV-3 cell, a breast cancer cell (e.g., a MCF-7 cell), a M07e cell, a human saphenous vascular endothelial cell (HSaVEC), a MT1-MMP cell, a primary hepatocyte cell (e.g., a Huh7 cell), an immune cell (e.g., a human T cell, e.g., a CD4+ T cell, e.g., a Th2 cell, e.g., a CAR T cell, e.g., a NK cell), a neuron cell (e.g., a LX-2 cell, e.g., a stellate cell e.g., a primary neuron cell, e.g., a neuroblastoma cell (e.g., a SH-SY5Y cell)), a lung cell (e.g., a lungDocket No.: 2017359-0072 (CAR-002.WO) fibroblast cell), a myoblast cell, a myotube cell, a primary cardiomyocyte, a skeletal muscle cell (e.g., a differentiated skeletal muscle cell), a human vein endothelial cell, a T84 cell, a ileum cell (intestinal), a primary human airway epithelia cell), a kidney cell (e.g., a human renal proximal tubule (HRCE) cell, e.g., a bile duct cell, e.g., an outer medullary cell, e.g., a mixed medullary cell, e.g., renal cortical epithelial cells, e.g., renal epithelial cells), a bone marrow MSC cell, a blood cell (e.g., a PBMC cell), a small intestine cell, a muscle cell, a heart cell, a spleen cell, a liver cell, a brain cell (e.g., a brain-striatum cell), an ocular cell, a testes cell, an oocyte, a medulla cell, a striatum cell, a spinal cord (or chord) cell, or a duodenum cell (e.g., wherein the one or more heterologous peptides comprises or is a heterologous targeting peptide).

132. The method of any one of claims 127-130, wherein a virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82 detargets a cell (e.g., a liver cell) relative to a virion or population of virions comprising an AAV capsid (e.g., detargets the liver about 50 times more than a virion comprising an AAV capsid, e.g., 100 times more than a virion comprising an AAV capsid, e.g., 150 times more than a virion comprising an AAV capsid. e.g., 200 times more than a virion comprising an AAV capsid, e.g., 150 times more than a virion comprising an AAV capsid, e.g., 200 times more than a virion comprising an AAV capsid, e.g., 250 times more than a virion comprising an AAV capsid, e.g., 300 times more than a virion comprising an AAV capsid).

133. A method of characterizing a virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82.

134. A method of manufacturing an intermediate (e.g., any intermediate that can be stored or shipped) of a virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82.Docket No.: 2017359-0072 (CAR-002.WO) 135. A method of providing a virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82, comprising assessing one or more characteristics of the virion or the population of virions and establishing one or more characteristics of the virion or population of virions (e.g., compared to a reference sample).

136. A system comprising a host cell according to any one of claims 85-107.

137. A method comprising contacting a cell with a construct of any one of claims 1-38.

138. A virion according to any one of claims 40-74 or a population of virions according to claim 75 or a pharmaceutical composition of claim 82 for use in the treatment of a disease or disorder.

139. Use of a construct of any one of claims 1-38 for the manufacture of a medicament to treat a disease or disorder.

140. Use of a virion of any one of claims 40-74 for the manufacture of a medicament to treat a disease or disorder.

141. Use of a population of virions of claim 75 for the manufacture of a medicament to treat a disease or disorder.

142. A kit comprising a construct of any one of claims 1-38, a parvovirus VP1 capsid polypeptide of claim 39, a virion of any one of claims 40-74, a population of virions of claim 75, a composition of any one of claims 78-83, or a host cell of any one of claims 85-107.Docket No.: 2017359-0072 (CAR-002.WO) 143. A virion of any one of claims 40-74, a population of virions according to claim 75, a method of any one of claims 127 or 128, wherein the virion or population of virions detargets the liver relative to a virion or population of virions comprising an AAV capsid (e.g., detargets the liver about 50 times more than a virion comprising an AAV capsid, e.g., 100 times more than a virion comprising an AAV capsid, e.g., 150 times more than a virion comprising an AAV capsid. e.g., 200 times more than a virion comprising an AAV capsid, e.g., 150 times more than a virion comprising an AAV capsid, e.g., 200 times more than a virion comprising an AAV capsid, e.g., 250 times more than a virion comprising an AAV capsid, e.g., 300 times more than a virion comprising an AAV capsid).

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