Engineered protein g fusogens and related lipid particles and methods thereof

Abstract

Provided herein are lipid particles, such as lenti viral particles, that incorporate or are pseudotyped with a retargeted paramyxovirus envelope attachment protein fused to a targeting moiety inserted within a peptide loop of the paramyxovirus envelope attachment protein, and in some aspects also a fusion (F) protein such as a NiV-F protein or a biologically active portion or variant thereof. The present disclosure also provides for preparation of the lipid particles, such as lentiviral particles, comprising a retargeted paramyxovirus envelope attachment protein fused to a targeting moiety inserted within a peptide loop of the paramyxovirus envelope attachment protein, as well as methods for preparing and using the lipid particles, such as lentiviral particles.

Description

This invention provides lipid particles, such as lentiviral particles, comprising or pseudotyped with retargeted paramyxoviral envelope attachment proteins, wherein the retargeted paramyxoviral envelope attachment protein is fused to a targeting portion, the targeting portion being inserted into the peptide ring of the paramyxoviral envelope attachment protein; and in some aspects, the lipid particles further comprise a fusion (F) protein, such as NiV-F protein, or a biologically active portion or variant thereof. This disclosure also provides methods for preparing said lipid particles (e.g., lentiviral particles) comprising retargeted paramyxoviral envelope attachment proteins fused to a targeting portion, wherein the targeting portion is inserted into the peptide ring of the paramyxoviral envelope attachment protein; and methods for preparing and using said lipid particles (e.g., lentiviral particles). Abstract

Claims

WHAT IS CLAIMED:

1. A lipid particle, comprising:(a) a retargeted paramyxovirus envelope attachment protein fused to a targeting moiety inserted within a peptide loop of the paramyxovirus envelope attachment protein; and(b) at least one paramyxovirus fusion (F) protein; wherein the protein in (a) and (b) are exposed on the outside of the lipid bilayer.

2. The lipid particle of claim 1 , wherein the targeting moiety is a first targeting moiety and the lipid particle comprises a second targeting moiety, wherein the second targeting moiety is inserted within a different peptide loop of the paramyxovirus envelope attachment protein, wherein the first targeting moiety and the second targeting moiety target the same or a different target cell.

3. The lipid particle of claim 1, wherein the targeting moiety is a first targeting moiety and the lipid particle comprises a second targeting moiety, wherein the second targeting moiety is attached to the C-terminus of the paramyxovirus envelope attachment protein, wherein the first targeting moiety and the second targeting moiety target the same or a different target cell.

4. The lipid particle of claim 1-3, wherein the fusion is via at least one flexible linker, optionally at least one peptide linker.

5. The lipid particle of claim 4, wherein the flexible linker is positioned at one of the N and C terminus of the targeting moiety.

6. The lipid particle of claim 4, wherein the flexible linker is positioned at each of the N and C terminus of the targeting moiety.

7. The lipid particle of any of claims 4-6, wherein the at least one linker independently comprises 4 to 12 amino acids.

8. The lipid particle of any of claims 4-7, wherein the at least one linker comprise GS, GGS, GGGGS, GGGGGS or combinations thereof.

9. The lipid particle of any of claims 4-8, wherein the at least one linker is selected from: (GGS)n, wherein n is 1 to 10; (GGGGS)n, wherein n is 1 to 10; or (GGGGGS)n, wherein n is 1 to 6.

10. The lipid particle any of claims 4-9, wherein the at least one linker is a poly-Glycine- Serine (G4S) linker, optionally wherein the linker is set forth in any one of SEQ ID NOs. 7-10.

11. The lipid particle of any of claims 1-10, wherein the paramyxovirus envelope attachment protein is an envelope attachment protein from a Nipah virus, Hendra virus, or Measles virus, or is a variant or biologically active portion thereof of any of the foregoing.

12. The lipid particle of claim 11, wherein the paramyxovirus envelope attachment protein is a variant Nipah G protein (NiV-G) that is a variant or a biologically active portion of a wild-type NiV-G.

13. The lipid particle of any of claims 1-12, wherein the peptide loop of the paramyxovirus envelope attachment protein is selected from the group consisting of:(i) amino acid residues 194-197;(ii) amino acid residues 208-213;(iii) amino acid residues 241-243;(iv) amino acid residues 242-243;(v) amino acid residues 388-391;(vi) amino acid residues 402-405;(vii) amino acid residues 489-491; and(viii) amino acid residues 495-497; optionally wherein the peptide loop is 388-391, 489-491, or 495-497.

14. The lipid particle of claim 12 or claim 13, wherein the variant NiV-G tis truncated by up to 40 contiguous amino acids at or near the N-terminus of the wild-type NiV-G set forth in SEQ ID NO:5.

15. The lipid particle of claim 12-14, wherein the variant NiV-G has a truncation of amino acids 2-34 of the wild-type NiV-G set forth in SEQ ID NO:5.

16. The lipid particle of any of claims 1-15, wherein the paramyxovirus envelope attachment protein comprises one or more mutations that reduces native tropism relative to the wild-type paramyxovirus envelope attachment protein not comprising the one or more mutations.

17. The lipid particle of any of claims 12-16, wherein the variant NiV-G exhibits reduced binding to Ephrin B2 or Ephrin B3.

18. The lipid particle of claim 17, wherein the variant NiV-G comprises: one or more amino acid substitutions corresponding to amino acid substitutions selected from the group consisting of E501A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:5.

19. The lipid particle of claim 17 or claim 18, wherein the variant NiV-G comprises amino acid substitutions E501A, W504A, Q530A and E533A with reference to numbering set forth in SEQ ID NO:5.

20. The lipid particle of any of claims 12-19, wherein the variant NiV-G has the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at or about 80%, at least at or about 81 %, at least at or about 82%, at least at or about 83%, at or about 84%, at least at or about 85%, at least at or about 86%, or at least at or about 87%, at least at or about 88%, or at least at or about 89%, at least at or about 90%, at least at or about 91 %, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO:6.

21. The lipid particle of any of claims 12-20, wherein the variant NiV-G has the amino acid sequence set forth in SEQ ID NO:6.

22. The lipid particle of any of claims 1-21, wherein the at least one paramyxovirus fusion (F) protein is a wild-type Nipah F protein (NiV-F) or is a functionally active variant or a biologically active portion thereof.

23. The lipid particle of claim 22, wherein the NiV-F protein or the biologically active portion is a truncated NiV-F that is truncated by at least 22 amino acids or at least 20 amino acids, at or near the C-terminus of wild-type NiV-F set forth in SEQ ID NO:1.

24. The lipid particle of any of claims 22 or 23, wherein the NiV-F protein or the biologically active portion thereof has a 22 amino acid truncation at or near the C-terminus of the wildtype NiV-F protein.

25. The lipid particle of any of claims 22-24, wherein the NiV-F protein comprises a deletion in it cytoplasmic tail and lacks amino acid residues 525-546 of SEQ ID NO:1.

26. The lipid particle of any of claims 22-25, wherein the NiV-F protein or the biologically active portion thereof has the sequence set forth in SEQ ID NO: 3 or 4, or a sequence of amino acids thatexhibits at least at or about 80%, 85%, 90% or 95% sequence identity to the sequence set forth in SEQ ID NO: 3 or 4.

27. The lipid particle of any of claims 1-26, wherein the paramyxovirus F protein is an F0 precursor or is a proteolytically cleaved form thereof comprising Fl and F2 subunits.

28. The lipid particle of claim 27, wherein the proteolytically cleaved form is a cathepsin E cleavage product.

29. The lipid particle of any of claims 1-28, wherein the targeting moiety is specific for a target molecule expressed on the surface of a target cell.

30. The lipid particle of any of claims 1-29, wherein the targeting moiety is selected from the group consisting of an antibody or antigen-binding fragment, an engineered binding domain, a nanobody, a DARPin, an Aptamer, an Affimer, an Affibody, a Knottin, an Avimer, a Monobody, an Anticalin, a Fynomer, and a targeting peptide.

31. The lipid particle of any of claims 1-30, wherein the targeting moiety is selected from the group consisting of a single domain antibody or a single chain variable fragment (scFv).

32. The lipid particle of claim 28 or 29, wherein the single domain antibody is a VHH.

33. The lipid particle of any of claims 29-32, wherein targeting the target molecule does not modulate or induce a signal in the target cell.

34. The lipid particle of claim 33, wherein the target molecule is a cell surface molecule is a protein, glycan, or lipid.

35. The lipid particle of any of claims 29-34, wherein the target cell is selected from the group consisting of tumor-infiltrating lymphocytes, T cells, neoplastic or tumor cells, virus-infected cells, stem cells, central nervous system (CNS) cells, hematopoietic stem cells (HSCs), and liver cells.

36. The lipid particle of any of claims 29-35, wherein the target cells is selected from the group consisting of a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a hepatocyte, a hematopoietic stem cell, a CD34+ hematopoietic stem cell, a CD105+ hematopoietic stem cell, a CD117+ hematopoietic stem cell, a CD105+ endothelial cell, a B cell, a CD20+ B cell, a CD19+ B cell, a cancer cell, aCD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, a NKG2D+ natural killer cell, a SLC1A3+ astrocyte, a SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

37. The lipid particle of any of claims 29-36, wherein the target cell is a hepatocyte.

38. The lipid particle of any of claims 29-37, wherein the target molecule is a cell surface molecule selected from the group consisting of ASGR1, ASGR2 and TM4SF5.

39. The lipid particle of any of claims 29-38, wherein the target cell is a T cell.

40. The lipid particle of claim 39, wherein the target molecule is a cell surface molecule selected from the group consisting of CD3, CD4, CD7 CD8, ASCT2, CD105, CD110, CD117, CD133, CD146, CD164, CD34, CD46, CD49f, CD90, EPCR, and ITGA3.

41. The lipid particle of claim 40, wherein the target cell is a CD4+ T cell or a CD 8+ T cell, optionally wherein the lipid particle comprises a first targeting moiety targeting a CD4+ T cell and a second targeting moiety targeting a CD 8+ T cell.

42. The lipid particle of any of claims 1-41, wherein the lipid particle comprises a viral nucleic acid.

43. The lipid particle of claim 42, wherein the viral nucleic acid comprises one or more of (e.g., all of) the following nucleic acid sequences: 5’ LTR (e.g., comprising U5 and lacking a functional U3 domain), Psi packaging element (Psi), Central polypurine tract (cPPT) / central termination sequence (CTS) (e.g. DNA flap), Poly A tail sequence, a posttranscriptional regulatory element (e.g. WPRE), a Rev response element (RRE), and 3’ LTR (e.g., comprising U5 and lacking a functional U3).

44. The lipid particle of any of claims 1-43, wherein the lipid particle is a viral vector.

45. The lipid particle of any of claims 1-44 that is a retroviral vector.

46. The lipid particle of any of claims 1-45 that is a lend viral vector.

47. The lipid particle of any of claims 1-41, wherein the lipid particle is devoid of viral genomic DNA.

48. The lipid particle of any of claims 1-41 and 47, that is a viral-like particle.

49. The lipid particle of any of claims 1-41, 47 and 48 that is a retroviral-like particle.

50. The lipid particle of any of claims 1-41, 47-49, that is a lentiviral-like particle.

51. The lipid particle of any of claims 1-50, further comprising an exogenous agent for delivery to a target cell.

52. The lipid particle of claim 51 , wherein the exogenous agent is present in the lumen.

53. The lipid particle of claim 51 or claim 52, wherein the exogenous agent is a protein or a nucleic acid, optionally wherein the nucleic acid is a DNA or RNA.

54. The lipid particle of any of claims 51-53, wherein the exogenous agent is a nucleic acid encoding a cargo for delivery to the target cell.

55. The lipid particle of any of claims 51-54, wherein the exogenous agent is or encodes a therapeutic agent, a diagnostic agent or a genome-modifying enzyme.

56. The lipid particle of any of claims 51-55, wherein the exogenous agent encodes a membrane protein, optionally wherein the membrane protein is an antigen receptor for targeting cells expressed by or associated with a disease or condition.

57. The lipid particle of claim 56, wherein the membrane protein is a chimeric antigen receptor (CAR).

58. A polynucleotide comprising a nucleic acid encoding the paramyxovirus attachment protein of the lipid particle of any of claims 1-57.

59. The polynucleotide of claim 58, wherein the nucleic acid sequence is a first nucleic acid sequence and the polynucleotide further comprises a second nucleic acid sequence encoding a paramyxovirus fusion (F) protein of the lipid particle of any of claims 1-57.

60. The polynucleotide of claim 58 or claim 59, wherein the polynucleotide comprises an IRES or a sequence encoding a linking peptide between the first and second nucleic acid sequences, optionally, wherein the linking peptide is a self-cleaving peptide or a peptide that causes ribosome skipping, optionally a T2A peptide.

61. The polynucleotide of any of claims 58-60, further comprising at least one promoter that is operatively linked to control expression of the nucleic acid, optionally expression of the first nucleic acid sequence and the second nucleic acid sequence.

62. A vector, comprising the polynucleotide of any of claims 58-61.

63. The vector of claim 62, wherein the vector is a mammalian vector, viral vector or artificial chromosome, optionally wherein the artificial chromosome is a bacterial artificial chromosome (BAC).

64. A plasmid, comprising the polynucleotide of any of claims 58-61.

65. The plasmid of claim 64, further comprising one or more nucleic acids encoding proteins for lentivirus production.

66. A cell comprising the polynucleotide of any of claims 58-61 or the vector of claim 62 or claim 63, or the plasmid of claim 64 or claim 65.

67. A method of making a lipid particle, comprising: a) providing a cell that comprises the polynucleotide of any of claims 58-61 or the vector of claim 62 or claim 63, or the plasmid of claim 64 or claim 65; b) culturing the cell under conditions that allow for production of a lipid particle, and c) separating, enriching, or purifying the targeted lipid particle from the cell, thereby making the targeted lipid particle.

68. A method of making a pseudotyped lentiviral vector, comprising: a) providing a producer cell that comprises a lentiviral viral nucleic acid(s), and the polynucleotide of any of claims 58-61 or the vector of claim 62 or claim 63, or the plasmid of claim 64 or claim 65; b) culturing the cell under conditions that allow for production of the lentiviral vector, andc) separating, enriching, or purifying the lentiviral vector from the cell, thereby making the pseudotyped lentiviral vector.

69. A producer cell comprising the polynucleotide of any of claims 58-61 or the vector of claim 62 or claim 63, or the plasmid of claim 64 or claim 65.

70. The producer cell of claim 69, further comprising nucleic acid encoding a paramyxovirus F protein or a biologically active portion thereof.

71. The producer cell of claim 69 or claim 70, wherein the cell further comprises a viral nucleic acid, optionally wherein the viral nucleic acid is a lentiviral nucleic acid.

72. A lipid particle produced by the method of claim 67 or claim 68 or from the producer cell of any of claims 69-71.

73. A composition comprising a plurality of lipid particles of any of claims 1-57 and 72.

74. The composition of claim 73 further comprising a pharmaceutically acceptable carrier.

75. A method of transducing a cell comprising contacting a cell with a lipid particle of any of claims 1-57 and 72 or a composition comprising a lipid particle of claim 73 or claim 74.

76. A method of delivering an exogenous agent to a subject (e.g., a human subject), the method comprising administering to the subject the lipid particle of any of claims 1-57 and 72 or a composition of claim 73 or claim 74, wherein the lipid particle or lentiviral vector comprise the exogenous agent.

77. A method of delivering an exogenous agent to a target cell, the method contacting a target cell with the lipid particle of any of claims 1-57 and 72 or a composition of claim 73 or claim 74, wherein the lipid particle or lentiviral vector comprise the exogenous agent.

78. The method of claim 77, wherein the contacting transduces the cell with lentiviral vector or the lipid particle.

79. The method of claim 77 or claim 78, wherein the contacting is in vivo in a subject.

80. The method of any of claims 76-79, wherein the method treats a disease or condition in the subject by the delivery of the exogenous agent.

81. A method of treating a disease or disorder in a subject (e.g., a human subject), the method comprising administering to the subject a lipid particle of any of claims 1-57 and 72 or the composition of claim 73 or claim 74.

82. A paramyxovirus envelope attachment protein comprising a targeting moiety inserted into a loop region of the paramyxovirus envelope attachment protein.

83. The paramyxovirus envelope attachment protein of claim 82, wherein the loop region is selected from the group consisting of:(i) amino acid residues 194-197;(ii) amino acid residues 208-213;(iii) amino acid residues 241-243;(iv) amino acid residues 242-243;(v) amino acid residues 388-391;(vi) amino acid residues 402-405;(vii) amino acid residues 489-491; and(viii) amino acid residues 495-497; optionally wherein the peptide loop is 388-391, 489-491, or 495-497.

84. The paramyxovirus envelope attachment protein of claim 82 or claim 83, wherein the targeting moiety is selected from the group consisting of an antibody or antigen-binding fragment, an engineered binding domain, a nanobody, a DARPin, an Aptamer, an Affimer, an Affibody, a Knottin, an Avimer, a Monobody, an Anticalin, a Fynomer, and a targeting peptide.

85. The paramyxovirus envelope attachment protein of any of claims 82-84, wherein the paramyxovirus envelope attachment protein is from Nipah.

86. The paramyxovirus envelope attachment protein of claim 85, wherein the paramyxovirus envelope attachment protein is a NiV-G or is a biologically active variant or truncated form thereof.

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