Methods and compositions for producing viral fusosomes

Abstract

To provide methods and compositions for producing fusosomes.SOLUTION: In some embodiments, a producer cell for producing fusosomes comprises an exogenous or overexpressed cathepsin molecule. In some embodiments, these cells generate an increased level of active fusogen, leading to a higher proportion of fusogenically active fusosomes. The present disclosure provides, at least in part, methods of making fusosomes that can be used for in vivo delivery. In some embodiments, the method comprises the step of expressing a cathepsin molecule in a producer cell, in order to increase levels of functional fusosomes produced by the cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 048,524, filed July 6, 2020, the contents of which are incorporated by reference in their entirety for all purposes.

[0002] Incorporation by reference of sequence listing This application is filed with an electronic Sequence Listing, which is provided as a file entitled 18615_2003540_SeqList.TXT, which is 97,896 bytes in size and was created on July 1, 2020. The information in the electronic format of this Sequence Listing is incorporated by reference in its entirety. [Background technology]

[0003] Complex biological agents are promising therapeutic candidates for various diseases. However, because the plasma membrane acts as a barrier between the cell and the extracellular space, it is difficult to deliver large biological agents into cells. There is a need in the art for new methods for delivering complex biological agents into target cells. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides, at least in part, methods for producing fusosomes that can be used for in vivo delivery. In some embodiments, the methods comprise expressing a cathepsin molecule in a producer cell to increase the level of functional fusosomes produced by the cell.

[0005] Enumeration of Embodiments The following embodiments are provided: 1. A method for producing a plurality of fusosomes, comprising: (a) providing a modified mammalian producer cell, e.g., a human cell, comprising: (i) a mature cathepsin molecule (e.g., cathepsin L or cathepsin B) that has increased levels or activity compared to a corresponding unmodified cell; (ii) optionally, an exogenous cargo molecule, e.g., a protein or nucleic acid, and (iii) a henipavirus F protein molecule, and (iv) Henipavirus G protein molecule; (b) maintaining (e.g., culturing) the modified mammalian cells under conditions that allow the production of a plurality of fusosomes comprising the Henipavirus F protein molecule and the Henipavirus G protein molecule.

[0006] 2. The method of embodiment 1, wherein the cargo molecule comprises a viral nucleic acid (e.g., a lentiviral nucleic acid).

[0007] 3. The method of embodiment 1, wherein the modified cell has been introduced with the exogenous cargo molecule (e.g., a nucleic acid encoding the exogenous cargo molecule has been introduced into the modified cell).

[0008] 4. The method of any of the preceding embodiments, wherein a cathepsin molecule or a nucleic acid encoding said cathepsin molecule has been introduced into said modified cell, e.g., under conditions for processing said cathepsin molecule into said mature cathepsin.

[0009] 5. The method of any of the preceding embodiments, wherein a cathepsin molecule or a nucleic acid encoding said cathepsin molecule has been introduced into said modified cell under conditions suitable for expression of said cathepsin molecule.

[0010] 6. A method for producing a modified mammalian producer cell, comprising: (i) introducing into a mammalian cell a nucleic acid molecule encoding a cathepsin molecule under conditions that increase expression of the mature form of the cathepsin molecule in the mammalian cell; (ii) optionally introducing an exogenous cargo molecule, e.g., a protein or nucleic acid, into said mammalian cell; (iii) introducing a Henipavirus F protein molecule into the mammalian cell (e.g., introducing a nucleic acid encoding the Henipavirus F protein molecule under conditions suitable for expression of the Henipavirus F protein molecule), and (iv) introducing a Henipavirus G protein molecule into the mammalian cell (e.g., introducing a nucleic acid encoding the Henipavirus G protein molecule under conditions suitable for expression of the Henipavirus G protein molecule), The method, wherein steps (i) to (iv) may be performed in any order, or one or more of steps (i) to (iv) may be performed simultaneously.

[0011] 7. A method of producing a plurality of fusosomes, comprising maintaining (e.g., culturing) the modified mammalian cells produced in embodiment 3a under conditions that allow the production of a plurality of fusosomes comprising said Henipavirus F protein molecule and said Henipavirus G protein molecule.

[0012] 8. The method of any preceding embodiment, further comprising separating at least one of said plurality of fusosomes from said modified cell.

[0013] 9. The method of any preceding embodiment, further comprising: a) assaying one or more fusosomes from said produced plurality to determine whether one or more (e.g., two, three, or more) criteria are met, said criterion(s) being selected from the following: i) in said fusosomes, at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein, or 1:2, 3:5, 7:10, 4:5, 9:10, or 1:1 1:1; ii) the fusosomes have a functional titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., in 293LX cells, as measured by detection of a GFP reporter in 293LX cells, e.g., in the assay of Example 1; iii) the fusosomes have a functional titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., in activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., as measured by detection of a GFP reporter in the activated T cells, e.g., in the assay of Example 3; iv) the plurality of fusosomes produced have a titer against target cells to non-target cells ratio of at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 500:1, 1000:1, 5000:1, 10,000:1, 50,000:1, or 100,000:1, e.g., the target cells overexpress a protein bound by the Henipavirus G protein molecule and the non-target cells are wild-type, e.g., the target cells overexpress CD8, e.g., in the assay of Example 1, and the non-target cells are wild-type; v) the fusosomes contain active Henipavirus F protein molecules at a level that is at least 10%, 20%, 30%, 40%, or 50% greater than the level of active Henipavirus F protein molecules in otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules. b) (optionally) approving the release of said produced plurality of fusosomes or fusosome compositions if one or more of said criteria are met.

[0014] 10. The method of any preceding embodiment, wherein said plurality of fusosomes have one, two, three, four, five, six, or all seven of the following characteristics: i) in said fusosomes, at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein; ii) the fusosomes have a functional titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1; iii) the fusosomes have a functional titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., in activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., as measured by detection of a GFP reporter in the activated T cells, e.g., in the assay of Example 3; iv) the plurality of fusosomes produced have a titer against target cells to non-target cells ratio of at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 500:1, 1000:1, 5000:1, 10,000:1, 50,000:1, or 100,000:1, e.g., the target cells overexpress a protein bound by the Henipavirus G protein molecule and the non-target cells are wild-type, e.g., the target cells overexpress CD8, e.g., in the assay of Example 1, and the non-target cells are wild-type; v) the fusosomes contain active Henipavirus F protein molecules at a level that is at least 10%, 20%, 30%, 40%, or 50% greater than the level of active Henipavirus F protein molecules in otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules.

[0015] 11. A modified cell produced by the method of embodiment 6.

[0016] 12. Modified mammalian cells, e.g., human cells, including: (i) a mature cathepsin molecule (e.g., cathepsin L or cathepsin B) that has increased levels or activity compared to a corresponding unmodified cell; (ii) optionally, an exogenous cargo molecule, e.g., a nucleic acid or protein, e.g., a viral nucleic acid, e.g., a lentiviral nucleic acid, and (iii) a henipavirus F protein molecule, and (iv) optionally, a Henipavirus G protein molecule.

[0017] 13. Modified mammalian cells, e.g., human cells, including: (i) optionally, an exogenous cargo molecule, e.g., a nucleic acid or protein, e.g., a viral nucleic acid, e.g., a lentiviral nucleic acid, and (ii) Henipavirus F protein molecules, wherein in the cell, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the Henipavirus F protein molecules are active Henipavirus F protein; and (iii) optionally, a Henipavirus G protein molecule.

[0018] 14. Fusosomes, which contain: (a) optionally, an exogenous cargo, e.g., a nucleic acid or protein, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) an activated henipavirus F protein molecule comprising a modified F1 form having a C-terminal truncation of up to 30 consecutive amino acids compared to a wild-type henipavirus protein F1 molecule, wherein at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the henipavirus F protein molecules in the fusosome are activated henipavirus F protein molecules; and (c) Henipavirus G protein molecule.

[0019] 15. The fusosome of embodiment 14, wherein the modified F1 form has a C-terminal truncation of 10 to 30, 15 to 30, 10 to 20, or 20 to 30 consecutive amino acids, such as 22 or 25 amino acids, compared to the wild-type henipavirus F1 protein.

[0020] 16. Fusosomes, which contain: (a) optionally, an exogenous cargo, e.g., a nucleic acid or protein, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) Henipavirus F protein molecules in said fusosomes, wherein at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein; and (c) Henipavirus G protein molecule.

[0021] 17. A fusosome described in any of embodiments 14 to 16, wherein the Henipavirus F protein molecule lacks an endocytic motif.

[0022] 18. The fusosome of embodiment 17, wherein the endocytic motif is a YXXφ motif.

[0023] 19. The fusosome of embodiment 17 or 18, wherein the endocytic motif is a YSRL motif.

[0024] 20. Fusosomes containing: (a) optionally, an exogenous cargo, e.g., a nucleic acid or protein, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) Henipavirus F protein molecules in said fusosomes, wherein at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein; and (c) Henipavirus G protein molecule, wherein the Henipavirus F protein molecule lacks an endocytic motif, e.g., a YXXφ motif, e.g., a YRSL motif.

[0025] 21. A fusosome according to embodiment 20, comprising a modified F1 form having a C-terminal truncation of up to 30 consecutive amino acids compared to the wild-type Henipavirus protein F1 molecule.

[0026] 22. The fusosome of embodiment 21, wherein the Henipavirus F protein molecule comprises a truncation of 10 to 30, 15 to 30, 10 to 20, or 20 to 30 amino acids, e.g., 22 or 25 amino acids, at the C-terminus compared to a wild-type Henipavirus F protein, e.g., compared to SEQ ID NO: 7.

[0027] 23. Fusosomes, which contain: (a) optionally, an exogenous cargo, e.g., a fusomal nucleic acid, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) Henipavirus F protein molecules in said fusosomes, wherein at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein; and (c) Henipavirus G protein molecule.

[0028] 24. A pharmaceutical composition comprising a fusosome according to any one of embodiments 14 to 23, and optionally a pharmaceutically acceptable excipient.

[0029] 25. A pharmaceutical composition comprising a plurality of fusosomes, wherein the fusosomes are (a) optionally, an exogenous cargo, e.g., a fusomal nucleic acid, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) a henipavirus F protein molecule, and (c) containing a henipavirus G protein molecule; The pharmaceutical composition has a titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1.

[0030] 26. A pharmaceutical composition comprising a plurality of fusosomes, wherein the fusosomes are (a) optionally, an exogenous cargo, e.g., a fusomal nucleic acid, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) a henipavirus F protein molecule comprising a modified F1 form having a C-terminal truncation of up to 30 consecutive amino acids compared to a wild-type henipavirus protein F1 molecule, or a henipavirus F protein molecule lacking an endocytic motif (e.g., a YXXφ motif, e.g., a YRSL motif); and (c) containing a henipavirus G protein molecule; The pharmaceutical composition has a titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1.

[0031] 27. A pharmaceutical composition comprising a plurality of fusosomes, wherein the fusosomes are (a) optionally, an exogenous cargo, e.g., a fusomal nucleic acid, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) a henipavirus F protein molecule, and (c) containing a henipavirus G protein molecule; The pharmaceutical composition has a titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., in target cells, e.g., activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., as measured by detection of a GFP reporter in activated T cells, e.g., in the assay of Example 3.

[0032] 28. A method for producing a pharmaceutical composition comprising a plurality of fusosomes, the method comprising: a) providing, e.g., producing, a plurality of fusosomes according to any one of embodiments 14 to 23, a pharmaceutical composition according to any one of embodiments 24 to 27, or fusosomes produced by the method according to any one of embodiments 1 to 10; b) assaying one or more fusosomes from said plurality to determine whether one or more (e.g., two, three, or more) criteria are met, wherein said criterion(s) are selected from the following: i) in said fusosomes, at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein; ii) the pharmaceutical composition has a titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1; iii) the pharmaceutical composition has a titer in activated T cells, e.g., primary T cells, e.g., Pan-T cells, of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured in activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., by detection of a GFP reporter in the T cells, e.g., as measured in the assay of Example 3; iv) the plurality of fusosomes have a titer on target cells to titer on non-target cells ratio of at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 500:1, 1000:1, 5000:1, 10,000:1, 50,000:1, or 100,000:1, e.g., the target cells overexpress a protein bound by the Henipavirus G protein molecule and are the non-target cells are wild-type, e.g., the target cells overexpress CD8, e.g., in the assay of Example 1, and the non-target cells are wild-type; v) the fusosomes contain active Henipavirus F protein molecules at a level that is at least 10%, 20%, 30%, 40%, or 50% greater than the level of active Henipavirus F protein molecules in otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules. c) (optionally) approving the release of said plurality of fusosomes or pharmaceutical composition if one or more of said criteria are met.

[0033] 29. A reaction mixture comprising: a) a plurality of target cells (e.g., human cells, e.g., primary human cells, e.g., cells derived from a subject), and b) A plurality of fusosomes according to any one of embodiments 14 to 23, a pharmaceutical composition according to any one of embodiments 24 to 27, or fusosomes prepared by the method according to any one of embodiments 1 to 10.

[0034] 30. Target cells (e.g., human cells, e.g., primary human cells, e.g., cells derived from a subject), including: a) an exogenous cargo molecule (e.g., an s protein or nucleic acid, e.g., a viral nucleic acid, e.g., a lentiviral nucleic acid), and b) Henipavirus F protein molecules, wherein in said target cell, at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules are active Henipavirus F protein; and c) Henipavirus G protein molecule.

[0035] 31. A method for delivering exogenous cargo (e.g., fusomal nucleic acid, e.g., viral nucleic acid, e.g., lentiviral nucleic acid) to a cell (e.g., in vivo or ex vivo), comprising contacting the cell with a plurality of fusosomes described in any of embodiments 14 to 23, the pharmaceutical composition described in any of embodiments 24 to 27, or fusosomes produced by the method of any of embodiments 1 to 10.

[0036] 32. A method for delivering exogenous cargo (e.g., fusomal nucleic acid, e.g., viral nucleic acid, e.g., lentiviral nucleic acid) to a subject, comprising administering to the subject an effective number of fusosomes described in any of embodiments 14-23, the pharmaceutical composition described in any of embodiments 24-27, or fusosomes produced by the method of any of embodiments 1-10.

[0037] 33. The pharmaceutical composition of any of embodiments 24-27, wherein the plurality of fusosomes have a titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1.

[0038] 34. The pharmaceutical composition of any of embodiments 24-27, wherein the plurality of fusosomes have a titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., in activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., as measured by detection of a GFP reporter in the T cells, e.g., in the assay of Example 3.

[0039] 35. The pharmaceutical composition of any of embodiments 24-27, wherein the plurality of fusosomes have a ratio of titer on target cells to titer on non-target cells of at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 500:1, 1000:1, 5000:1, 10,000:1, 50,000:1, or 100,000:1, e.g., the target cells overexpress a protein bound by the Henipavirus G protein molecule and the non-target cells are wild-type, e.g., the target cells overexpress CD8 in the assay of Example 1 and the non-target cells are wild-type.

[0040] 36. The pharmaceutical composition of any of embodiments 24-27, wherein the plurality of fusosomes contain active Henipavirus F protein molecules at a level that is at least 10%, 20%, 30%, 40%, or 50% greater than otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules.

[0041] 37. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein the cathepsin molecule comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, or a sequence having at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity thereto.

[0042] 38. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein the cathepsin molecule comprises a fusion or chimera.

[0043] 39. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said modified cell comprises at least 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 copies of exogenous cathepsin molecules.

[0044] 40. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said modified cell comprises at least 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 50,000, 100,000, 500,000, or 1,000,000 copies of total cathepsin L molecules.

[0045] 41. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein the elevated level of cathepsin molecules comprises at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, or 100,000-fold or more cathepsin molecules than the amount of endogenous cathepsin L in a corresponding unmodified cell.

[0046] 42. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the increased activity of said cathepsin molecules comprises cathepsin molecule activity per cell that is at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, or 100,000-fold or more greater than the activity of cathepsin molecules in a corresponding unmodified cell, e.g., as measured in the Diederich et al. 2012 assay.

[0047] 43. The method of making or modified cell of any preceding embodiment, wherein some or all of the cathepsin molecules are located in lysosomes and / or endosomes of the modified cell.

[0048] 44. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the fusosomes contain active Henipavirus F protein molecules at levels that are at least 10%, 20%, 30%, 40%, or 50% greater than otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules.

[0049] 45. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein at least 33%, 35%, 40%, 45%, 50%, 55%, or 60% of the Henipavirus F protein molecules in said fusosomes are active Henipavirus F protein.

[0050] 46. ​​The method, modified cell, or pharmaceutical composition of any preceding embodiment, wherein the fusosomes have a functional titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1.

[0051] 47. The method, modified cell, or pharmaceutical composition of any preceding embodiment, wherein the fusosomes have a functional titer of at least about 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 TU / mL, e.g., in activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., as measured by detection of a GFP reporter in the activated T cells, e.g., in the assay of Example 3.

[0052] 48. The method, modified cell, or pharmaceutical composition of any preceding embodiment, wherein the plurality of fusosomes produced have a titer on target cells to titer on non-target cells ratio of at least 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 500:1, 1000:1, 5000:1, 10,000:1, 50,000:1, or 100,000:1, e.g., the target cells overexpress a protein bound by the Henipavirus G protein molecule and the non-target cells are wild-type, e.g., the target cells overexpress CD8, e.g., in the assay of Example 1, and the non-target cells are wild-type.

[0053] 49. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the fusosomes comprise a level of total Henipavirus protein F that is 70% to 130%, 80% to 120%, 90% to 110%, 95% to 105%, or about 100% of the level of total Henipavirus protein F comprised in otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules.

[0054] 50. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said Henipavirus F protein molecule comprises a Nipah virus or Hendra virus protein F sequence.

[0055] 51. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus F protein molecule comprises the amino acid sequence of a wild-type Nipah virus of SEQ ID NO: 7, or a sequence having at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity thereto.

[0056] 52. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said Henipavirus F protein molecule comprises a Henipavirus protein F of Table 4.

[0057] 53. The method, engineered cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus F protein molecule comprises a truncation of 10 to 30, 15 to 30, 10 to 20, or 20 to 30 amino acids, e.g., 22 or 25 amino acids, at the C-terminus compared to a wild-type Henipavirus F protein, e.g., a protein in Table 4.

[0058] 54. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus F protein molecule lacks an endocytic motif, e.g., a YXXφ motif, e.g., a YRSL motif.

[0059] 55. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said Henipavirus F protein molecule comprises a Nipah virus or Hendra virus protein F sequence.

[0060] 56. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus G protein molecule comprises the amino acid sequence of a wild-type Nipah virus of SEQ ID NO: 9, or a sequence having at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity thereto.

[0061] 57. The method, engineered cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus G protein molecule comprises a truncation of 10 to 50, 10 to 40, 20 to 50, 20 to 40, 20 to 30, 30 to 50, or 30 to 40 amino acids, e.g., 34 amino acids, at the N-terminus compared to a wild-type Henipavirus G protein, e.g., a protein in Table 5.

[0062] 58. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus G protein molecule comprises one or more mutations (e.g., at least 1, 2, 3, 4, 5, 6, or 7 mutations) at a glycosylation site, e.g., an N-linked glycosylation site, e.g., an N-linked glycosylation site in the extracellular domain, e.g., at the G1, G2, G3, G4, G5, G6, and / or G7 sites described in Biering et al. (2012) J.Virol. 86(22):11991-12002.

[0063] 59. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus F protein molecule comprises one or more mutations (e.g., at least 1, 2, 3, or 4 mutations) at a glycosylation site, e.g., an N-linked glycosylation site, e.g., at the F2 (e.g., N67), F3 (e.g., N99), F4 (e.g., N414), and / or F5 (e.g., N464) sites described in Lee et al. (2011) Trends Microbiol. 19(8):389-399.

[0064] 60. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein said Henipavirus G protein molecule is a retargeted Henipavirus G protein molecule.

[0065] 61. The method, engineered cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the Henipavirus G protein molecule has a reduced affinity for ephrin B2 and / or ephrin B3 compared to a wild-type Henipavirus G protein, e.g., wherein the Henipavirus G protein molecule comprises a mutation (e.g., a mutation to alanine) at one or more of E501, W504, Q530, and E533.

[0066] 62. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said Henipavirus G protein molecule further comprises a targeting domain that is exogenous to a wild-type Henipavirus G protein.

[0067] 63. The method, modified cell, fusosome, or pharmaceutical composition of embodiment 62, wherein the targeting domain comprises an antibody molecule.

[0068] 64. The method, modified cell, fusosome, or pharmaceutical composition according to embodiment 62 or 63, wherein the targeting domain binds to CD8, CD105, EpCAM, or Gria4.

[0069] 65. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein said fusomal nucleic acid comprises at least one, e.g., at least two, plasmids.

[0070] 66. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said fusomal nucleic acid is not a henipavirus nucleic acid and does not comprise a henipavirus gene.

[0071] 67. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said fusomal nucleic acid is not a Hendra virus nucleic acid and does not comprise a Hendra virus gene.

[0072] 68. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein said fusomal nucleic acid is a lentiviral nucleic acid.

[0073] 69. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein said fusosomal nucleic acid encodes a therapeutic payload.

[0074] 70. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein said modified cell is a human cell.

[0075] 71. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein said modified cell, fusosome, or pharmaceutical composition is produced in accordance with GMP standards.

[0076] 72. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said modified cell is a canine cell, a primate (e.g., a non-human primate, e.g., an African green monkey) cell, or a murine cell.

[0077] 73. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein the modified cell is a kidney cell or an epithelial cell (e.g., a kidney epithelial cell).

[0078] 74. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein said modified cell is other than an epithelial cell.

[0079] 75. The method, modified cell, fusosome, or pharmaceutical composition of any preceding embodiment, wherein the modified cell comprises the Henipavirus F protein molecule in one or more of the endosome, lysosome, or cell membrane.

[0080] 76. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding embodiments, wherein the modified cell comprises the cathepsin molecule in one or more of the endosome, lysosome, or cell membrane.

[0081] 77. Fusosomes containing: a) a lipid bilayer comprising a fusogen (e.g., a henipavirus fusogen, e.g., a henipavirus protein G molecule) retargeted to bind to CD105, and b) A lumen containing nucleic acid, e.g., fusomal nucleic acid, e.g., lentiviral nucleic acid.

[0082] 78. Fusosomes containing: a) a lipid bilayer comprising a fusogen (e.g., a henipavirus fusogen, e.g., a henipavirus protein G molecule) retargeted to bind to EpCAM; and b) A lumen containing nucleic acid, e.g., fusomal nucleic acid, e.g., lentiviral nucleic acid.

[0083] 79. Fusosomes containing: a) a lipid bilayer comprising a fusogen (e.g., a henipavirus fusogen, e.g., a henipavirus protein G molecule) retargeted to bind Gria4; and b) A lumen containing nucleic acid, e.g., fusomal nucleic acid, e.g., lentiviral nucleic acid.

[0084] 80. The fusosome of any of the preceding embodiments, which is one or more of the following: i) the fusosomes fuse with target cells at, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher rates than non-target cells; ii) the fusosome fuses with a target cell at a rate that is, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater than another fusosome; iii) the fusosomes fuse with target cells at a rate such that the agent contained in the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target cells after 24, 48, or 72 hours; iv) the fusosomes deliver the nucleic acid to target cells at a rate that is, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater than that of non-target cells; v) the fusosome delivers the nucleic acid to a target cell at a rate that is, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold greater than another fusosome; or vi) the fusosomes deliver the nucleic acid to target cells at a rate such that the agent contained in the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells after 24, 48, or 72 hours.

[0085] 81. The fusosome of any of the preceding embodiments, wherein the nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, e.g., a nucleic acid encoding an exogenous agent, a payload gene, e.g., a nucleic acid encoding the exogenous agent (optionally including an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., comprising a U5 and lacking a functional U3).

[0086] 82. The fusosome of any of the preceding embodiments, comprising one or more (e.g., all) of a polymerase (e.g., a reverse transcriptase, e.g., pol or a portion thereof), an integrase (e.g., pol or a portion thereof, e.g., a functional or non-functional variant), a matrix protein (e.g., gag or a portion thereof), a capsid protein (e.g., gag or a portion thereof), a nucleocapsid protein (e.g., gag or a portion thereof), and a protease (e.g., pro).

[0087] 83. The fusosome of any of the preceding embodiments, wherein the fusosome, when administered to a subject, is one or more of the following: i) less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent detectably present in the subject is contained in non-target cells; ii) at least 90%, 95%, 96%, 97%, 98%, or 99% of the cells of the subject that detectably contain the exogenous agent are target cells (e.g., cells of a single cell type, e.g., T cells); iii) fewer than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 cells of the subject detectably containing the exogenous agent are non-target cells; or iv) the average level of the exogenous agent in all target cells of the subject is at least 100-fold, 200-fold, 500-fold, or 1,000-fold greater than the average level of the exogenous agent in all non-target cells of the subject; or v) the exogenous agent is not detectable in any non-target cells of the subject.

[0088] 84. The fusosome of any of the preceding embodiments, wherein said retargeting fusogen comprises sequences selected from Nipah virus F and G proteins, measles virus F and H proteins, tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0089] 85. The fusosome of any of the preceding embodiments, wherein the fusogen comprises a domain of at least 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids in length having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a wild-type paramyxovirus fusogen, e.g., a sequence in Table 4 or Table 5.

[0090] 86. The fusosome of embodiment 85, wherein the paramyxovirus is a Nipah virus, for example a Henipa virus.

[0091] 87. The fusosome of any of the previous embodiments, wherein the target cells are cancer cells and the non-target cells are non-cancerous cells.

[0092] 88. The fusosome of any of the preceding embodiments, which does not deliver the nucleic acid to a non-target cell, such as an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, an atypical antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancerous cell.

[0093] 89. The fusosome of any of the preceding embodiments, wherein less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% of a non-target cell type (e.g., one or more of antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) comprise said nucleic acid, e.g., retroviral nucleic acid, e.g., using quantitative PCR.

[0094] 90. The fusosome of any of the preceding embodiments, wherein the target cell comprises 0.00001-10, .0001-10, .001-10, .01-10, .1-10, .5-5, 1-4, 1-3, or 1-2 copies of the nucleic acid, e.g., a retroviral nucleic acid or portion thereof, per host cell genome, e.g., the copy number of the nucleic acid is assessed in vivo after administration.

[0095] 91. The fusosome of any of the preceding embodiments, The fusosomes wherein less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01% of non-target cells (e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) comprise the exogenous agent, or the exogenous agent (e.g., a protein) is not detectably present in non-target cells, e.g., antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0096] 92. The fusosome of any of the preceding embodiments, wherein the fusosome delivers the nucleic acid, e.g., a retroviral nucleic acid, to a target cell, e.g., a T cell, 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, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, an NKG2D+ natural killer cell, an SLC1A3+ astrocyte, an SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0097] 93. For example, quantitative PCR can be used to identify target cells (e.g., T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells, B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, Glu 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes (one or more of: SLC1A3+ neurons, NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells) comprise the nucleic acid.

[0098] 94. Target cells (e.g., T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, hepatocytes, hematopoietic stem cells, CD34+ hematopoietic stem cells, CD105+ hematopoietic stem cells, CD117+ hematopoietic stem cells, CD105+ endothelial cells) , B cells, CD20+ B cells, CD19+ B cells, cancer cells, CD133+ cancer cells, EpCAM+ cancer cells, CD19+ cancer cells, Her2 / Neu+ cancer cells, GluA2+ neurons, GluA4+ neurons 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes (e.g., NKG2D+ natural killer cells, SLC1A3+ astrocytes, SLC7A10+ adipocytes, or CD30+ lung epithelial cells) comprise the exogenous agent.

[0099] 95. The fusosome of any of the preceding embodiments, wherein after administration, the ratio of target cells containing said nucleic acid to non-target cells containing said nucleic acid is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a quantitative PCR assay.

[0100] 96. The fusosome of any of the preceding embodiments, wherein the ratio of the average copy number of the nucleic acid or a portion thereof contained in target cells to the average copy number of the nucleic acid or a portion thereof contained in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a quantitative PCR assay.

[0101] 97. The fusosome of any of the preceding embodiments, wherein the ratio of the median copy number of the nucleic acid or portion thereof contained in target cells to the median copy number of the nucleic acid or portion thereof contained in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a quantitative PCR assay.

[0102] 98. The fusosome of any of the preceding embodiments, wherein the ratio of target cells comprising the exogenous RNA agent to non-target cells comprising the exogenous RNA agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a reverse transcription quantitative PCR assay.

[0103] 99. The fusosome of any of the preceding embodiments, wherein the ratio of the average level of the exogenous RNA agent in the target cells to the average level of the exogenous RNA agent in the non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a reverse transcription quantitative PCR assay.

[0104] 100. The fusosome of any of the preceding embodiments, wherein the ratio of the median level of the exogenous RNA agent in said target cells to the median level of the exogenous RNA agent in said non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a reverse transcription quantitative PCR assay.

[0105] 101. The fusosome of any of the preceding embodiments, wherein the ratio of target cells comprising the exogenous protein agent to non-target cells comprising the exogenous protein agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a FACS assay.

[0106] 102. The fusosome of any of the preceding embodiments, wherein the ratio of the average level of the exogenous protein agent in the target cells to the average level of the exogenous protein agent in the non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a FACS assay.

[0107] 103. The fusosome of any of the preceding embodiments, wherein the ratio of the median level of the exogenous protein agent in the target cells to the median level of the exogenous protein agent in the non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., according to a FACS assay.

[0108] 104. The fusosome of any of the preceding embodiments, comprising one or both of the following: i) exogenous or overexpressed immunosuppressive proteins on the lipid bilayer, e.g., envelope, and ii) immunostimulatory proteins that are absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to fusosomes produced from an otherwise unmodified, similar cell of origin.

[0109] 105. The fusosome of any of the preceding embodiments, comprising one or more of the following: i) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer, e.g., envelope, and a second exogenous or overexpressed immunosuppressive protein on the lipid bilayer, e.g., envelope; ii) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer, e.g., envelope, and a second immunostimulatory protein that is absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to fusosomes produced from an otherwise unmodified, similar cell of origin; or iii) a first immunostimulatory protein that is absent or present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to fusosomes produced from an otherwise similar cell of origin, and a second immunostimulatory protein that is absent or present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to fusosomes produced from an otherwise similar cell of origin, but unmodified.

[0110] 106. The fusosome of any of the previous embodiments, wherein the nucleic acid comprises one or more insulator sequences.

[0111] 107. The fusosome of any of the preceding embodiments, which, when administered to a subject (e.g., a human subject or a mouse), is one or more of the following: i) the fusosomes do not elicit a detectable antibody response (e.g., after a single or multiple doses), or antibodies to the fusosomes are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by a FACS antibody detection assay; ii) the fusosomes do not elicit a detectable cellular immune response (e.g., a T cell response, an NK cell response, or a macrophage response), or a cellular immune response against the fusosomes is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by a PBMC lysis assay, an NK cell lysis assay, a CD8 killer T cell lysis assay, or a macrophage phagocytosis assay; iii) the fusosomes do not elicit a detectable innate immune response, e.g., complement activation (e.g., after a single dose or multiple doses), or a innate immune response against the fusosomes is present at a level less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by a complement titer assay; iv) less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.002%, or 0.001% of the fusosomes are inactivated by serum, e.g., by a serum inactivation assay; v) target cells that receive the exogenous agent from the fusosomes do not develop a detectable antibody response (e.g., after a single or multiple doses), or antibodies to the target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by a FACS antibody detection assay; or vi) target cells that receive the exogenous agent from the fusosomes do not mount a detectable cellular immune response (e.g., a T cell response, an NK cell response, or a macrophage response), or there is a cellular response against the target cells at a level that is less than 10%, 5%, 4%, 3%, 2%, or 1% above background levels, e.g., by macrophage phagocytosis assay, PBMC lysis assay, NK cell lysis assay, or CD8 killer T cell lysis assay.

[0112] 108. The fusosome of any of the preceding embodiments, wherein one or more (e.g., two or all three) of the following apply: the fusosome is a retroviral vector, the lipid bilayer is contained in an envelope, e.g., a viral envelope, and the nucleic acid is a retroviral nucleic acid.

[0113] 109. The fusosome of embodiment 107 or 108, wherein the background level is the corresponding level in the same subject before administration of the particle or vector.

[0114] 110. The fusosome according to any of embodiments 107 to 109, wherein the immunosuppressive protein is a complement regulatory protein or CD47.

[0115] 111. The fusosome of any of embodiments 107-110, wherein the immunostimulatory protein is an MHC (e.g., HLA) protein.

[0116] 112. The fusosome of any of embodiments 107-111, wherein one or both of the following are true: the first exogenous or overexpressed immunosuppressive protein is other than CD47, and the second immunostimulatory protein is other than MHC.

[0117] 113. The fusosome of any of the preceding embodiments, wherein MHC I (e.g., HLA-A, HLA-B, or HLA-C) or MHC II (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR) is absent from said fusosomes or is present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to fusosomes produced from an otherwise similar, unmodified cell of origin.

[0118] 114. The fusosome of any of the preceding embodiments, comprising one or both of the following: (i) an exogenous or overexpressed immunosuppressive protein, or (ii) an immunostimulatory protein that is absent or present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) relative to fusosomes produced from an otherwise unmodified similar cell of origin.

[0119] 115. The fusosome of any of the preceding embodiments, which circulates for at least 0.5, 1, 2, 3, 4, 6, 12, 18, 24, 36, or 48 hours after administration to said subject.

[0120] 116. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate 30 minutes after administration.

[0121] 117. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate one hour after administration.

[0122] 118. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate for 2 hours after administration.

[0123] 119. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate for 4 hours after administration.

[0124] 120. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate 8 hours after administration.

[0125] 121. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate for 12 hours after administration.

[0126] 122. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate for 18 hours after administration.

[0127] 123. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate for 24 hours after administration.

[0128] 124. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate 36 hours after administration.

[0129] 125. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes circulate for 48 hours after administration.

[0130] 126. The fusosome of any of the preceding embodiments, which has reduced immunogenicity compared to a reference retrovirus, e.g., a fusosome similar to the fusosome but unmodified, as measured by a reduced humoral response after one or more administrations of the fusosome to a suitable animal model, e.g., an animal model described herein.

[0131] 127. The fusosome of any of the previous embodiments, wherein the reduced humoral response is measured in a serum sample by anti-cellular antibody titers, e.g., anti-retroviral antibody titers, e.g., by ELISA.

[0132] 128. The fusosome of any of the preceding embodiments, wherein serum samples from animals administered the fusosomes have a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more reduction in anti-fusosome antibody titers compared to serum samples from subjects administered unmodified cells.

[0133] 129. The fusosomes of any of the preceding embodiments, wherein a serum sample from a subject administered the fusosomes has an increased anti-cellular antibody titer, e.g., an increase of 1%, 2%, 5%, 10%, 20%, 30%, or 40% from baseline, e.g., the baseline refers to a serum sample from the same subject prior to administration of the fusosomes.

[0134] 130. The fusosome of any of the preceding embodiments, The subject to whom the fusosomes are administered has, is known to have, or is being tested for pre-existing antibodies (e.g., IgG or IgM) that react with the fusosomes; the subject to whom the fusosomes are administered does not have detectable levels of pre-existing antibodies that react with the fusosomes; The subject receiving the fusosomes has, is known to have, or is being tested for antibodies (e.g., IgG or IgM) that react with the fusosomes; subjects who have received (e.g., at least 1, 2, 3, 4, 5, or more) administrations of the fusosomes do not have detectable levels of antibodies reactive with the fusosomes, or The fusosomes, wherein the level of the antibody does not increase by more than 1%, 2%, 5%, 10%, 20%, or 50% between two time points, the first time point being before the first administration of the fusosomes and the second time point being after one or more administrations of the fusosomes.

[0135] 131. The fusosome of any of the previous embodiments, wherein the fusosome is a retroviral vector produced from a cell expressing exogenous or overexpressed HLA-G or HLA-E, e.g., a cell transfected with a nucleic acid encoding HLA-G or HLA-E.

[0136] 132. The fusosome of any of the preceding embodiments, wherein the fusosome is a retroviral vector produced from NMC-HLA-G cells, and the percentage of lysis, e.g., PBMC-mediated lysis, NK cell-mediated lysis, and / or CD8+ T cell-mediated lysis, is reduced at a particular time point compared to a retroviral vector produced from NMC or an NMC-empty vector.

[0137] 133. The fusosome of any of the previous embodiments, wherein said modified fusosome avoids phagocytosis by macrophages.

[0138] 134. The fusosome of any of the previous embodiments, wherein said fusosome is produced from a cell expressing exogenous or overexpressed CD47, e.g., a cell transfected with a nucleic acid encoding CD47.

[0139] 135. The fusosome of any of the preceding embodiments, wherein the fusosome is a retroviral vector and the phagocytic index of macrophages incubated with the retroviral vector derived from NMC-CD47 is reduced compared to that derived from NMC or an NMC-empty vector.

[0140] 136. The fusosome of any of the preceding embodiments, wherein phagocytosis by macrophages is reduced compared to a reference fusosome, e.g., a fusosome similar to said fusosome but unmodified, e.g., wherein phagocytosis by macrophages is reduced by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, wherein said reduced phagocytosis by macrophages is measured by assaying an in vitro phagocytic index.

[0141] 137. The fusosome of any preceding embodiment, wherein a composition comprising a plurality of said fusosomes has a phagocytic index of 0, 1, 10, 100, or more when incubated with macrophages in an in vitro assay of phagocytosis by macrophages.

[0142] 138. The fusosome of any of the preceding embodiments, which has been modified to have a reduced complement titer compared to an unmodified retroviral vector.

[0143] 139. The fusosome of any of the preceding embodiments, produced from cells comprising an exogenous or overexpressed complement regulatory protein (e.g., DAF), e.g., from cells transfected with a nucleic acid encoding a complement regulatory protein, e.g., DAF.

[0144] 140. The fusosome of any preceding embodiment, wherein the fusosome is a retroviral vector, and the amount of retroviral vector present at 200 pg / ml of C3a is greater for the modified retroviral vector (e.g., HEK293-DAF) incubated with corresponding mouse serum (e.g., HEK-293 DAF mouse serum) than for a reference retroviral vector (e.g., HEK293 retroviral vector) incubated with corresponding mouse serum (e.g., HEK293 mouse serum).

[0145] 141. The fusosome of any preceding embodiment, wherein the fusosome is a retroviral vector and the amount of retroviral vector present at 200 pg / ml of C3a is greater for the modified retroviral vector (e.g., HEK293-DAF) incubated with naive mouse serum than for a reference retroviral vector (e.g., HEK293 retroviral vector) incubated with naive mouse serum.

[0146] 142. The fusosome of any of the preceding embodiments, which is resistant to complement-mediated inactivation in a patient's serum 30 minutes after administration.

[0147] 143. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are resistant to complement-mediated inactivation.

[0148] 144. The fusosome of any of the previous embodiments, wherein the complement regulatory protein comprises one or more of a protein that binds to decay accelerating factor (DAF, CD55), e.g., factor H (FH)-like protein-1 (FHL-1), e.g., C4b-binding protein (C4BP), e.g., complement receptor 1 (CD35), e.g., membrane cofactor protein (MCP, CD46), e.g., protectin (CD59), e.g., a protein that inhibits CD / C5 convertase enzymes of the classical and alternative complement pathways, e.g., a protein that regulates MAC assembly.

[0149] 145. The fusosome of any of the preceding embodiments, produced by cells with reduced levels of MHC class I, e.g., derived from cells transfected with DNA encoding shRNA targeting MHC class I, e.g., wherein retroviral vector derived NMC-shMHC class I has reduced expression of MHC class I compared to NMC and NMC-vector controls.

[0150] 146. The fusosome of any of the previous embodiments, wherein the measure of immunogenicity of the fusosome (e.g., retroviral vector) is serum inactivation.

[0151] 147. The fusosome of any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples incubated with serum from fusosome-naive mice and heat-inactivated serum.

[0152] 148. The fusosome of any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent is not different between fusosome samples incubated with serum from fusosome-naive mice and fusosome samples from serum-free control incubations.

[0153] 149. The fusosome of any of the preceding embodiments, wherein the percentage of cells receiving the exogenous agent is less in fusosome samples incubated with positive control serum than in fusosome samples incubated with serum from fusosome-naive mice.

[0154] 150. The fusosome of any of the preceding embodiments, wherein, e.g., a modified retroviral vector modified by a method described herein exhibits reduced serum inactivation (e.g., reduced compared to administration of an unmodified retroviral vector) following multiple (e.g., more than one, e.g., two or more) administrations of said modified retroviral vector.

[0155] 151. The fusosome of any of the preceding embodiments, which is not inactivated by serum after multiple administrations.

[0156] 152. The fusosome of any of the preceding embodiments, wherein the measure of the immunogenicity of the fusosome is, for example, serum inactivation after multiple administrations.

[0157] 153. The fusosome of any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples incubated with serum from mice treated with modified (e.g., HEK293-HLA-G) fusosomes and heat-inactivated serum.

[0158] 154. The fusosome of any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples incubated with serum from mice treated 1, 2, 3, 5, or 10 times with a modified (e.g., HEK293-HLA-G) retroviral vector.

[0159] 155. The fusosome of any of the preceding embodiments, wherein the percentage of cells receiving the exogenous agent does not differ between fusosome samples incubated with serum from vehicle-treated mice and serum from mice treated with modified (e.g., HEK293-HLA-G) fusosomes.

[0160] 156. The fusosome of any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent is lower in fusosomes derived from a reference cell (e.g., HEK293) than in modified (e.g., HEK293-HLA-G) fusosomes.

[0161] 157. The fusosome of any of the preceding embodiments, wherein the measure of the immunogenicity of the fusosome is an antibody response.

[0162] 158. The fusosome of any of the previous embodiments, wherein the subject to whom the fusosome described herein is administered has a pre-existing antibody that binds to and recognizes said fusosome.

[0163] 159. The fusosome of any of the preceding embodiments, wherein serum from fusosome-naive mice shows more signal (e.g., fluorescence) than a negative control, e.g., serum from mice depleted of IgM and IgG, indicating, e.g., that immunogenicity has occurred.

[0164] 160. The fusosome of any of the preceding embodiments, wherein serum from fusosome-naive mice exhibits a similar signal (e.g., fluorescence) compared to said negative control, indicating, for example, that no detectable immunogenicity occurred.

[0165] 161. The fusosome of any of the preceding embodiments, comprising a modified retroviral vector, e.g., modified by a method described herein, wherein after multiple (e.g., more than one, e.g., two or more) administrations of said modified retroviral vector, the humoral response is reduced (e.g., reduced compared to administration of an unmodified retroviral vector).

[0166] 162. The fusosome of any of the previous embodiments, wherein the humoral response is assessed by measuring a value for the level of anti-fusosome antibodies (e.g., IgM, IgG1, and / or IgG2 antibodies).

[0167] 163. The fusosome of any of the preceding embodiments, wherein the modified (e.g., NMC-HLA-G) fusosome, e.g., retroviral vector, after injection, reduces anti-viral IgM or IgG1 / 2 antibody titers (e.g., as measured by fluorescence intensity on a FACS) compared to a control, e.g., an NMC retroviral vector or an NMC-empty retroviral vector.

[0168] 164. The fusosome of any of the preceding embodiments, wherein the recipient cell is not targeted by an antibody response or the antibody response is below a reference level.

[0169] 165. The fusosome of any of the preceding embodiments, wherein the signal (e.g., mean fluorescence intensity) is similar in recipient cells from mice treated with the retroviral vector and mice treated with PBS.

[0170] 166. The fusosome of any of the preceding embodiments, wherein the measure of immunogenicity of the recipient cells is a macrophage response.

[0171] 167. The fusosome of any of the preceding embodiments, wherein the recipient cells are not targeted by macrophages or are targeted at below reference levels.

[0172] 168. The fusosome of any of the preceding embodiments, wherein the phagocytic index is similar in recipient cells derived from fusosome-treated and PBS-treated mice.

[0173] 169. The fusosome of any of the previous embodiments, wherein the measure of immunogenicity of the recipient cells is a PBMC response.

[0174] 170. The fusosome of any of the preceding embodiments, wherein the recipient cells do not induce a PBMC response.

[0175] 171. The fusosome of any of the preceding embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated and PBS-treated mice.

[0176] 172. The fusosome of any of the previous embodiments, wherein the measure of immunogenicity of the recipient cells is a natural killer cell response.

[0177] 173. The fusosome of any of the preceding embodiments, wherein the recipient cells do not induce a natural killer cell response or induce a reduced natural killer cell response, e.g., lower than a reference value.

[0178] 174. The fusosomes of any of the preceding embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated and PBS-treated mice.

[0179] 175. The fusosome of any of the previous embodiments, wherein the measure of immunogenicity of the recipient cells is a CD8+ T cell response.

[0180] 176. The fusosome of any of the preceding embodiments, wherein the recipient cells do not induce a CD8+ T cell response or induce a lesser CD8+ T cell response, e.g., lower than a reference value.

[0181] 177. The fusosomes of any of the preceding embodiments, wherein the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated and PBS-treated mice.

[0182] 178. The fusosome of any of the preceding embodiments, wherein the fusogen is a retargeted fusogen.

[0183] 179. The fusosome of any of the preceding embodiments, comprising a retroviral nucleic acid encoding one or both of the following: (i) a positive target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous agent, or (ii) a non-target cell-specific regulatory element operably linked to a nucleic acid encoding said exogenous agent.

[0184] 180. The fusosome described in embodiment 179, wherein the nucleic acid comprises two insulator sequences, e.g., a first insulator sequence upstream of the region encoding the exogenous agent and a second insulator sequence downstream of the region encoding the exogenous agent, e.g., the first insulator sequence and the second insulator sequence comprise the same or different sequences.

[0185] 181. The median level of the exogenous agent in a sample of cells isolated after administering the fusosomes to the subject at a first time point is at least 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 181. The fusosome of any of embodiments 179-180, wherein the fusosome is less than or about 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, or 5%, or is less than or about 10,000%, 5,000%, 2,000%, 1,000%, 500%, 200%, 100%, 50%, 20%, 10%, or 5%.

[0186] 182. The fusosome of embodiment 181, wherein the median expression level per cell is assessed only in cells having a retroviral genome copy number of at least 1.0.

[0187] 183. A fusosome described in any of embodiments 179 to 182, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in the subject detectably contain the exogenous agent.

[0188] 184. A fusosome described in any of embodiments 181 to 182, wherein the median expression level of the payload gene is assessed across cells isolated from the subject at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, and 1095 days after administration of the fusosome to the subject.

[0189] 185. The fusosome of any of embodiments 179 to 184, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells in the subject that detectably contain the exogenous agent at a first time point still detectably contain the exogenous agent at a second, i.e., later, time point, wherein the first time point is 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days after administration of the fusosome to the subject.

[0190] 186. The fusosomes of embodiment 185, wherein the second time point is 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days after the first time point.

[0191] 187. Fusosomes according to any of embodiments 179 to 186, which are not genotoxic or do not increase the rate of tumor formation in target cells compared to target cells not treated with said fusosomes.

[0192] 188. A fusosome according to any of embodiments 179 to 187, wherein the median level of the exogenous agent is assessed in a cell population from a subject administered the fusosome.

[0193] 189. A fusosome described in any of embodiments 179 to 188, wherein the median level of the exogenous agent assessed in cell populations recovered (e.g., isolated) from the subject on different days after administration differs by less than about 10,000%, 1000%, 100%, or 10%, e.g., 10,000% to 1000%, 1000% to 100%, or 100% to 10%, from the median level of the exogenous agent in cell populations assessed on days 7, 14, 28, or 56, and wherein the cells within the populations have a vector copy number of at least 1.0.

[0194] 190. A fusosome according to any of embodiments 179 to 189, wherein the level of the exogenous agent is assessed across cells from a subject administered the fusosome.

[0195] 191. A fusosome described in any of embodiments 179 to 190, wherein the percentage of cells containing the exogenous agent is assessed in a plurality of cells recovered (e.g., isolated) from the subject at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, and 1095 days after administration of the fusosome.

[0196] 192. A fusosome described in any of embodiments 179 to 191, wherein the difference in the percentage of cells containing the exogenous agent assessed in cells isolated on two different days after administration is less than 1%, 5%, 10%, 20%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 1500%, or 2000%.

[0197] 193. A fusosome described in any of embodiments 179 to 192, wherein the percentage of target cells positive for the exogenous agent is similar across cells recovered at 7 days, 14 days, 28 days, 56 days, 112 days, 365 days, 730 days, or 1095 days.

[0198] 194. Fusosomes according to any of embodiments 179 to 193, at least the same number of target cells are positive for the exogenous agent on days 14, 28, 56, 112, 365, 730, or 1095 as on day 7; at least as many target cells are positive for the exogenous agent on day 28, day 56, day 112, day 365, day 730, or day 1095 as on day 14, or at least as many target cells are positive for the exogenous agent on day 56, day 112, day 365, day 730, or day 1095 as on day 28; at least the same number of target cells are positive for the exogenous agent on days 112, 365, 730, or 1095 as on day 56; at least the same number of target cells are positive for the exogenous agent on days 365, 730, or 1095 as on day 112; at least the same number of target cells are positive for the exogenous agent on day 730 or day 1095 as on day 365; or The fusosomes, wherein at least the same number of target cells are positive for the exogenous agent at day 1095 as at day 730.

[0199] 195. Fusosomes according to any of embodiments 179 to 194, the median level of the exogenous agent in target cells containing the exogenous agent is similar in cells harvested at day 7, day 14, day 28, day 56, day 112, day 365, day 730, or day 1095; the median level of the exogenous agent in target cells containing the exogenous agent at day 14, day 28, day 56, day 112, day 365, day 730, or day 1095 is at least as high as at day 7; or the median level of the exogenous agent in target cells containing the exogenous agent at day 28, day 56, day 112, day 365, day 730, or day 1095 is at least as high as at day 14; the median level of the exogenous agent in target cells containing the exogenous agent at day 56, day 112, day 365, day 730, or day 1095 is at least as high as at day 28; or the median level of the exogenous agent in target cells containing the exogenous agent at day 112, day 365, day 730, or day 1095 is at least as high as at day 56; or the median level of the exogenous agent in target cells containing the exogenous agent at day 365, day 730, or day 1095 is at least as high as at day 112; the median level of the exogenous agent in target cells containing the exogenous agent at day 730 or day 1095 is at least as high as at day 365; or The fusosomes, wherein the median level of the exogenous agent in target cells containing the exogenous agent at day 1095 is at least as high as at day 730.

[0200] 196. A method of delivering an exogenous agent to a subject (e.g., a human subject), comprising administering to the subject a fusosome of any of the preceding embodiments, thereby delivering the exogenous agent to the subject.

[0201] 197. A method for regulating a function in a subject (e.g., a human subject), a target tissue, or a target cell, comprising contacting the subject, the target tissue, or the target cell with a fusosome described in any of the preceding embodiments, e.g., administering the fusosome to the subject, the target tissue, or the target cell.

[0202] 198. The method of embodiment 197, wherein the target tissue or the target cell is present in a subject.

[0203] 199. A method of treating or preventing a disorder, e.g., cancer, in a subject (e.g., a human subject), comprising administering to the subject a fusosome according to any of the preceding embodiments.

[0204] 200. A method of producing fusosomes according to any of the preceding embodiments, comprising: a) providing a source cell comprising the nucleic acid and the fusogen (e.g., a retargeted fusogen); b) culturing the source cells under conditions that allow the production of the fusosomes; and c) isolating, enriching, or purifying said fusosomes from said source cells, thereby producing said fusosomes.

[0205] 201. The method of any of the preceding embodiments, wherein the source cells for producing the fusosomes lack fusogenic receptors or the fusogenic receptors are present at a reduced level (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to an otherwise similar, unmodified source cell.

[0206] 202. The origin cell of embodiment 201, wherein the fusogen causes fusion of the fusosome with the target cell after binding to the fusogen receptor.

[0207] 203. An origin cell according to any one of embodiments 201-202, which binds to the second similar origin cell, for example, a fusogen of the origin cell binds to a fusogen receptor of the second origin cell.

[0208] 204. A population of source cells according to any of embodiments 201-203.

[0209] 205. The method of any of embodiments 201-204, wherein less than 10%, 5%, 4%, 3%, 2%, or 1% of the cells of origin are multinucleated.

[0210] 206. The method of any of embodiments 201-205, wherein the source cells are modified to reduce (e.g., prevent) fusion with other source cells during the production of fusosomes as described herein.

[0211] 207. The method of any of embodiments 201-206, wherein the fusogen (e.g., the retargeted fusogen) does not bind to a protein contained in the source cell, e.g., a protein on the surface of the source cell.

[0212] 208. The method of any of embodiments 201-207, wherein the fusogen (e.g., the retargeted fusogen) binds to a protein contained in the cell of origin, but does not fuse with the cell.

[0213] 209. The method of any of embodiments 201-208, wherein the fusogen does not induce fusion with the source cell.

[0214] 210. The method of any of embodiments 201-209, wherein the source cells do not express a protein (e.g., an antibody) that binds to the fusogen.

[0215] 211. The method of any of embodiments 201-210, wherein a plurality of cells of origin do not form syncytia when expressing the fusogen, or less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated (e.g., containing two or more nuclei).

[0216] 212. The method of any of embodiments 201-211, wherein a plurality of cells of origin do not form syncytia when producing fusosomes, or less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the cells in the population are multinucleated.

[0217] 213. The method of any of embodiments 201-212, wherein less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the nuclei in the population are in syncytia.

[0218] 214. The method of any of embodiments 201-213, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the nuclei in said population are in mononuclear cells.

[0219] 215. The method of any of embodiments 201 to 214, wherein the percentage of cells that are multinucleated is lower in the population of modified source cells compared to a population of otherwise similar, but unmodified, source cells, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower.

[0220] 216. The method of any of embodiments 201 to 215, wherein the percentage of nuclei present in syncytia is lower in the population of modified source cells compared to a population of otherwise similar, but unmodified, source cells, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% lower.

[0221] 217. The method of any of embodiments 201-216, wherein multinucleated cells (e.g., cells with two or more nuclei) are detected by a microscopic assay, e.g., using a DNA stain.

[0222] 218. The method of any of embodiments 201-217, wherein the number of functional fusosomes (e.g., viral particles) obtained from the modified cell of origin is at least 10%, 20%, 40%, 40%, 50%, 60%, 70%, 8%, 90%, 2-fold, 5-fold, or 10-fold greater than the number of fusosomes obtained from a similar but unmodified cell of origin.

[0223] 219. The fusosome of any of the preceding embodiments, which lacks fusogen receptors or which have fusogen receptors present at reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% reduced) compared to fusosomes from an otherwise similar, unmodified cell of origin.

[0224] 220. The method of any of embodiments 201-219, comprising knocking down or knocking out the fusogen receptor in the origin cell or its precursor.

[0225] Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0226] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, for example, in any table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including any tables herein, refer to database entries as of July 6, 2020. When a gene or protein refers to multiple sequence accession numbers, all of the sequence variants are encompassed. Additionally, the materials, methods, and examples are merely illustrative and not intended to be limiting. [Brief explanation of the drawings]

[0227] [Figure 1] Figures A-B are a series of diagrams outlining the processing of henipavirus F proteins. Figure A shows the inactive precursor (F0), which results from initial translation of the henipavirus F protein. This precursor is transported to the plasma membrane (PM) and then recycled to endosomes and lysosomes, where it is cleaved by cathepsin L to form the fusion-active F1 / F2 subunits. This active form forms a complex with protein G and initiates membrane fusion. Figure B shows two motifs in the cytoplasmic tail of the henipavirus F protein (Y(525)RSL and Y(542)Y) that were identified as important for endocytosis of the henipavirus F protein and exposure to cathepsin L for cleavage. These motifs are missing in the NivFd22 truncated protein used to enhance lentiviral pseudotyping. [Figure 2](A-B) Data from a series of experiments showing the titer of fusosomes targeting CD8 or other cell surface markers after overexpression of cathepsin L. (A) Quantification of functional virus titer measured after transduction of CD8-overexpressing cells as described in Example 1. (B) Quantification of virus titer in target cells transduced with Niv protein G constructs bearing targeting moieties recognizing different cell surface moieties, with and without overexpression of cathepsin L as described in Example 2. [Figure 3] A-B show quantification of functional titers of CD8-targeting fusosomes on Pan T cells. Pan T cells were transduced with either concentrated (A) or crude (B) pseudotyped lentiviral lysate as described in Example 3. From left to right, each bar represents: 1) NivF without HA and without overexpression of CathL, Xfect transfection reagent; 2) NivF with HA and without overexpression of CathL, Xfect transfection reagent; 3) NivF without HA and overexpression of CathL, Xfect transfection reagent; and 4) NivF with overexpression of HA and CathL, Xfect transfection reagent. [Figure 4] Figure 4 shows flow cytometric quantification of transduced GFP-positive Pan T cells. GFP expression in Pan T cells indicates successful transduction of target cells by fusosomes. Pan T cells were transduced with pseudotyped lentiviral lysates isolated from 293LX producer cells transfected as described in Example 3 and Table 6. This flow cytometry plot shows GFP levels on the X-axis, indicating successful transduction of Pan T cells, and CD8 levels on the Y-axis, indicating which cells within the population are CD8-positive and therefore targeted by fusosomes. All experiments used a pseudotyped lentiviral dilution of 0.04. The percentage of CD8- and GFP-double-positive cells for each transduction shown in Figure 4 is included in Table 7. [Figure 5]Figures A to C show the effect of cathepsin L overexpression on henipavirus F protein processing in producer cells and their respective isolated pseudotyped lentiviral samples. 293LX producer cells were transfected as previously described to produce CD8-targeted Nipah G and F pseudotyped lentiviral vectors. Their respective supernatants containing pseudotyped lentivirus were isolated as described in Example 3 and Table 6. In Figure A, the Western blot band intensities of the F precursor inactive protein and cleaved fusion-active F1 subunit detected in producer cells and pseudotyped lentiviral samples were quantified as in Example 4A. The X axis indicates the samples (producer cells-CathL and +CathL, LV-CathL and +CathL), and the Y axis indicates the AUC (area under the curve) of protein signal intensity from the Western blot. Producer cells-CathL showed AUC values ​​of approximately 12-13,000 for both F0 and F1, producer cells+CathL showed AUC values ​​of approximately 2,000 for F0 and approximately 9,000 for F1, LV-CathL showed AUC values ​​of approximately 20,000 for F0 and approximately 9,000 for F1, and LV+CathL showed AUC values ​​of approximately 12,000 for both F0 and F1. In B, the percentage of cleaved F1 subunit to total F protein (F1+F0) was measured as in Example 4A. The X-axis shows samples (producer cells-CathL and +CathL, LV- and +CathL), and the Y-axis shows the percentage of cleaved F1 subunit to total F protein. The percentage of cleaved F1 subunit to total F protein was approximately 45% for producer cells minus CathL, approximately 80% for producer cells plus CathL, approximately 30% for LV-CathL, and approximately 50% for LV plus CathL. C contains a schematic diagram of the henipavirus F protein, showing the active F1 / F2 subunits with the cleavage site and the entire inactive F0 subunit for reference. [Figure 6]The production and processing of mature (proteolytically processed) cathepsin L in producer cell samples is measured. 293LX producer cells were transfected and their respective supernatants containing pseudotyped lentiviruses were isolated as described in Example 3 and Table 6. Producer cells were lysed to obtain protein samples, which were analyzed by Western blot using an anti-cathepsin L antibody as described in Example 4B. The image shows the protein band corresponding to mature cathepsin L. [Figure 7] p24 production was measured in isolated pseudotyped lentivirus samples. 293LX producer cells were transfected, and their respective supernatants containing pseudotyped lentivirus were isolated as described in Example 3 and Table 6. Pseudotyped lentivirus samples were analyzed by Western blot using an anti-p24 antibody as described in Example 4C. [Figure 8] Expression of Henipavirus G protein in producer cell samples was measured. 293LX producer cells were transfected and their respective supernatants containing pseudotyped lentiviruses were isolated as described in Example 3 and Table 6. Producer cells were lysed to obtain protein samples, which were analyzed by Western blot using an anti-Henipavirus G protein antibody as described in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0228] The present disclosure provides, at least in part, fusosome methods and compositions for in vivo delivery. In particular, the present disclosure provides methods for producing a plurality of fusosomes using mammalian producer cells that contain elevated levels or activity of a mature cathepsin molecule (e.g., cathepsin L or cathepsin B), a Henipavirus F protein, a Henipavirus G protein, and optionally an exogenous cargo molecule. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. A method for producing a plurality of fusosomes, comprising: (a) providing a modified mammalian producer cell, e.g., a human cell, comprising: (i) a mature cathepsin molecule (e.g., cathepsin L or cathepsin B) that has increased levels or activity compared to a corresponding unmodified cell; (ii) optionally, an exogenous cargo molecule, e.g., a protein or nucleic acid, and (iii) a henipavirus F protein molecule, and (iv) Henipavirus G protein molecule; (b) maintaining (e.g., culturing) the modified mammalian cells under conditions that allow the production of a plurality of fusosomes comprising the Henipavirus F protein molecule and the Henipavirus G protein molecule. (Item 2) 1. A method for producing a modified mammalian producer cell, comprising: (i) introducing into a mammalian cell a nucleic acid molecule encoding a cathepsin molecule under conditions that increase expression of the mature form of the cathepsin molecule in the mammalian cell; (ii) optionally introducing an exogenous cargo molecule, e.g., a protein or nucleic acid, into said mammalian cell; (iii) introducing a Henipavirus F protein molecule into the mammalian cell (e.g., introducing a nucleic acid encoding the Henipavirus F protein molecule under conditions suitable for expression of the Henipavirus F protein molecule), and (iv) introducing a Henipavirus G protein molecule into the mammalian cell (e.g., introducing a nucleic acid encoding the Henipavirus G protein molecule under conditions suitable for expression of the Henipavirus G protein molecule), The method, wherein steps (i) to (iv) may be performed in any order, or one or more of steps (i) to (iv) may be performed simultaneously. (Item 3) Modified mammalian cells, e.g., human cells, including: (i) a mature cathepsin molecule that has increased levels or activity compared to a corresponding unmodified cell; (e.g., cathepsin L or cathepsin B), (ii) optionally, an exogenous cargo molecule, e.g., a nucleic acid or protein, e.g., a viral nucleic acid, e.g., a lentiviral nucleic acid, and (iii) a henipavirus F protein molecule, and (iv) optionally, a Henipavirus G protein molecule. (Item 4) Fusosomes containing: (a) optionally, an exogenous cargo, e.g., a nucleic acid or protein, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) an activated henipavirus F protein molecule comprising a modified F1 form having a C-terminal truncation of up to 30 consecutive amino acids compared to a wild-type henipavirus protein F1 molecule, wherein at least 33% of the henipavirus F protein molecules in the fusosome are activated henipavirus F protein molecules; and (c) Henipavirus G protein molecule. (Item 5) Fusosomes containing: (a) optionally, an exogenous cargo, e.g., a fusomal nucleic acid, e.g., a viral nucleic acid (e.g., a lentiviral nucleic acid); (b) Henipavirus F protein molecules, wherein at least 33% of the Henipavirus F protein molecules in the fusosomes are active Henipavirus F protein; and (c) Henipavirus G protein molecule. (Item 6) A pharmaceutical composition comprising the fusosomes according to item 4 or 5, and optionally a pharmaceutically acceptable excipient. (Item 7) 1. A method for delivering exogenous cargo (e.g., fusomal nucleic acid, e.g., viral nucleic acid, e.g., lentiviral nucleic acid) to a cell (e.g., in vivo or ex vivo), comprising contacting the cell with a plurality of fusosomes described in any of items 4 or 5, the pharmaceutical composition described in item 6, or fusosomes produced by the method described in item 1. (Item 8) 1. A method for delivering exogenous cargo (e.g., fusomal nucleic acid, e.g., viral nucleic acid, e.g., lentiviral nucleic acid) to a subject, comprising administering to the subject an effective number of fusosomes described in either item 4 or 5, the pharmaceutical composition described in item 6, or fusosomes produced by the method described in item 1. (Item 9) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the Henipavirus F protein molecule lacks an endocytic motif. (Item 10) 10. The method, modified cell, fusosome, or pharmaceutical composition according to item 9, wherein the endocytosis motif is a YXXφ motif or a YSRL motif. (Item 11) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the cathepsin molecule comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having at least 80% identity thereto. (Item 12) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the cathepsin molecule comprises the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38 or SEQ ID NO: 39, or a sequence having at least 80% identity thereto. (Item 13) The method, modified cell, fusosome, or pharmaceutical composition according to any of the preceding items, wherein the elevated level of cathepsin molecules comprises at least 50% more cathepsin molecules than the amount of endogenous cathepsin L in a corresponding unmodified cell. (Item 14) The method, modified cell, fusosome, or pharmaceutical composition according to any of the preceding items, wherein the fusosomes contain active Henipavirus F protein molecules at a level that is at least 10% higher than otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules. (Item 15) The method, modified cell, fusosome, or pharmaceutical composition of any preceding item, wherein in said fusosome, at least 33% of the Henipavirus F protein molecules are active Henipavirus F protein. (Item 16) The method, modified cell, or pharmaceutical composition of any of the preceding items, wherein the fusosomes have a functional titer of at least about 200,000 TU / mL, e.g., as measured by detection of a GFP reporter in 293LX cells, e.g., in 293XL cells, e.g., in the assay of Example 1. (Item 17) The method, modified cell, or pharmaceutical composition of any of the preceding items, wherein the fusosomes have a functional titer of at least about 200,000 TU / mL, e.g., in activated T cells, e.g., primary T cells, e.g., Pan-T cells, e.g., as measured by detection of a GFP reporter in the activated T cells, e.g., in the assay of Example 3. (Item 18) The method, modified cell, or pharmaceutical composition of any of the preceding items, wherein the plurality of fusosomes produced has a titer on target cells to titer on non-target cells ratio of at least 2:1, e.g., the target cells overexpress a protein bound by the Henipavirus G protein molecule and the non-target cells are wild-type, e.g., the target cells overexpress CD8 and the non-target cells are wild-type, e.g., in the assay of Example 1. (Item 19) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the fusosomes comprise a level of total Henipavirus protein F that is 70% to 130% of the level of total Henipavirus protein F comprised in otherwise similar fusosomes produced from cells that do not have elevated levels or activity of cathepsin molecules. (Item 20) 7. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the Henipavirus F protein molecule comprises the amino acid sequence of a wild-type Nipah virus of SEQ ID NO: 7 or a sequence having at least 80% identity thereto. (Item 21) The method, modified cell, fusosome, or pharmaceutical composition of any preceding item, wherein the Henipavirus F protein molecule comprises a Henipavirus protein F of Table 4. (Item 22) The Henipavirus F protein molecule comprises a truncation of 10 to 30, 15 to 30, 10 to 20, or 20 to 30 amino acids, e.g., 22 or 25 amino acids, at the C-terminus compared to a wild-type Henipavirus F protein, e.g., a protein of Table 4, and optionally the Henipavirus F protein has a C-terminal amino acid sequence that is at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97% of SEQ ID NO: 17. 98%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to the Henipavirus F protein; and optionally, the Henipavirus F protein is set forth in SEQ ID NO: 17. (Item 23) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the Henipavirus F protein molecule lacks an endocytic motif, e.g., a YXXφ motif, e.g., a YRSL motif. (Item 24) 10. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the Henipavirus G protein molecule comprises the amino acid sequence of a wild-type Nipah virus of SEQ ID NO: 9 or a sequence having at least 80% identity thereto. (Item 25) 10. The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the Henipavirus G protein molecule comprises a truncation of 10 to 50 amino acids at the N-terminus compared to a wild-type Henipavirus G protein, e.g., a protein in Table 5; and optionally, the Henipavirus F protein comprises an amino acid sequence having at least or about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 18; and optionally, the Henipavirus F protein is set forth in SEQ ID NO: 18. (Item 26) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the Henipavirus G protein molecule is a retargeted Henipavirus G protein molecule. (Item 27) The method, modified cell, fusosome, or pharmaceutical composition of any preceding item, wherein the fusosomal nucleic acid is a lentiviral nucleic acid. (Item 28) The method, modified cell, fusosome, or pharmaceutical composition of any of the preceding items, wherein the fusosomal nucleic acid encodes a therapeutic payload. (Item 29) 2. The method, modified cell, fusosome, or pharmaceutical composition of any preceding item, wherein the modified cell is a human cell.

[0229] definition Terms used in the claims and specification are defined as follows, unless otherwise specified.

[0230] As used herein, the term "antibody molecule" refers to a polypeptide that contains sufficient sequence(s) from an immunoglobulin heavy chain variable region and / or sufficient sequence(s) from an immunoglobulin light chain variable region to confer antigen-specific binding. An antibody molecule can include a full-length antibody and / or fragments thereof, e.g., Fab fragments, that support antigen binding. In some embodiments, an antibody molecule contains heavy chain CDR1, CDR2, and CDR3 sequences and light chain CDR1, CDR2, and CDR3 sequences. Antibody molecules include, for example, human, humanized, and CDR-grafted antibodies and antigen-binding fragments thereof. In some embodiments, an antibody molecule includes at least one immunoglobulin variable region segment, e.g., a protein containing amino acid sequences providing an immunoglobulin variable domain or an immunoglobulin variable domain sequence. Examples of antibody molecules include humanized antibody molecules, intact IgA, IgG, IgE, or IgM antibodies, bi- or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies, e.g., IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), small modular antibodies, and the like. ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAbs®), Anticalin®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, microproteins, Fynomers®, Centyrin®, and KALBITOR®.

[0231] As used herein, "cargo molecule" refers to a molecule (e.g., a nucleic acid molecule or a polypeptide, e.g., a protein) contained in a fusosome. In some embodiments, the cargo molecule is packaged into the fusosome by a cell, e.g., a cell of origin described herein. In some embodiments, the cargo molecule is an agent exogenous to the fusosome or cell of origin.

[0232] As used herein, the term "cathepsin molecule" refers to a molecule having the structure and / or function of a cathepsin (e.g., cathepsin B or cathepsin L described herein). A cathepsin molecule, in some embodiments, can be a cysteine ​​protease. In some embodiments, a cathepsin molecule comprises the amino acid sequence of a cathepsin protein described herein (e.g., cathepsin B or cathepsin L, e.g., human cathepsin B or human cathepsin L). In some embodiments, (without being bound by theory) increasing the level or activity of a cathepsin molecule can result in increased functional titer of fusosomes (e.g., as described in Example 3), for example, by increased processing of F protein (e.g., henipavirus F protein). In some embodiments, a cathepsin molecule increases the ratio of active F protein (e.g., as measured by F1 levels) to inactive F protein (e.g., F0) in producer cells, for example, as described in Example 4. As used herein, "total cathepsin molecules" generally refers to the total number of cathepsin molecules in a cell, e.g., a source cell. Total cathepsin molecules may, in some cases, include both cathepsin molecules exogenous to the cell and cathepsin molecules endogenous to the cell. As used herein, "exogenous cathepsin molecules" are cathepsin molecules that are exogenous to a fusosome, a source cell, and / or a target cell. In some embodiments, the exogenous cathepsin molecules contain one or more differences (e.g., mutations) relative to a wild-type cathepsin molecule (e.g., expressed by a source cell, e.g., a producer cell). In some embodiments, the exogenous cathepsin molecule has, e.g., the sequence of a wild-type cathepsin molecule and is expressed by a nucleic acid molecule exogenously provided to the source cell (e.g., a producer cell).

[0233] As used herein, "fusosome" refers to an amphipathic lipid bilayer surrounding a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. In embodiments, the fusosome contains nucleic acid. In some embodiments, the fusosome is a membrane-enclosed preparation. In some embodiments, the fusosome is derived from a cell of origin.

[0234] As used herein, a "fusosome composition" refers to a composition comprising one or more fusosomes.

[0235] As used herein, "fusogen" refers to a substance or molecule that creates an interaction between two membrane-enclosed cavities. In embodiments, the fusogen promotes fusion of the membranes. In other embodiments, the fusogen creates a connection, e.g., a pore, between two cavities (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, the fusogen comprises a complex of two or more proteins, e.g., where neither protein has fusion activity alone. In some embodiments, the fusogen comprises a targeting domain.

[0236] As used herein, "fusogen receptor" refers to an entity (e.g., a protein) contained in a target cell, and binding of a fusogen on a fusosome (e.g., a retrovirus) to a fusogen receptor on the target cell facilitates delivery of a nucleic acid (e.g., a retroviral nucleic acid) (and optionally an exogenous agent encoded therein) to the target cell.

[0237] As used herein, "insulator sequence" refers to a nucleotide sequence that blocks an enhancer or prevents heterochromatin spreading. Insulator sequences may be wild-type or mutant.

[0238] As used herein, the term "effective amount" refers to that amount of a pharmaceutical composition sufficient to significantly and favorably modify the symptoms and / or condition being treated (e.g., result in a favorable clinical response). The effective amount of an active ingredient for use in a pharmaceutical composition will vary according to the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concurrent therapy, the particular active ingredient(s) employed, the particular pharmaceutically acceptable excipient(s) and / or carrier(s) used, and similar factors, within the knowledge and expertise of the attending physician.

[0239] As used herein, an "exogenous agent" with respect to fusosomes refers to an agent that is not contained in or encoded by a fusogen produced from a corresponding wild-type virus or a corresponding wild-type originating cell. In some embodiments, the exogenous agent is not naturally occurring, e.g., a protein or nucleic acid having a sequence that is altered (e.g., by insertion, deletion, or substitution) relative to a naturally occurring protein. In some embodiments, the exogenous agent is not naturally occurring in the originating cell. In some embodiments, the exogenous agent is naturally occurring in the originating cell but is exogenous to the virus. In some embodiments, the exogenous agent is not naturally occurring in the recipient cell. In some embodiments, the exogenous agent is naturally present in the recipient cell but not at a desired level or at a desired time. In some embodiments, the exogenous agent comprises RNA or a protein.

[0240] As used herein, the term "henipavirus F protein molecule" refers to a polypeptide having the structure and / or function of a henipavirus fusion protein (e.g., encoded by a henipavirus F gene). In some embodiments, the henipavirus F protein molecule participates in (e.g., induces, in combination with a henipavirus G protein molecule) fusion between the membrane of the fusosome and the membrane of a target cell. In some embodiments, the henipavirus F protein molecule is part of a polypeptide trimer, e.g., a homotrimer. In some embodiments, a fusosome comprises multiple henipavirus F protein molecules on its surface. In some embodiments, the henipavirus F protein molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a henipavirus F protein or a protein encoded by a henipavirus F gene. In some embodiments, the henipavirus F protein molecule is capable of promoting fusion between the membrane of a fusosome and the membrane of a target cell (e.g., in combination with a henipavirus G protein molecule). Henipavirus F protein molecules can be active or inactive. Typically, henipavirus F proteins are produced in an inactive form and then processed to an active form. More specifically, henipavirus F proteins are typically produced as an F0 chain and then cleaved to produce F1 and F2 chains (which are connected to each other by disulfide bridges), which are active. As used herein, an "active" henipavirus F protein molecule refers to a henipavirus F protein molecule containing an F1 chain produced, for example, by cleaving the F0 chain to produce F1 and F2 chains. As used herein, an "inactive" henipavirus F protein molecule refers to a henipavirus F protein molecule having an F0 chain. "Whole" henipavirus F protein includes both active and inactive henipavirus F protein.

[0241] As used herein, the term "henipavirus G protein molecule" refers to a polypeptide having the structure and / or function of a henipavirus G protein (e.g., encoded by a henipavirus G gene). In some embodiments, the henipavirus G protein molecule is capable of binding to a polypeptide on the surface of a target cell. In some embodiments, a fusosome comprises multiple henipavirus G protein molecules on its surface. In some embodiments, the henipavirus G protein molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a henipavirus G protein or a protein encoded by a henipavirus G gene. In some embodiments, the henipavirus G protein molecule is a fusion protein comprising, for example, a heterologous targeting moiety. In some embodiments, the henipavirus G protein molecule is a retargeted fusogen.

[0242] As used herein, the term "pharmaceutically acceptable" refers to excipients, compositions and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0243] As used herein, a "positive target cell-specific regulatory element" (or positive TCSRE) refers to a nucleic acid sequence that increases the level of an exogenous agent in a target cell compared to a non-target cell, wherein a nucleic acid encoding the exogenous agent is operably linked to the positive TCSRE. In some embodiments, the positive TCSRE is a functional nucleic acid sequence, e.g., the positive TCSRE can comprise a promoter or enhancer. In some embodiments, the positive TCSRE encodes a functional RNA sequence, e.g., the positive TCSRE can encode a splice site that promotes correct splicing of the RNA in the target cell. In some embodiments, the positive TCSRE encodes a functional protein sequence, or the positive TCSRE can encode a protein sequence that promotes correct post-translational modification of a protein. In some embodiments, the positive TCSRE reduces the level or activity of a downregulator or inhibitor of the exogenous agent.

[0244] As used herein, a "non-target cell-specific regulatory element" (or NTCSRE) refers to a nucleic acid sequence that reduces the level of an exogenous agent in non-target cells compared to target cells, wherein a nucleic acid encoding the exogenous agent is operably linked to the NTCSRE. In some embodiments, the NTCSRE is a functional nucleic acid sequence, e.g., an miRNA recognition site, that causes degradation or inhibition of a retroviral nucleic acid in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, e.g., the nucleic acid encodes an miRNA sequence present in an mRNA encoding an exogenous protein agent, causing the mRNA to be degraded or inhibited in non-target cells. In some embodiments, the NTCSRE increases the level or activity of a down-regulator or inhibitor of the exogenous agent. The terms "negative TCSRE" and "NTCSRE" are used interchangeably herein.

[0245] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting moiety with a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a targeting moiety that is different from the targeting moiety in the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting moiety that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to include a targeting moiety. In embodiments, the fusogen includes one or more sequence changes outside the targeting moiety relative to the naturally occurring form of the fusogen, for example, in the transmembrane domain, fusogenically active domain, or cytoplasmic domain.

[0246] As used herein, "target cell" refers to a cell type to which it is desired that a fusosome (e.g., a lentiviral vector) deliver an exogenous agent. In embodiments, the target cell is a particular tissue type or class of cell, e.g., an immune effector cell, e.g., a T cell. In some embodiments, the target cell is a diseased cell, e.g., a cancer cell.

[0247] As used herein, "non-target cells" refers to cell types to which it is not desired for fusosomes (e.g., lentiviral vectors) to deliver exogenous agents. In some embodiments, non-target cells are cells of a particular tissue type or class. In some embodiments, non-target cells are non-abnormal cells, e.g., non-cancerous cells.

[0248] As used herein, the terms "treat," "treating," or "treatment" refer to ameliorating a disease or disorder, e.g., slowing or halting or reducing the progression of a disease or disorder, e.g., the primary cause of the disorder or at least one of its clinical symptoms.

[0249] Cathepsins Described herein are methods and compositions involving fusosomes containing elevated levels or activity of cathepsin molecules, e.g., mature cathepsin molecules. In some embodiments, the cathepsin molecule is cathepsin L or cathepsin B. Generally, cathepsins are protease enzymes that are generally active in organelles characterized by low pH (e.g., low compared to the pH of the cytoplasm), e.g., lysosomes. In some cases, cathepsins, e.g., cathepsin L and cathepsin B, are cysteine ​​proteases involved in intracellular protein degradation (e.g., lysosomal proteolysis).

[0250] In some embodiments, cathepsin molecules are initially produced as preproenzymes, commonly referred to as procathepsins, which are subsequently processed intracellularly to form "mature" cathepsin molecules. In some embodiments, mature cathepsin molecules may comprise heavy and light chain polypeptides. Mature cathepsins may exist as single chains (e.g., approximately 28 kDa) and / or as two-chain heavy and light chains (e.g., approximately 24 and 4 kDa, respectively). In some embodiments, the heavy and light chain polypeptides are linked, for example, by one or more disulfides. In some embodiments, mature cathepsin molecules comprise the amino acid sequence of a cathepsin L1 protein, e.g., a human cathepsin L1 protein (e.g., the amino acid sequence of SEQ ID NO: 1, below). In some embodiments, the cathepsin molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary cathepsin L1 sequence of SEQ ID NO: 1. In some embodiments, the mature cathepsin molecule comprises the amino acid sequence of a cathepsin B protein, e.g., a human cathepsin B protein (e.g., the amino acid sequence of SEQ ID NO: 2 below). In some embodiments, the cathepsin molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary cathepsin B sequence of SEQ ID NO: 2. Exemplary cathepsin L1 sequence (SEQ ID NO:1): MDYAFQYVQDNGGLDSEESYPYEATEESCKYNPKYSVANDTGFVDIPKQEKALMKAVATVGPISVAIDAGHESFLFYKEGIYFEPDCSSEDMDHGVLVVGYGFESTESDNNKYWLVKNSWGEEWGMGGYVKMAKDRRNHCGIASAASYPTV Exemplary Cathepsin B sequence (SEQ ID NO:2): MHGNNGHSVPPSKRSETRAPVAPAGCNGGYPAEAWNFWTRKGLVSGGLYESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPTYKQDKHYGYNSYS VSNSEKDIMAEIYKNGPVEGAFSVYSDFLLYKSGVYQHVTGEMMGGHAIRILGWGVENGTPYWLVANSWNTDWGDNGFFKILRGQDHCGIESEVVAGIPRTDQYWEKI

[0251] In some embodiments, the mature cathepsin molecule comprises the amino acid sequence of a cathepsin L1 protein, e.g., a human cathepsin L1 protein (e.g., the amino acid sequence of SEQ ID NO: 37). In some embodiments, the cathepsin molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary cathepsin L1 sequence of SEQ ID NO: 37.

[0252] In some embodiments, the mature cathepsin molecule comprises the amino acid sequence of a cathepsin B protein, e.g., a human cathepsin B protein (e.g., the amino acid sequence of SEQ ID NO: 38). In some embodiments, the cathepsin molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary cathepsin B sequence of SEQ ID NO: 38.

[0253] In some embodiments, the mature cathepsin molecule comprises the amino acid sequence of a cathepsin B protein, e.g., a human cathepsin B protein (e.g., the amino acid sequence of SEQ ID NO: 39). In some embodiments, the cathepsin molecule has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary cathepsin B sequence of SEQ ID NO: 39.

[0254] In some embodiments, a nucleic acid encoding a cathepsin molecule is introduced into a host cell used in connection with the production of fusosomes provided herein. For example, a nucleic acid encoding a cathepsin (e.g., cathepsin L or cathepsin B) is introduced into a packaging cell line (producer cell) used in connection with the methods of producing retroviral vectors described below. In some embodiments, the nucleic acid molecule encodes a propeptide form of cathepsin, which includes the coding sequence for the mature cathepsin. Cleavage of the propeptide produces the mature cathepsin. In other embodiments, the nucleic acid molecule encodes a mature cathepsin set forth, for example, in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39. In some embodiments, the nucleic acid encodes a cathepsin L that exhibits at least 85%, 90%, 95%, 98%, or more sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid encodes the cathepsin L set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid encodes a cathepsin L that exhibits at least 85%, 90%, 95%, 98%, or more sequence identity to SEQ ID NO: 37. In some embodiments, the nucleic acid encodes a cathepsin L set forth in SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encodes a cathepsin B that exhibits at least 85%, 90%, 95%, 98%, or more sequence identity to SEQ ID NO: 2. In some embodiments, the nucleic acid molecule encodes a cathepsin B that exhibits at least 85%, 90%, 95%, 98%, or more sequence identity to SEQ ID NO: 38. In some embodiments, the nucleic acid molecule encodes a cathepsin B that exhibits at least 85%, 90%, 95%, 98%, or more sequence identity to SEQ ID NO: 39. In some embodiments, the nucleic acid molecule encodes a cathepsin B that exhibits at least 85%, 90%, 95%, 98%, or more sequence identity to SEQ ID NO: 39.

[0255] In some embodiments, the producer cells are mammalian cells. Any suitable cell line can be used as a producer or packaging cell line for the production of fusosomes, e.g., retroviral vector particles, e.g., lentiviral vectors. In some embodiments, the cell line comprises a mammalian cell, e.g., a human cell. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT cells, and the like. 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0256] In some embodiments, the cathepsin molecule is expressed in a host cell (e.g., a producer cell for producing fusosomes described herein). Any suitable method for expressing an exogenous polypeptide can be used to express the cathepsin molecule in such a host cell.

[0257] In some embodiments, the cathepsin molecule is transiently expressed in host cells, for example, by transfecting the host cell with a nucleic acid construct comprising the sequence encoding the cathepsin molecule under the control of a suitable promoter (for example, a constitutive promoter or an inducible promoter).In some embodiments, the nucleic acid molecule is introduced into the cell for episomal delivery.Methods for providing transgene as an episome include expression plasmid, virus-like particle, or adenovirus (AAV) delivery.

[0258] In some embodiments, expression is achieved using a site-specific activator of a cathepsin locus in a cell, e.g., a mammalian cell. For example, a fusion protein can be introduced into the cell comprising a cathepsin gene (e.g., CTSB or CTSL) and a site-specific binding domain specific for the transcription activator. In some embodiments, the site-specific binding domain is selected from the group consisting of a zinc finger, a transcription activation-like (TAL) effector, a meganuclease, and a CRISPR / Cas9 system component, or a modified form thereof. In some embodiments, the encoded regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the site-specific binding domain is a CRISPR / Cas system, which comprises a modified Cas nuclease lacking nuclease activity and a guide RNA (gRNA). In some embodiments, the modified nuclease is a catalytically inactive form of Cas9 (dCas9). In some optional embodiments, the transcriptional activator is selected from a herpes simplex-derived transactivation domain, a Dnmt3a methyltransferase domain, p65, VP16, and VP64, hi some optional embodiments, the transcriptional activator is the tripartite activator VP64-p65-Rta (VPR).

[0259] In some embodiments, the cathepsin molecule is introduced into cells under conditions for stable expression of the cathepsin. For example, in some embodiments, the cathepsin molecule is introduced into cells to be integrated into the chromosome of the cell. Any of a variety of methods can be used for the stable integration of the delivered nucleic acid molecule into cells. In some embodiments, the nucleic acid encoding the cathepsin is delivered into cells using a lentiviral vector. In other embodiments, the nucleic acid encoding the cathepsin is delivered into cells by targeted integration into a selected locus in the cell.

[0260] Methods for targeted integration are known. For example, any of a variety of site-specific nucleases can be used to mediate targeted cleavage of host cell DNA, biasing insertion to a selected genomic locus (see, for example, U.S. Patent No. 7,888,121 and U.S. Patent Publication No. 20110301073). Specific nucleases that cleave within or near the endogenous locus can be used, and the transgene can be integrated at or near the cleavage site by homology-directed repair (HDR) or by end-capture during non-homologous end joining (NHEJ). This integration process is influenced by the use or non-use of homologous regions in the transgene donor. These chromosomal homologous regions in the donor flank the transgene cassette and are homologous to the sequence of the endogenous locus at the cleavage site.

[0261] In some embodiments, the target locus is a non-cognate locus, e.g., one selected for a desired beneficial property. In some cases, a nucleic acid encoding a cathepsin may be inserted into a specific "safe harbor" location in the genome, either utilizing a promoter found at the safe harbor locus or regulating expression of the transgene via an exogenous promoter fused to the transgene prior to insertion. Several such "safe harbor" loci have been described, including the AAVS1 (also known as PPP1R12C) and CCR5 genes, Rosa26, and albumin in human cells (see co-owned U.S. Patent Publication Nos. 20080299580, 20080159996, and 201000218264, and U.S. Application Nos. 13 / 624,193 and 13 / 624,217). As noted above, nucleases specific to the safe harbor can be used to insert the transgene construct via either HDR or NHEJ-driven processes.

[0262] In some embodiments, other components used to produce the fusosomes can also be expressed in producer cells or packaging cells, e.g., as described below, for retroviral or virus-like particle production methods. In some embodiments, a transfer vector can be used that is a retroviral (e.g., lentiviral) transfer plasmid encoding a transgene of interest (e.g., an exogenous agent), where the transgene sequence is flanked by long terminal repeat (LTR) sequences to facilitate integration of the transfer plasmid sequence into the host genome, which is otherwise replication-deficient due to the lack of viral sequences. The transfer vector can then be introduced into a packaging cell line containing the gag, pol, and env genes but lacking the LTRs and packaging components. The recombinant retroviral particles are secreted into the culture medium and then collected, optionally concentrated, and used for gene transfer.

[0263] In certain embodiments, the packaging cell line comprises genes encoding a henipavirus F protein molecule (e.g., any of those described) and a henipavirus G protein molecule (e.g., any of those described) such that the retroviral vector (e.g., lentiviral vector) is pseudotyped with an envelope protein from a henipavirus. In certain embodiments, the packaging cell line comprises genes encoding a henipavirus F protein molecule (e.g., any of those described) and a henipavirus G protein molecule (e.g., any of those described) such that the virus-like particle (e.g., lentivirus-like particle) is pseudotyped with an envelope protein from a henipavirus. In some embodiments, the henipavirus is a nipahvirus. As described herein, the G protein molecule can be modified to incorporate a targeting / binding ligand to retarget the pseudotyped fusosome (e.g., a lentiviral vector or virus-like particle) to any desired target cell. In some embodiments, production of the retroviral particle (e.g., lentiviral vector or lentivirus-like particle) using the provided producer cells that exhibit elevated or increased expression of a cathepsin (e.g., due to delivery of an exogenous nucleic acid encoding a cathepsin) results in a retroviral vector pseudotyped with the F or G protein molecule, where improved processing of the F protein results in increased expression of active F protein.

[0264] In some embodiments, increasing the level or activity of a cathepsin molecule can promote increased functional titer of fusosomes (e.g., as described in Examples 1-3), for example, by increasing processing of F protein (e.g., henipavirus F protein). For example, a cathepsin molecule can increase the ratio of active F protein (F1+F2) to inactive protein (F0) in producer cells, for example, as described in Example 4. In some embodiments, the ratio of active to inactive F protein is increased by decreasing the level of inactive protein, for example, as described in Example 4.

[0265] Fusosomes, e.g., cell-derived fusosomes Fusosomes can take a variety of forms. Generally, fusosomes described herein comprise cathepsin molecules with increased activity and / or increased levels (e.g., as described herein). In some embodiments, the fusosomes comprise Henipavirus F protein molecules and Henipavirus G protein molecules. In some embodiments, the fusosomes described herein are derived from a source cell (e.g., a producer cell described herein). Fusosomes can comprise, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, apoptotic bodies (from apoptotic cells), microparticles (e.g., derived from platelets), ectosomes (e.g., derived from neutrophils and monocytes in serum), prostatosomes (obtainable from prostate cancer cells), cardiosomes (derived from cardiac cells), or any combination thereof. In some embodiments, fusosomes are naturally released from a source cell; in some embodiments, the source cell is treated to promote fusosome formation. In some embodiments, the fusosomes are about 10 to 10,000 nm in diameter, for example, about 30 to 100 nm in diameter. In some embodiments, the fusosomes comprise one or more synthetic lipids.

[0266] In some embodiments, the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus. In some embodiments, the fusosome comprising a lipid bilayer comprises a retroviral vector comprising an envelope. For example, in some embodiments, the amphipathic lipid bilayer of the fusosome is or comprises a viral envelope. The viral envelope can comprise a fusogen, e.g., a fusogen endogenous to the virus or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome comprises viral nucleic acid, e.g., retroviral nucleic acid, e.g., lentiviral nucleic acid. The viral nucleic acid can be a viral genome. In some embodiments, the fusosome further comprises one or more viral nonstructural proteins, e.g., in its cavity or lumen.

[0267] Fusosomes can have various properties that facilitate delivery of a payload, such as a desired transgene or exogenous agent, to a target cell. For example, in some embodiments, the fusosome and cell of origin together contain sufficient nucleic acid(s) to create a particle capable of fusing with a target cell. In embodiments, these nucleic acid(s) encode a protein having one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0268] Fusosomes can also contain various structures that facilitate delivery of a payload to a target cell. For example, in some embodiments, the fusosome (e.g., a virus, e.g., a retrovirus, e.g., a lentivirus) contains one or more (e.g., all) of the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., a functional or non-functional variant), protease, and fusogen. In some embodiments, the fusosome further contains rev. In some embodiments, one or more of the foregoing proteins are encoded by the retroviral genome, and in some embodiments, one or more of the foregoing proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) comprises one or more (e.g., all) of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., comprising a U5 and lacking a functional U3). In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more insulator sequences. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more miRNA recognition sites. In some embodiments, the one or more miRNA recognition sites are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and the WPRE.

[0269] In some embodiments, the fusosomes provided herein are administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject may be at risk for, have symptoms of, or be diagnosed or identified as having a particular disease or condition (e.g., a disease or condition described herein). In one embodiment, the subject has cancer. In one embodiment, the subject has an infectious disease. In some embodiments, the fusosomes comprise a nucleic acid sequence encoding an exogenous agent for treating a disease or condition.

[0270] Fusomal components and helper cells In some embodiments, the fusosomal nucleic acid comprises one or more (e.g., all) of a 5' promoter (e.g., for controlling expression of the entire packaged RNA), a 5' LTR (e.g., a U5 containing an R (including a polyadenylation tail signal) and / or a primer activation signal), a primer binding site, a Psi packaging signal, an RRE element for nuclear export, a promoter directly upstream of the transgene to control transgene expression, the transgene (or other exogenous agent element), a polypurine tract, and a 3' LTR (e.g., containing a mutated U3, R, and U5). In some embodiments, the fusosomal nucleic acid further comprises one or more cPPT, WPRE, and / or insulator sequences.

[0271] In some embodiments, the fusosome contains elements of one or more retroviruses. Retroviruses typically replicate by reverse transcribing their genomic RNA into a linear, double-stranded DNA copy, which then covalently integrates into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spuma virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses. In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus.

[0272] In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0273] Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1, and HIV type 2), Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0274] In some embodiments, a vector herein refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The nucleic acid to be transferred is typically linked, e.g., inserted, into the nucleic acid molecule of the vector. A vector may contain a sequence that induces autonomous replication in a cell or may contain a sequence sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses. A viral vector may, for example, contain a nucleic acid molecule (e.g., a transfer plasmid) that typically contains virus-derived nucleic acid elements that facilitate the transfer of the nucleic acid molecule or its integration into the cellular genome or a viral particle that mediates nucleic acid transfer. Viral particles typically contain various viral and sometimes host cell components in addition to the nucleic acid(s). A viral vector may, for example, include a virus or viral particle capable of transferring a nucleic acid into a cell or the transferred nucleic acid (e.g., as naked DNA). Viral vectors and transfer plasmids may contain structural and / or functional genetic elements primarily derived from viruses. Retroviral vectors may contain viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. Lentiviral vectors may contain viral vectors or plasmids containing structural and functional genetic elements, or portions thereof, including long terminal repeats, primarily derived from lentiviruses.

[0275] In embodiments, a lentiviral vector (e.g., a lentiviral expression vector) can comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of these elements can be present in the lentiviral particle in RNA form or in the DNA plasmid in DNA form.

[0276] In some vectors described herein, at least a portion of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild-type virus, rendering the viral vector replication-deficient. In some embodiments, the vector is capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0277] The structure of wild-type retroviral genomes often includes a 5' long terminal repeat (LTR) and a 3' long terminal repeat (LTR), between which or within which are located a packaging signal for enabling genome packaging, a primer binding site, an integration site for enabling integration into the host cell genome, and the gag, pol, and env genes encoding packaging components that promote viral particle assembly. More complex retroviruses have additional features, such as the rev and RRE sequences in HIV, which enable efficient transport of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells. In the provirus, the viral genes are flanked on both sides by regions called long terminal repeats (LTRs). The LTRs are involved in proviral integration and transcription. LTRs can also function as enhancer-promoter sequences and control the expression of the viral genes. Encapsidation of the retroviral RNA is mediated by a psi sequence located at the 5' end of the viral genome.

[0278] The LTRs themselves are usually similar (e.g., identical) sequences that can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements can vary greatly between different retroviruses.

[0279] For the viral genome, the site of transcription initiation is usually the boundary between U3 and R in one LTR, and the site of poly(A) addition (termination) is the boundary between R and U5 in the other LTR. U3 contains most of the proviral transcriptional regulatory elements, including a promoter and multiple enhancer sequences that respond to cellular and, in some cases, viral transcriptional activator proteins. Some retroviruses contain any one or more of the following genes that encode proteins involved in the control of gene expression: tot, rev, tax, and rex.

[0280] Regarding the structural genes gag, pol, and env themselves, gag encodes the internal structural protein of the virus. The Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), which contains DNA polymerase, associated RNase H, and integrase (IN), which mediate genome replication. The env gene encodes the virion's surface (SU) glycoprotein and transmembrane (TM) protein, which form a complex that interacts specifically with cellular receptor proteins. This interaction facilitates infection, for example, by fusion of the viral and cellular membranes.

[0281] In replication-defective retroviral vector genomes, gag, pol, and env may be absent or non-functional. The R regions at both ends of the RNA are usually repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome, respectively.

[0282] Retroviruses may also contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include (in HIV) one or more of vif, vpr, vpx, vpu, tat, rev, and nef. EIAV has (among other things) an additional gene, S2. The proteins encoded by the additional genes perform various functions, some of which may overlap with functions provided by cellular proteins. In EIAV, for example, tat acts as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). It binds to a stable stem-loop RNA secondary structure called TAR. Rev regulates and modulates viral gene expression via the rev-responsive element (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11). The mechanisms of action of these two proteins are thought to be largely similar to those of their primate counterparts. Furthermore, an EIAV protein, Ttm, has been identified, which is encoded by the first exon of tat spliced ​​into the env coding sequence at the start of the transmembrane protein.

[0283] In addition to protease, reverse transcriptase, and integrase, non-primate lentiviruses contain a fourth pol gene product that encodes a dUTPase, which may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

[0284] In embodiments, a recombinant lentiviral vector (RLV) is a vector that carries sufficient retroviral genetic information to enable packaging of an RNA genome into viral particles capable of infecting target cells in the presence of packaging components. Infection of the target cell can involve reverse transcription and integration into the target cell genome. The RLV typically carries non-viral coding sequences delivered to the target cell by the vector. In embodiments, the RLV is incapable of independent replication to produce infectious retroviral particles within the target cell. Typically, the RLV lacks functional gag-pol and / or env genes and / or other genes involved in replication. The vector can be configured as a split-intron vector, for example, as described in PCT Patent Application No. WO 99 / 15683, which is incorporated herein by reference in its entirety.

[0285] In some embodiments, the lentiviral vector comprises a minimal viral genome, e.g., the viral vector has been engineered to remove non-essential elements and retain essential elements to provide the functionality required to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98 / 17815, which is incorporated herein by reference in its entirety.

[0286] A minimal lentiviral genome may comprise, for example, (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to produce the lentiviral genome in the source cell may also comprise transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the source cell. These regulatory sequences may include natural sequences associated with the transcribed retroviral sequence, e.g., the 5'U3 region, or they may include a heterologous promoter, e.g., another viral promoter, e.g., a CMV promoter. Some lentiviral genomes contain additional sequences to promote efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively, or in combination, codon optimization may be used; for example, the gene encoding the exogenous agent may be codon-optimized, as described, for example, in WO 01 / 79518, which is incorporated herein by reference in its entirety. Alternative sequences that perform similar or identical functions to the rev / RRE system may also be used. For example, a functional analog of the rev / RRE system is found in the Mason-Pfizer monkey virus. This virus contains an RRE-type sequence in its genome known as a CTE, which is thought to interact with a factor in infected cells. This cellular factor can be considered a rev analog. Thus, the CTE can be used as a substitute for the rev / RRE system. Furthermore, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have similar effects on IRE-BP.

[0287] In some embodiments, the fusomal nucleic acid (e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid, e.g., a primate or non-primate lentiviral nucleic acid) (1) has a gag gene deleted, where the gag deletion removes one or more nucleotides downstream of approximately nucleotide 350 or 354 of the gag coding sequence; (2) has one or more accessory genes not present in the retroviral nucleic acid; (3) lacks a tat gene but includes a leader sequence between the end of the 5' LTR and the ATG of gag; and (4) a combination of (1), (2), and (3). In embodiments, the lentiviral vector comprises all of the features of (1), (2), and (3). This strategy is described in more detail, for example, in WO 99 / 32646, which is incorporated herein by reference in its entirety.

[0288] In some embodiments, primate lentiviral minimal systems do not require any of the additional HIV / SIV genes vif, vpr, vpx, vpu, tat, rev, and nef for either vector production or transduction of dividing and non-dividing cells, hi some embodiments, EIAV minimal vector systems do not require S2 for either vector production or transduction of dividing and non-dividing cells.

[0289] The deletion of these additional genes may allow the vector to be produced without genes associated with diseases in lentivirus (e.g., HIV) infections. In particular, tat is associated with diseases. Secondly, the deletion of these additional genes allows the vector to package more heterologous DNA. Thirdly, genes with unknown functions, such as S2, may not be included, thereby reducing the risk of causing unwanted effects. Examples of minimal lentiviral vectors are disclosed in WO99 / 32646 and WO98 / 17815.

[0290] In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (if it is an EIAV vector system), and optionally also vif, vpr, vpx, vpu, and nef. In some embodiments, the retroviral nucleic acid also lacks rev, RRE, or both.

[0291] In some embodiments, the retroviral nucleic acid comprises vpx. The Vpx polypeptide binds to and induces the degradation of SAMHD1, a restriction factor that degrades free dNTPs in the cytoplasm. Thus, the concentration of free dNTPs in the cytoplasm increases as Vpx degrades SAMHD1, increasing reverse transcription activity, thereby promoting reverse transcription of the retroviral genome and integration into the target cell genome.

[0292] Different cells have different frequencies of specific codon usage.This codon bias corresponds to the bias in the relative abundance of specific tRNA in cell type.By changing the codons in the sequence and adjusting them to match the relative abundance of corresponding tRNA, it is possible to increase expression.Similarly, it is possible to decrease expression by deliberately selecting codons whose corresponding tRNAs are known to be rare in specific cell type.Therefore, further translational control is possible.Further explanation of codon optimization is described in, for example, WO99 / 41397, which is incorporated herein by reference in its entirety.

[0293] In some embodiments, the retroviral nucleic acid lacks all nonstructural genes. In some embodiments, the fusosome is a virus-like particle (VLP) derived from a virus. In some embodiments, the viral envelope can contain a fusogen, e.g., a fusogen endogenous to the virus or a pseudotyped fusogen. The VLP can be derived from a retrovirus or a lentivirus. The VLP mimics the natural virion structure but lacks the viral genomic information necessary for independent replication within a host cell. Thus, in some aspects, the VLP is noninfectious. In certain embodiments, the VLP does not contain a viral genome. In some embodiments, the amphipathic lipid bilayer of the VLP is or comprises the viral envelope. In some embodiments, the VLP comprises at least one type of structural protein derived from a virus. In most cases, this protein forms a proteinaceous capsid. In some cases, the capsid is also surrounded by a lipid bilayer derived from the cell from which the assembled VLP was released (e.g., a VLP comprising a human immunodeficiency virus structural protein, such as GAG). In some embodiments, the VLP further comprises a targeting moiety as an envelope protein within the lipid bilayer.

[0294] In some embodiments, the fusosome comprises a supramolecular complex formed by viral proteins that self-assemble into a capsid. In some embodiments, the fusosome is a virus-like particle derived from a viral capsid protein. In some embodiments, the fusosome is a virus-like particle derived from a viral nucleocapsid protein. In some embodiments, the fusosome comprises nucleocapsid-derived proteins that retain the ability to package nucleic acids. In some embodiments, the fusosome, e.g., virus-like particle, comprises only viral structural glycoproteins among proteins from the viral genome. In some embodiments, the fusosome does not comprise a viral genome.

[0295] In some embodiments, the fusosome packages nucleic acid from the host cell during the expression process, e.g., a nucleic acid encoding an exogenous agent. In some embodiments, the nucleic acid does not encode any genes involved in viral replication. In certain embodiments, the fusosome is a virus-like particle, e.g., a retrovirus-like particle such as a replication-deficient lentivirus-like particle.

[0296] In some embodiments, the fusosomes are virus-like particles that contain sequences that are free of or lack viral RNA, which may be the result of removing or eliminating viral RNA from the sequences. In some embodiments, this can be achieved by using the endogenous packaging signal binding site on gag. In some embodiments, the endogenous packaging signal binding site is on pol. In some embodiments, the delivered RNA contains a cognate packaging signal. In some embodiments, a heterologous binding domain (heterologous to gag) located on the delivered RNA and a cognate binding site located on gag or pol can be used to ensure packaging of the delivered RNA. In some embodiments, the heterologous sequence can be non-viral or viral, and in such cases, can be derived from a different virus. In some embodiments, the fusosomes can be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. In some embodiments, the fusosomes can also be used to deliver a therapeutic gene of interest, in which case pol is typically included.

[0297] In some embodiments, the VLP comprises a supramolecular complex formed by viral proteins that self-assemble into a capsid. In some embodiments, the VLP is derived from a viral capsid. In some embodiments, the VLP is derived from a viral nucleocapsid. In some embodiments, the VLP is derived from a nucleocapsid and retains the ability to package nucleic acids. In some embodiments, the VLP comprises only viral structural glycoproteins. In certain embodiments, the VLP does not comprise a viral genome.

[0298] Many viruses, including HIV and other lentiviruses, use a number of rare codons, and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved.

[0299] Codon optimization has several other advantages. Due to changes in their sequences, the nucleotide sequences encoding the packaging components may have reduced or eliminated RNA instability sequences (INS). At the same time, the amino acid sequences coding for the packaging components are maintained so that the viral components encoded by the sequences remain the same, or at least similar enough, so that the function of the packaging components is not impaired. In some embodiments, codon optimization also overcomes the Rev / RRE requirement for transport, rendering the optimized sequences Rev-independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within a vector system (e.g., between regions of overlap in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titer and / or improved safety.

[0300] In some embodiments, only the codons for the INS are codon optimized, while in other embodiments, the entire sequence is codon optimized, except for the sequence encompassing the gag-pol frameshift site.

[0301] The gag-pol gene contains two overlapping reading frames encoding the gag-pol proteins. Expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused, at least in part, by a ribosome-stalling RNA secondary structure. Such a secondary structure is present downstream of the frameshift site in the gag-pol gene. In HIV, the region of overlap extends from 1222 nucleotides downstream from the start of gag (nucleotide 1 is A of the gag ATG) to the end of gag (nt 1503). Consequently, the 281-bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. In some embodiments, retention of this fragment results in more efficient expression of the gag-pol proteins. In EIAV, the start of the overlap is at nt 1262 (nucleotide 1 is A of the gag ATG). The end of the overlap is at nt 1461. The wild-type sequence can be retained from nt 1156 to 1465 to ensure that the frameshift site and the gag-pol overlap are preserved.

[0302] For example, derivations from optimal codon usage may be made to provide convenient restriction sites and conservative amino acid changes may be introduced into the gag-pol protein.

[0303] In some embodiments, codon optimization is based on codons with low codon usage in mammalian systems. The third and sometimes the second and third bases may be changed.

[0304] It will be appreciated that due to the degenerate nature of the genetic code, numerous gag-pol sequences can be achieved by those skilled in the art. Additionally, many retroviral variants have been described that can be used as a starting point for generating codon-optimized gag-pol sequences. Lentiviral genomes can be highly variable. For example, there are many quasispecies of HIV-I that are still functional. This is also true for EIAV. These variants can be used to improve specific parts of the transduction process. Examples of HIV-I variants can be found in the HIV database maintained by Los Alamos National Laboratory. Details of EIAV clones can be found in the NCBI database maintained by the National Institutes of Health.

[0305] The strategy for codon-optimizing gag-pol sequences can be used with any retrovirus, such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I, and HIV-2. This method can also be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERVs), MLV, and other retroviruses.

[0306] As described above, the packaging components of the retroviral vector can include the expression products of the gag, pol, and env genes. Additionally, packaging can use a short four-stem-loop sequence followed by partial sequences from gag and env as a packaging signal. Thus, inclusion of a missing gag sequence in the retroviral vector genome (in addition to the complete gag sequence on the packaging construct) can be used. In embodiments, the retroviral vector contains a packaging signal comprising 255-360 nucleotides of gag in vectors that still retain the env sequence, or approximately 40 nucleotides of gag in certain combinations of splice donor mutations, gag, and env deletions. In some embodiments, the retroviral vector contains a gag sequence containing one or more deletions, e.g., a gag sequence comprising approximately 360 nucleotides derivable from the N-terminus.

[0307] The retroviral vector, helper cell, helper virus, or helper plasmid can include retroviral structural and accessory proteins, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef proteins, or other retroviral proteins. In some embodiments, the retroviral proteins are derived from the same retrovirus. In some embodiments, the retroviral proteins are derived from multiple retroviruses, such as two, three, four, or more retroviruses.

[0308] The gag and pol coding sequences are usually organized as a Gag-Pol precursor in natural lentiviruses. The gag sequence encodes a 55 kD Gag precursor protein, also called p55. The p55 is cleaved by virally encoded protease 4 (the product of the pol gene) during maturation into four smaller proteins designated MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved from Gag by virally encoded protease and further digested to separate protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities.

[0309] Native Gag-Pol sequences can be used in helper vectors (e.g., helper plasmids or helper viruses), or modifications can be made, including chimeric Gag-Pols, where the Gag and Pol sequences are derived from different viruses (e.g., different species, subspecies, strains, clades, etc.), and / or the sequences have been modified to improve transcription and / or translation and / or to reduce recombination.

[0310] In various embodiments, the fusomal nucleic acid comprises a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of the gag protein, which (i) contains a mutated INS1 inhibitory sequence that reduces restriction of RNA nuclear export relative to wild-type INS1, (ii) contains a two-nucleotide insertion that results in a frameshift and premature termination, and / or (iii) does not contain the INS2, INS3, and INS4 inhibitory sequences of gag.

[0311] In some embodiments, the vectors described herein are hybrid vectors that contain both retroviral (e.g., lentiviral) and non-lentiviral viral sequences, hi some embodiments, the hybrid vectors contain retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0312] According to certain embodiments, most or all of the backbone sequence of the viral vector is derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used and combined, and numerous substitutions and modifications in a given lentiviral sequence can be provided without impairing the ability of the transfer vector to perform the functions described herein. A variety of lentiviral vectors are described in Naldini et al. (1996a, 1996b, and 1998), Zufferey et al. (1997), Dull et al., 1998, U.S. Patent Nos. 6,013,516 and 5,994,136, many of which can be adapted to produce retroviral nucleic acids.

[0313] Long terminal repeats (LTRs) are usually found at each end of the provirus. LTRs are usually direct repeats in their natural sequence context and contain domains located at the ends of retroviral nucleic acids, including the U3, R, and U5 regions. LTRs generally promote retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. The LTRs may contain multiple regulatory signals, including transcriptional regulators, polyadenylation signals, and sequences for viral genome replication and integration. The viral LTR is usually divided into three regions, designated U3, R, and U5. The U3 region usually contains enhancer and promoter elements. The U5 region is usually a sequence between the primer binding site and the R region and may contain a polyadenylation sequence. The R (repeat) region may be flanked by the U3 and U5 regions. The LTR is usually composed of the U3, R, and U5 regions and may appear at both the 5' and 3' ends of the viral genome. In some embodiments, adjacent to the 5'LTR are sequences for reverse transcription of the genome (tRNA primer binding site) and for efficient packaging of viral RNA into particles (Psi site).

[0314] The packaging signal can comprise a sequence located within the retroviral genome that mediates the insertion of the viral RNA into the viral capsid or particle.See, for example, Clever et al., 1995.J.of Virology, Vol.69, No.4; pp.2101-2109.Some retroviral vectors use a minimal packaging signal (psi [Ψ] sequence) for the encapsidation of the viral genome.

[0315] In various embodiments, the fusomal nucleic acid comprises a modified 5'LTR and / or 3'LTR. Either or both of the LTRs may contain one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modification of the 3'LTR is often performed to improve the safety of lentiviral or retroviral systems by rendering the virus replication-deficient, e.g., incapable of complete and efficient replication so that infectious virions are not produced (e.g., replication-deficient lentiviral progeny).

[0316] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the enhancer-promoter region known as the U3 region of the right (3') LTR has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the U3 region of the right (3') LTR may be used as a template for the U3 region of the left (5') LTR during viral replication, and thus the absence of the U3 enhancer-promoter inhibits viral replication. In embodiments, the 3' LTR is modified so that the U5 region has been removed, altered, or replaced, for example, with an exogenous poly(A) sequence. The 3' LTR, the 5' LTR, or both the 3' and 5' LTRs can be modified LTRs.

[0317] In some embodiments, the U3 region of the 5'LTR is replaced with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter can drive high levels of transcription in a Tat-independent manner. In certain embodiments, the heterologous promoter has the additional advantage of controlling the manner in which the viral genome is transcribed. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more compounds or physiological conditions, such as temperature or pH, under which host cells are cultured.

[0318] In some embodiments, the viral vector comprises a TAR (transactivation response) element, located, for example, in the R region of the lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral transactivator (tat) genetic element to enhance viral replication. However, this element is not required, for example, in embodiments in which the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0319] The R region, e.g., the region within a retroviral LTR beginning at the start of the capping group (i.e., the start of transcription) and ending just before the start of the polyA tract, may be flanked by the U3 and U5 regions and plays a role in the transfer of nascent DNA from one end of the genome to the other during reverse transcription.

[0320] The fusomal nucleic acid may also include a FLAP element, e.g., a nucleic acid whose sequence includes the central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., which are incorporated herein by reference in their entireties. al., 2000, Cell, 101:173. During HIV-1 reverse transcription, the central initiation of positive-strand DNA at the central polypurine tract (cPPT) and the central termination at the central termination sequence (CTS) can result in the formation of a triple-stranded DNA structure: the HIV-1 central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone contains one or more FLAP elements upstream or downstream of the gene encoding the exogenous agent. For example, in some embodiments, a transfer plasmid contains a FLAP element, e.g., a FLAP element derived from or isolated from HIV-1.

[0321] In embodiments, retroviral or lentiviral nucleic acids contain one or more transport elements, such as cis-acting posttranscriptional regulatory elements that regulate the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA transport elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, for example, Cullen et al., 1991. J. Virol. 65:1053 and Cullen et al., 1991. Cell 58:423) and the hepatitis B virus posttranscriptional regulatory element (HPRE), which are incorporated herein by reference in their entirety. Generally, the RNA transport element is located within the 3'UTR of a gene and can be inserted as one or more copies.

[0322] In some embodiments, expression of heterologous sequences in viral vectors is increased by incorporating one or more, for example, all of the following into the vector: a posttranscriptional regulatory element, a polyadenylation site, and a transcription termination signal. Various posttranscriptional regulatory elements, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE, Zufferey et al., 1999, J. Virol., 73:2886), the posttranscriptional regulatory element (HPRE) present in hepatitis B virus (Huang et al., Mol. Cell. Biol., 5:3864), and analogs (Liu et al., 1995, Genes Dev., 9:1766) (each of which is incorporated herein by reference in its entirety), can increase expression of heterologous nucleic acids in proteins. In some embodiments, the retroviral nucleic acids described herein include a posttranscriptional regulatory element, such as a WPRE or HPRE.

[0323] In some embodiments, the fusosomal nucleic acids described herein lack or do not include a post-transcriptional regulatory element, eg, a WPRE or HPRE.

[0324] Elements directing termination and polyadenylation of heterologous nucleic acid transcripts can be included, for example, to increase expression of exogenous agents. A transcription termination signal can be found downstream of the polyadenylation signal. In some embodiments, the vector contains a polyadenylation sequence 3' to the polynucleotide encoding the exogenous agent. A polyA site can contain a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. A polyadenylation sequence can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to increased translation efficiency. Helpful examples of polyA signals that can be used in retroviral nucleic acids include AATAAA, ATTAAA, AGTAAA, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), or another suitable heterologous or endogenous polyA sequence.

[0325] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator sequences, eg, an insulator sequence described herein.

[0326] In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous agent. The vector may have one or more LTRs, any of which may contain one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector may further comprise one or more accessory elements to increase transduction efficiency (e.g., cPPT / FLAP), one or more accessory elements to increase viral packaging (e.g., psi (Ψ) packaging signal, RRE), and / or other elements to increase expression of the exogenous gene (e.g., poly(A) sequence), and may optionally include a WPRE or HPRE.

[0327] In some embodiments, the lentiviral nucleic acid includes, e.g., from 5' to 3', one or more, e.g., all, of a promoter (e.g., CMV), an R sequence (e.g., including TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a Psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter for inducing expression of an exogenous substance, a gene encoding the exogenous substance, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).

[0328] Vectors engineered to remove splice sites Some lentiviral vectors have strong splicing and polyadenylation signals that can be integrated within active genes, leading to the formation of aberrant, possibly truncated, transcripts.

[0329] The mechanism of proto-oncogene activation may involve the generation of chimeric transcripts resulting from the interaction of promoter elements or splice sites contained in the genome of the insertional mutagen with the cellular transcription unit targeted by the integration (Gabriel et al. 2009. Nat Med 15:1431-1436, Bokhoven, et al. J Virol 83:283-29). Chimeric fusion transcripts comprising vector sequences and cellular mRNAs can be generated either by read-through transcription, initiating from the vector sequences and proceeding to adjacent cellular genes, or vice versa.

[0330] In some embodiments, the lentiviral nucleic acids described herein comprise a lentiviral backbone in which at least two of the splice sites have been removed, e.g., to improve the safety profile of the lentiviral vector. The types and methods of identifying such splice sites are described in WO2012156839A2, which is incorporated by reference in its entirety.

[0331] Methods for producing retroviruses Large-scale production of viral particles is often useful to achieve a desired viral titer. Viral particles can be produced by transfecting a transfer vector into a packaging cell line containing viral structural and / or accessory genes, such as the gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes.

[0332] In embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Typically, the packaging vector is contained in packaging cells and introduced into the cells via transfection, transduction, or infection. A retroviral, e.g., lentiviral, transfer vector can be introduced into a packaging cell line via transfection, transduction, or infection to generate an original cell or cell line. The packaging vector can be introduced into human cells or cell lines by standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into the cells with a dominant selectable marker, e.g., neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug to isolate clones. The selectable marker gene may be physically linked to the gene encoding it by the packaging vector, for example, by an IRES or a self-cleaving viral peptide.

[0333] Packaging cell lines include cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replicative enzymes (e.g., gag, pol, and env) that are capable of packaging viral particles. Any suitable cell line, for example, mammalian cells, e.g., human cells, can be used. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0334] Source cell lines include cell lines capable of producing recombinant retroviral particles, including packaging cell lines and transfer vector constructs containing packaging signals. Methods for preparing virus stock solutions are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and NRL Landau et al. (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference. Infectious virus particles can be recovered from packaging cells, for example, by cell lysis or by collecting the cell culture supernatant. Optionally, the recovered virus particles can be concentrated or purified.

[0335] Plasmid and cell line packaging In some embodiments, source cells used as packaging cell lines contain one or more plasmids encoding viral structural proteins and replicative enzymes (e.g., gag, pol, and env) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences encoding the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids encoding the viral structural proteins and replicative enzymes are transfected simultaneously or at different times. In some embodiments, the plasmids encoding the viral structural proteins and replicative enzymes are transfected simultaneously with the packaging vector or at different times.

[0336] In some embodiments, the source cell line contains one or more stably integrated viral structural genes, hi some embodiments, expression of the stably integrated viral structural genes is inducible.

[0337] In some embodiments, expression of the viral structural genes is regulated at the transcriptional level, in some embodiments, expression of the viral structural genes is regulated at the translational level, in some embodiments, expression of the viral structural genes is regulated at the post-translational level.

[0338] In some embodiments, expression of the viral structural genes is regulated by a tetracycline (Tet)-dependent system, in which the Tet-regulated transcription repressor (Tet-R) binds to DNA sequences contained in the promoter and represses transcription through steric hindrance (Yao et al., 1998; Jones et al., 2005). Addition of doxycycline (dox) releases Tet-R, allowing transcription. Several other suitable transcriptionally regulated promoters, transcription factors, and small molecule inducers are suitable for regulating transcription of viral structural genes.

[0339] In some embodiments, the third-generation lentiviral components human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and envelope proteins under the control of a Tet-regulated promoter and linked to an antibiotic resistance cassette are separately integrated into the origin cell genome, in some embodiments, the origin cell has only one copy of each of the Rev, Gag / Pol, and envelope proteins integrated into its genome.

[0340] In some embodiments, a nucleic acid encoding the exogenous agent (e.g., a retroviral nucleic acid encoding the exogenous agent) is also integrated into the origin cell genome. In some embodiments, the nucleic acid encoding the exogenous agent is maintained episomally. In some embodiments, the nucleic acid encoding the exogenous agent is transfected into the origin cell with Rev, Gag / Pol, and envelope proteins stably integrated into the genome. See, e.g., Milani et al. EMBO Molecular Medicine, 2017, incorporated herein by reference in its entirety.

[0341] In some embodiments, the retroviral nucleic acid described herein is incapable of reverse transcription. Such nucleic acids, in embodiments, are capable of transiently expressing exogenous agents. The retrovirus or fusosome may comprise a non-functional reverse transcriptase protein or may be free of reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a non-functional primer binding site (PBS) and / or att site. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, and S2, or functional equivalents thereof, are non-functional or absent from the retroviral nucleic acid. In embodiments, one or more accessory genes selected from S2, rev, and tat are non-functional or absent from the retroviral nucleic acid.

[0342] Strategies for packaging retroviral nucleic acids Typically, modern retroviral vector systems consist of (1) a viral genome with cis-acting vector sequences for transcription, reverse transcription, integration, translation, and packaging of viral RNA into the viral particle; and (2) a producer cell line expressing the trans-acting retroviral gene sequences (e.g., gag, pol, and env) necessary for the production of viral particles. Complete separation of the cis- and trans-acting vector sequences prevents the virus from sustaining replication for multiple cycles of infection. The generation of live virus can be avoided by several strategies, for example, by minimizing overlap between cis- and trans-acting sequences to avoid recombination.

[0343] Viral vector particles containing sequences that lack or are devoid of viral RNA may be the result of removing or eliminating viral RNA from the sequence. In one embodiment, this can be achieved by using the endogenous packaging signal binding site on gag. Alternatively, the endogenous packaging signal binding site is on pol. In this embodiment, the delivered RNA contains a cognate packaging signal. In another embodiment, a heterologous binding domain (heterologous to gag) located on the delivered RNA and a cognate binding site located on gag or pol can be used to ensure packaging of the delivered RNA. The heterologous sequence can be non-viral or viral, and in that case, can be derived from a different virus. The vector particles can be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. These vector particles can also be used to deliver therapeutic genes of interest, in which case pol is typically included.

[0344] In one embodiment, gag-pol is modified, and this packaging signal is replaced with corresponding packaging signal.In this embodiment, this particle can package this RNA with new packaging signal.The advantage of this approach is that it can package the RNA sequence that does not have viral sequence, for example, RNAi.

[0345] An alternative approach is to rely on the overexpression of the RNA to be packaged.In one embodiment, the RNA to be packaged is overexpressed in the absence of the RNA containing the packaging signal.This can result in a significant level of therapeutic RNA being packaged, and this amount is sufficient to transduce cells and produce biological effects.

[0346] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or retroviral gag and pol proteins, wherein the gag protein or pol protein comprises a heterologous RNA-binding domain capable of recognizing a corresponding sequence in an RNA sequence and facilitating packaging of the RNA sequence into a viral vector particle.

[0347] In some embodiments, the heterologous RNA-binding domain comprises an RNA-binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U1 small nuclear ribonucleoprotein particle, a Nova protein, a TFl 11A protein, a TISl protein, a trp RNA-binding attenuation protein (TRAP), or a pseudouridine synthase.

[0348] In some embodiments, the methods herein include detecting or confirming the absence of a replication-competent retrovirus. The methods may include assessing the RNA level of one or more target genes, such as structural or packaging genes, whose gene products are expressed in certain cells infected with a replication-competent retrovirus, such as a gammaretrovirus or lentivirus, but are not present in the viral vector used to transduce the cells with a heterologous nucleic acid, and are not present and / or expressed, or would not be expected to be present and / or expressed, in cells that do not contain a replication-competent retrovirus. A replication-competent retrovirus may be determined to be present if the RNA level of one or more target genes is higher than a reference value, which can be measured directly or indirectly, for example, from a positive control sample containing the target gene. For further disclosure, see, for example, WO2018023094A1.

[0349] In some embodiments, assembly of the fusosome (i.e., VLP) is initiated by binding of the core protein to a unique encapsidation sequence (e.g., a UTR with a stem-loop structure) within the viral genome, and in some embodiments, the interaction of the core with the encapsidation sequence promotes oligomerization.

[0350] In some embodiments, the source cells for VLP production contain one or more plasmids (i.e., packaging plasmids) encoding viral structural proteins (e.g., gag, pol) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag and pol precursors are on the same plasmid. In some embodiments, the sequences encoding the gag and pol precursors are on different plasmids. In some embodiments, the sequences encoding the gag and pol precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag and pol precursors have different expression signals, e.g., different promoters. In some embodiments, expression of the gag and pol precursors is inducible.

[0351] In some embodiments, the formation of the VLPs or any viral vectors described above can be detected by any suitable technique known in the art, including, for example, electron microscopy, dynamic light scattering, selective chromatographic separation and / or density gradient centrifugation.

[0352] Repression of genes encoding exogenous agents in the cell of origin The (over)expressed proteins in the cell of origin may indirectly or directly affect the assembly and / or infectivity of the vector virion, and the incorporation of the exogenous agent into the vector virion may also affect downstream processing of the vector particle.

[0353] In some embodiments, a tissue-specific promoter is used to restrict the expression of the exogenous agent contained in the source cell. In some embodiments, a heterologous translational control system is used in eukaryotic cell culture to suppress the translation of the exogenous agent contained in the source cell. More specifically, the retroviral nucleic acid may contain a binding site operably linked to a gene encoding the exogenous agent, the binding site being capable of interacting with an RNA-binding protein such that translation of the exogenous agent is suppressed or prevented in the source cell.

[0354] In some embodiments, the RNA-binding protein is a tryptophan RNA-binding attenuation protein (TRAP), e.g., a bacterial tryptophan RNA-binding attenuation protein. The use of an RNA-binding protein (e.g., a bacterial trp operon regulatory protein, tryptophan RNA-binding attenuation protein, TRAP) and its bound RNA target represses or prevents translation of a transgene in a source cell. This system is referred to as a Transgene Repression In Vector Production cell system, or TRIP system.

[0355] In embodiments, placing a binding site for an RNA-binding protein (e.g., a TRAP binding sequence, tbs) upstream of the NOI translation start codon allows for specific repression of translation of mRNA derived from an internal expression cassette, while not adversely affecting the production or stability of vector RNA. The number of nucleotides between the tbs and the translation start codon of the gene encoding the exogenous agent can vary from 0 to 12 nucleotides. The tbs can be placed downstream of an internal ribosome entry site (IRES) to repress translation of the gene encoding the exogenous agent in a multicistronic mRNA.

[0356] Kill Switches and Amplification In some embodiments, the polynucleotide or cell carrying the gene encoding the exogenous agent utilizes a suicide gene, e.g., an inducible suicide gene, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain aspects, the suicide gene is not immunogenic to the host cell carrying the exogenous agent. Examples of suicide genes include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using a specific chemical inducer of dimerization (CID).

[0357] In certain embodiments, the vector comprises a gene segment that renders target cells, e.g., immune effector cells, e.g., T cells, susceptible to negative selection in vivo. For example, transduced cells may be eliminated as a result of changes in an individual's in vivo conditions. A negatively selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, e.g., a compound. Negatively selectable genes are known in the art and include, among others, the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 11:223, 1977), the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA 89:33 (1992)).

[0358] In some embodiments, transduced cells, e.g., immune effector cells such as T cells, comprise a polynucleotide that further comprises a positive marker that allows for the selection of cells with a negatively selectable phenotype in vitro. A positively selectable marker can be a gene that, when introduced into a target cell, expresses a dominant phenotype that allows for the positive selection of cells carrying the gene. Genes of this type include, among others, the hygromycin B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and multidrug resistance (MDR) genes.

[0359] In some embodiments, the positive selectable marker and negative selectable element are linked such that loss of the negative selectable element necessarily results in loss of the positive selectable marker. For example, the positive and negative selectable markers can be fused so that loss of one necessarily results in loss of the other. An example of a fusion polynucleotide whose expression product is a polypeptide that confers both the desired positive and negative selection functions is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene results in a polypeptide that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection. See Lupton SD, et al., Mol. and Cell. Biology 1 1:3374-3378, 1991. Further, in embodiments, the polynucleotide encoding the chimeric receptor is a retroviral vector containing the fusion gene, particularly one that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection, e.g., Lupton, SD et al., supra. (1991) are present in the HyTK retroviral vector. See also publications PCT U591 / 08442 and PCT / U594 / 05601, which describe the use of bifunctional selectable fusion genes derived from the fusion of a dominant positive selectable marker and a negative selectable marker.

[0360] Suitable positive selectable markers may be derived from genes selected from the group consisting of hph, nco, and gpt, and suitable negative selectable markers may be derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. Other suitable markers are bifunctional selectable fusion genes in which the positive selectable marker is derived from hph or neo and the negative selectable marker is derived from the cytosine deaminase or TK genes or selectable markers.

[0361] Strategies for modulating lentiviral integration Retroviral and lentiviral nucleic acids lacking or non-functional essential proteins / sequences have been disclosed to prevent retroviral or lentiviral genome integration into target cell genomes. For example, viral nucleic acids lacking each of the amino acids constituting the highly conserved DDE motif of retroviral integrase (Engelman and Craigie (1992) J. Virol. 66:6361-6369; Johnson et al. (1986) Proc. Natl. Acad. Sci. USA 83:7648-7652; Khan et al. (1991) Nucleic Acids Res. 19:851-860) allow the production of integration-deficient retroviral nucleic acids.

[0362] For example, in some embodiments, a retroviral nucleic acid herein comprises a lentiviral integrase containing a mutation that renders such integrase unable to catalyze the integration of the viral genome into the cellular genome. In some embodiments, such mutations are type I mutations that directly affect the integration, or type II mutations that induce pleiotropic defects that affect virion morphogenesis and / or reverse transcription. A non-limiting example of a helpful type I mutation is the mutation of three residues involved in the catalytic core domain of integrase: DX 39-58 DX 35 The mutation affecting any of residues D64, D116, and E152 of HIV-1 integrase is in particular an integrase-dependent mutation that renders the integrase unable to catalyze the integration of the viral genome into the cellular genome. In particular embodiments, the mutation is a substitution of one or more amino acid residues of the DDE motif of the catalytic core domain of the integrase, preferably a substitution of the first aspartic acid residue of the DDE motif with an asparagine residue. In some embodiments, the retroviral vector does not comprise an integrase protein.

[0363] In some embodiments, the retrovirus integrates into an active transcription unit. In some embodiments, the retrovirus does not integrate near the transcription start site, the 5' end of a gene, or a DNAse 1 cleavage site. In some embodiments, the retrovirus integration does not activate proto-oncogenes or inactivate tumor suppressor genes. In some embodiments, the retrovirus is not genotoxic. In some embodiments, the lentivirus integrates into an intron.

[0364] In some embodiments, the retroviral nucleic acid is integrated into the genome of the target cell at a specific copy number. The average copy number can be measured from a single cell, a cell population, or an individual cell colony. Exemplary methods for measuring copy number include polymerase chain reaction (PCR) and flow cytometry.

[0365] In some embodiments, the DNA encoding the exogenous agent is integrated into the genome. In some embodiments, the DNA encoding the exogenous agent is maintained episomally. In some embodiments, the ratio of the exogenous agent integrated into the episomal DNA encoding the exogenous agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, 100.

[0366] In some embodiments, the DNA encoding the exogenous agent is linear. In some embodiments, the DNA encoding the exogenous agent is circular. In some embodiments, the ratio of linear copies to circular copies of the DNA encoding the exogenous agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, 100.

[0367] In some embodiments, the DNA encoding the exogenous agent is circular and has one LTR. In some embodiments, the DNA encoding the exogenous agent is circular and has two LTRs. In some embodiments, the ratio of the circular DNA encoding the exogenous agent containing one LTR to the circular DNA encoding the exogenous agent containing two LTRs is at least 0.1, 0.5, 1.0, 2, 5, 10, 20, 50, 100.

[0368] Episomal virus maintenance In retroviruses that are poorly integrated, circular cDNA by-products of reverse transcription (e.g., 1-LTR and 2-LTR) can accumulate in the cell nucleus without being integrated into the host genome (see Yanez-Munoz RJ et al., Nat. Med. 2006, 12:348-353). As with other exogenous DNA, these intermediates are subsequently integrated into cellular DNA at the same frequency (e.g., 10 3 ~10 5 / cell).

[0369] In some embodiments, episomal retroviral nucleic acids do not replicate. Episomal viral DNA can be modified to be maintained in replicating cells through the inclusion of a eukaryotic origin of replication and a scaffold / matrix attachment region (S / MAR) for association with the nuclear matrix.

[0370] Thus, in some embodiments, the retroviral nucleic acids described herein comprise a eukaryotic origin of replication or a variant thereof. Examples of eukaryotic origins of replication of interest include the origin of replication of the β-globin gene described by Aladjem et al. (Science, 1995, 270:815-819), the consensus sequence derived from an autonomously replicating sequence associated with an alpha-satellite sequence previously isolated from monkey CV-1 cells and human skin fibroblasts described by Price et al. Journal of Biological Chemistry, 2003, 278(22):19649-59, and the origin of replication of the human c-myc promoter region described by McWinney and Leffak (McWinney C. and Leffak M., Nucleic Acid Research 1990, 18(5):1233-42). In embodiments, the variants substantially retain the ability to initiate replication in eukaryotes. The ability of a particular sequence to initiate replication can be measured by any suitable method, for example, by autonomous replication assays based on bromodeoxyuridine incorporation and density shift (Araujo FD et al., supra; Frappier L. et al., supra).

[0371] In some embodiments, the retroviral nucleic acid comprises a scaffold / matrix attachment region (S / MAR) or a variant thereof, e.g., a non-consensus-like AT-rich DNA element several hundred base pairs in length, which organizes the nuclear DNA of the eukaryotic genome into chromatin domains by periodic attachment to the protein scaffold or matrix of the cell nucleus. They are usually found in non-coding regions such as flanking regions, chromatin boundary regions, and introns. An example of an S / MAR region is the S / MAR of the 1.8 kbp human IFN-γ gene (hIFN-γ) described by Bode et al. (Bode J. et al., Science, 1992, 255:195-7). large ), the 0.7 Kbp S / MAR minimal region of the human IFN-γ gene (hIFN-γ) described by Ramezani (Ramezani A. et al., Blood 2003, 101:4717-24) short), Mesner LD et al., Proc Natl Acad Sci USA, 2003, 100:3281-86 (the 0.2 Kbp minimal region of the S / MAR of the human dehydrofolate reductase gene (hDHFR). In an embodiment, the functionally equivalent variant of the S / MAR is a sequence selected based on a set of six rules that, together or individually, have been suggested to contribute to the function of the S / MAR (Kramer et al. (1996) Genomics 33, 305; Singh et al. (1997) Nucl. Acids Res 25, 1419). These rules have been integrated into the MAR-Wiz computer program, which is freely available at genomecluster.secs.oakland.edu / MAR-Wiz. In an embodiment, the variant substantially maintains the same function of the S / MAR from which it is derived, in particular, its ability to specifically bind to the nuclear matrix. Those skilled in the art can determine whether a particular variant can specifically bind to the nuclear matrix by, for example, an in vitro or in vivo MAR assay, as described by Mesner et al. (Mesner LD et al., supra). In some embodiments, a particular sequence is an S / MAR variant if the particular variant exhibits a tendency for DNA strand separation. This property can be measured using specific programs based on the method of equilibrium statistical mechanics. The stress-induced duplex destabilization (SIDD) analysis method is defined in Bode et al. (2005) J. Mol. Biol. 358, 597 as "calculating the degree to which an imposed level of superhelical stress reduces the free energy required to open the duplex at each position along the DNA sequence. The results are displayed as a SIDD profile, in which sites of strong destabilization appear as deep minima."The SIDD algorithm and mathematical basis (Bi and Benham (2004) Bioinformatics 20, 1477) and analysis of SIDD profiles can be performed using the freely available internet resource WebSIDD (www.genomecenter.ucdavis.edu / benham). Thus, in some embodiments, a polynucleotide is considered to be a variant of an S / MAR if it exhibits a similar SIDD profile as the S / MAR.

[0372] Fusogens and pseudotyping For example, fusogens, including viral envelope proteins (env), generally determine the range of host cells that can be infected and transformed by fusosomes. In some embodiments, the fusosomes herein comprise a henipavirus F protein molecule and a henipavirus G protein molecule. In some embodiments, the henipavirus F protein molecule and / or the henipavirus G protein molecule contribute to the fusion of the fusosome with a cell membrane. For example, the henipavirus F protein molecule can mediate fusion between the membrane of the fusosome and a cell membrane, such as the cell membrane of a desired target cell. The henipavirus G protein can, for example, bind to a molecule (e.g., a polypeptide) on the surface of the target cell.

[0373] Examples of retroviral env genes that can be used as fusogens include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (fowl plague virus), and influenza virus envelopes. Similarly, genes encoding envelopes from RNA viruses (e.g., Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, and RNA virus families of Retroviridae) and DNA viruses (Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Iridoviridae) can be used. Representative examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV. For lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, native env proteins include gp41 and gp120. In some embodiments, the viral env proteins expressed by the source cells described herein are encoded on a separate vector from the viral gag and pol genes, as previously described.

[0374] In some embodiments, envelope proteins for presentation on fusosomes include, but are not limited to, any of the following origins: influenza A, e.g., H1N1, H1N2, H3N2, and H5N1 (avian influenza), influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any of the Norwalk virus group viruses, enteric adenovirus, parvovirus, dengue virus , monkeypox, Mononegavirales, Lyssaviruses such as rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 and 2 and Australian bat virus, Ephemelovirus, Vesiculovirus, Vesicular stomatitis virus (VSV), Herpesviruses such as herpes simplex virus types 1 and 2, varicella zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus Viruses 6 and 8, human immunodeficiency virus (HIV), papillomaviruses, murine gammaherpesviruses, arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridae such as Crimean-Congo hemorrhagic fever virus, hantaviruses, viruses causing hemorrhagic fever with renal syndrome, Rift Valley fever virus, Ebola Filoviridae (filoviruses), including hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae (including Kyasanur Forest disease virus, Omsk hemorrhagic fever virus, and viruses that cause tick-borne encephalitis), and Paramyxoviridae, such as Hendra virus and Nipah virus, variola major and variola minor (smallpox), Alphaviruses, such as Venezuelan equine encephalitis virus, eastern equine encephalitis virus, western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephalitis-causing virus.

[0375] Fusosomes or pseudotyped viruses generally have modifications in one or more of their envelope proteins, e.g., the envelope protein is replaced with an envelope protein from another virus. For example, HIV can be pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope protein, allowing HIV to infect a wider range of cells. This is because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4+ presenting cells. In some embodiments, lentiviral envelope proteins are pseudotyped with VSV-G. In one embodiment, the source cell produces a recombinant retrovirus, e.g., a lentivirus, pseudotyped with the VSV-G envelope glycoprotein. In some embodiments, the source cell described herein produces a fusosome, e.g., a recombinant retrovirus, e.g., a lentivirus, pseudotyped with the VSV-G glycoprotein.

[0376] Furthermore, fusogens or viral envelope proteins can be modified or engineered to contain polypeptide sequences that enable the transduction vector to target and infect host cells outside its normal range or to more specifically restrict transduction to a cell or tissue type. For example, the fusogens or envelope proteins can be linked in-frame to targeting sequences, such as receptor ligands, antibodies (using antigen-binding portions of antibodies or recombinant antibody-type molecules, e.g., single-chain antibodies), and polypeptide moieties or modifications thereof (e.g., glycosylation sites present in the targeting sequence), which, when presented on the transduction vector coat, facilitate the directional delivery of virion particles to the intended target cells. Furthermore, envelope proteins can further contain sequences that regulate cellular function. Modulation of cellular function by transduction vectors can increase or decrease the transduction efficiency of certain cell types in a mixed population of cells. For example, stem cells can be more specifically transduced with envelope sequences containing ligands or binding partners that specifically bind to stem cells but not other cell types found in blood or bone marrow. Non-limiting examples are stem cell factor (SCF) and Flt-3 ligand. Other examples include, for example, antibodies (e.g., cell type-specific single chain antibodies) and essentially any antigen (including receptors) that bind to tissues such as lung, liver, pancreas, heart, endothelial, smooth, breast, prostate, epithelial, vascular cancer, etc.

[0377] Exemplary Fusogens In some embodiments, the fusosome comprises one or more fusogens, e.g., that promote fusion of the fusosome to a membrane, e.g., a cell membrane, hi some embodiments, the one or more fusogens comprise a Henipavirus F protein molecule (e.g., an activated Henipavirus F protein molecule) and / or a Henipavirus G protein molecule.

[0378] In some embodiments, the retroviral vector or fusosome contains one or more fusogens on its envelope to target specific cell or tissue types. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. In some embodiments, the retroviral vector or fusosome contains a first fusogen that is a protein fusogen and a second fusogen that is a lipid fusogen or a chemical fusogen. The fusogen can bind to a fusogen-binding partner on the surface of the target cell. In some embodiments, the fusogen-containing fusosome integrates its membrane into the lipid bilayer of the target cell.

[0379] In some embodiments, one or more of the fusogens described herein may be included in the fusosomes.

[0380] Protein fusogens In some embodiments, the fusogen is a protein fusogen, e.g., a mammalian protein or a homolog of a mammalian protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a non-mammalian protein, e.g., a viral protein or a homolog of a viral protein (e.g., having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a naturally occurring protein or a derivative of a naturally occurring protein, a synthetic protein, a fragment thereof, a variant thereof, a protein fusion or fragment comprising one or more fusogens, and any combination thereof. In some embodiments, the protein fusogen comprises a Henipavirus F protein molecule (e.g., an activated Henipavirus F protein molecule) and / or a Henipavirus G protein molecule.

[0381] In some embodiments, the fusogen causes intermixing between lipids in the retroviral vector or fusosome and lipids in the target cell, hi some embodiments, the fusogen causes the formation of one or more pores between the interior of the retroviral vector or fusosome and the cytoplasm of the target cell.

[0382] Mammalian proteins In some embodiments, the fusogen comprises a mammalian protein. See, e.g., Table 1. Examples of mammalian fusogens include SNARE family proteins, e.g., vSNAREs and tSNAREs, syncytin proteins, e.g., syncytin-1 (DOI: 10.1128 / JVI.76.13.6442-6452.2002), and syncytin-2, myomaker (biorxiv.org / content / early / 2017 / 04 / 02 / 123158, doi.org / 10.1101 / 123158, doi:10.1096 / fj.201600945R, doi:10.10 38 / nature12343), myomixer (www.nature.com / nature / journal / v499 / n7458 / full / nature12343.html, doi:10.1038 / nature12343), myomerger (science.sciencemag.org / content / early / 2017 / 04 / 05 / science.aam9361, DOI:10.1126 / science.aam9361), FGFRL1 (fibroblast growth factor receptor 1 (FGFRL) receptor-like 1), Minion (doi.org / 10.1101 / 122697), isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (e.g., those disclosed in US 6,099,857A), gap junction proteins, such as connexin 43, connexin 40, connexin 45, connexin 32, or connexin 37 (e.g., those disclosed in US 2007 / 0224176), Hap2, any protein capable of inducing syncytium formation between heterologous cells (see Table 2), Examples of fusogenic proteins include, but are not limited to, any protein having fusogenic properties (see Table 3), its homologs, fragments, variants, and protein fusions comprising one or more proteins or fragments thereof. In some embodiments, the fusogen is encoded by a human endogenous retroviral element (hERV) found in the human genome. Additional exemplary fusogens are disclosed in US 6,099,857A and US 2007 / 0224176, the entire contents of which are incorporated herein by reference. [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3]

[0383] In some embodiments, the retroviral vector or fusosome comprises a curvature-generating protein, such as Epsin1, dynamin, or a protein containing a BAR domain. See, e.g., Kozlovet et al., CurrOp StrucBio 2015, Zimmerberg et al. Nat Rev 2006, Richard et al. See al, Biochem J 2011.

[0384] Non-mammalian proteins viral proteins In some embodiments, the fusogen can include a non-mammalian protein, such as a viral protein. In some embodiments, the viral fusogen is a henipavirus F protein (e.g., an activated henipavirus F protein). In some embodiments, the viral fusogen is a class I viral membrane fusion protein, a class II viral membrane protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion protein, or a homolog thereof, a fragment thereof, a variant thereof, or a protein fusion comprising one or more proteins or fragments thereof.

[0385] In some embodiments, class I viral membrane fusion proteins include, but are not limited to, baculovirus F proteins, such as F proteins of the genus nuclear polyhedrosis virus (NPV), for example, Spodoptera exigua MNPV (SeMNPV) F protein and Lymantria dispar MNPV (LdMNPV), and paramyxovirus F proteins.

[0386] In some embodiments, class II viral membrane proteins include, but are not limited to, tick-borne encephalitis E (TBEV E), Semliki Forest virus E1 / E2.

[0387] In some embodiments, class III viral membrane fusion proteins include, but are not limited to, rhabdovirus G (e.g., vesicular stomatitis virus fusion protein G (VSV-G)), herpesvirus glycoprotein B (e.g., herpes simplex virus 1 (HSV-1) gB)), Epstein-Barr virus glycoprotein B (EBV gB), Thogotovirus G, baculovirus gp64 (e.g., Autographa California multiple NPV (AcMNPV) gp64), and Borna disease virus (BDV) glycoprotein (BDV G).

[0388] Examples of other viral fusogens, such as membrane glycoproteins and viral fusion proteins, include, but are not limited to, viral syncytial proteins such as influenza hemagglutinin (HA) or mutants, or fusion proteins thereof, human immunodeficiency virus type 1 envelope protein (HIV-1 ENV), HIV binding LFA-1-derived gp120 to form lymphocyte syncytia, HIV gp41, HIV gp160, or transactivator of transcription (TAT) of HIV, viral glycoprotein VSV-G, viral glycoprotein from vesicular stomatitis virus of the Rhabdoviridae family, glycoproteins gB and gH-gL of varicella-zoster virus (VZV), murine leukemia virus (MLV)-10A1, gibbon ape leukemia virus glycoprotein (GaLV), G-type glycoproteins of rabies, Mokola, vesicular stomatitis, and togaviruses, mouse hepatitis virus JHM surface spike protein, porcine respiratory coronavirus spike and membrane glycoproteins, avian infectious bronchitis spike glycoprotein and its precursor, bovine enteric coronavirus spike protein, measles virus F and H, HN, or G genes of canine distemper virus, Newcastle disease virus, human parainfluenza virus 3, simian virus 41, Sendai virus, and human respiratory syncytial virus, human herpesvirus 1 and simian varicella virus gH with chaperone protein gL, human, bovine, and cercopithecoid herpesvirus gB, envelope glycoproteins of Friend murine leukemia virus and Mason-Pfizer monkey virus, mumps virus hemagglutinin neuraminidase, and glioproteins F1 and F2, membrane glycoprotein from Venezuelan equine encephalomyelitis, paramyxovirus F protein, SIV gp160 protein, Ebola virus G protein, or Sendai virus fusion protein, or homologs, fragments, variants, and protein fusions comprising one or more proteins or fragments thereof.

[0389] Non-mammalian fusogens include viral fusogens, their homologs, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof. Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In embodiments, class I fusogens, such as human immunodeficiency virus (HIV) gp41, have a characteristic post-fusion conformation with a signature trimer of α-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, orthomyxovirus hemagglutinin, paramyxovirus F proteins (e.g., measles (Katoh et al. BMC Biotechnology 2010, 10:37)), retrovirus ENV proteins, and filovirus and coronavirus fusogens. In embodiments, class II viral fusogens, such as dengue E glycoprotein, have the structural characteristics of a beta sheet that refolds to form an elongated ectodomain resulting in a trimer of hairpins. In embodiments, the class II viral fusogen lacks a central coiled-coil. Class II viral fusogens can be found in alphaviruses (e.g., E1 proteins) and flaviviruses (e.g., E glycoproteins). Class II viral fusogens include fusogens from Semliki Forest virus, Sinbis virus, rubella virus, and dengue virus. In embodiments, class III viral fusogens, such as vesicular stomatitis virus G glycoprotein, combine structural features found in classes I and II. In embodiments, class III viral fusogens resemble class II viral fusogens, comprising an alpha helix (e.g., the protein folds back to form a six-helix bundle, similar to class I viral fusogens) and a beta sheet with an amphipathic fusion peptide at its terminus. Class III viral fusogens can be found in rhabdoviruses and herpesviruses.In embodiments, the class IV viral fusogen is a fusion-associated small transmembrane (FAST) protein (doi:10.1038 / sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins" (2012). Electronic Thesis and Dissertation Repository. Paper 388), which is encoded by non-enveloped reovirus. In embodiments, the class IV viral fusogen is sufficiently small to not form a hairpin (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0390] In some embodiments, the fusogen is a paramyxovirus fusogen. In some embodiments, the fusogen is a henipavirus fusogen, such as from any of the viruses listed in Table 3A. In some embodiments, the fusogen is Nipah virus protein F, measles virus F protein, tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, henipavirus F protein, morbillivirus F protein, respirovirus F protein, Sendai virus F protein, rubulavirus F protein, or avulavirus F protein.

[0391] In some embodiments, the fusogen is a Poxviridae fusogen.

[0392] Further exemplary fusogens are disclosed in US9,695,446, US2004 / 0028687, US6,416,997, US7,329,807, US2017 / 0112773, US2009 / 0202622, WO2006 / 027202, and US2004 / 0009604, the entire contents of all of which are incorporated herein by reference. [Table 4]

[0393] In some embodiments, the fusogen comprises a protein having a hydrophobic fusogenic peptide domain. In some embodiments, the fusogen comprises a Henipavirus F protein molecule or a biologically active portion thereof. In some embodiments, the Henipavirus F protein is Hendra (Hev) virus F protein, Nipah (NiV) virus F protein, Cedar (CedPV) virus F protein, Mojiang virus F protein, or a bat paramyxovirus F protein, or a biologically active portion thereof.

[0394] Table 4 provides non-limiting examples of F proteins. In some embodiments, the N-terminal hydrophobic fusion peptide domain of the F protein molecule or biologically active portion thereof is exposed to the outside of the lipid bilayer.

[0395] Henipavirus F proteins are encoded as F0 precursors containing a signal peptide (e.g., corresponding to amino acid residues 1-26 of SEQ ID NO:7). After cleavage of the signal peptide, mature F0 (e.g., SEQ ID NO:13) is transported to the cell surface and then internalized. It is cleaved by cathepsin L (e.g., between amino acids 109-110 of SEQ ID NO:7) into mature fusogenic subunits F1 (e.g., corresponding to amino acids 110-546 of SEQ ID NO:7, shown in SEQ ID NO:15) and F2 (e.g., corresponding to amino acid residues 27-109 of SEQ ID NO:7, shown in SEQ ID NO:14). The F1 and F2 subunits associate via disulfide bonds and are recycled back to the cell surface. The F1 subunit contains a fusion peptide domain (e.g., corresponding to amino acids 110-129 of SEQ ID NO:7) located at its N-terminus, which can insert into the cell membrane to drive fusion. In certain cases, fusion activity is blocked by association of the F protein with the G protein; when G engages a target molecule and dissociates from F, the fusion peptide is exposed and mediates membrane fusion.

[0396] The sequence and activity of the F protein is highly conserved among different henipavirus species. For example, the F proteins of NiV and HeV viruses share 89% amino acid sequence identity. Furthermore, in some cases, henipavirus F proteins show compatibility with G proteins from other species to induce fusion (Brandel-Tretheway et al., 2003). et al. Journal of Virology. 2019. 93(13):e00577-19). In some embodiments or provided fusosomes, the F protein is heterologous to the G protein, i.e., the F and G proteins or biologically active portions are from different Henipavirus species. For example, the F protein is from Hendra virus and the G protein is from Nipah virus. In other embodiments, the F protein can be a chimeric F protein comprising regions of F proteins from different Henipavirus species. In some embodiments, exchanging a region of amino acid residues of the F protein from one Henipavirus species with another can result in fusion to the G protein of the species containing the amino acid insertion. (Brandel-Tretheway et al. 2019). In some cases, the chimeric F protein comprises an extracellular domain from one Henipavirus species and a transmembrane and / or cytoplasmic domain from a different Henipavirus species. For example, the F protein comprises the extracellular domain of Hendra virus and the transmembrane / cytoplasmic domain of Nipah virus. The F protein sequences disclosed herein are primarily disclosed as expressed sequences that include an N-terminal signal sequence. Because such N-terminal signal sequences are generally cleaved co- or post-translationally, the mature protein sequences for all F protein sequences disclosed herein are also contemplated as lacking an N-terminal signal sequence.

[0397] In some embodiments, the F protein is encoded by a nucleotide sequence that encodes the sequence set forth in any one of SEQ ID NOs: 3-7, or a functionally active variant or biologically active portion thereof having a sequence that is at least or about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 3-7. In certain embodiments, the F protein or functionally active variant or biologically active portion thereof retains fusion activity in conjunction with a Henipavirus G protein, e.g., a G protein set forth in Table 5 (e.g., NiV-G or HeV-G). Fusion activity includes the activity of the F protein, in conjunction with the Henipavirus G protein, to promote or facilitate fusion of two membrane spaces, e.g., the space of a targeted lipid particle having Henipavirus F and G proteins embedded in its lipid bilayer, and the cytoplasm of a target cell, e.g., a cell containing a surface receptor or molecule recognized by or bound by the targeting envelope protein. In some embodiments, the F protein and G protein are from the same Henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different Henipavirus species (e.g., NiV-G and HeV-F). In certain embodiments, the F protein or functionally active variant or biologically active portion retains a cleavage site cleaved by cathepsin L (e.g., corresponding to the cleavage site between amino acids 109 and 110 of SEQ ID NO: 7).

[0398] Reference to retaining fusion activity includes between or exceeding 10% or about 10% and 150% or about 150% of the level or degree of binding of the corresponding wild-type F protein as set forth in SEQ ID NOs: 3-7, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 35% or at least about 35% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 40% or at least about 40% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type F protein, e. at least 45% or at least about 45% of the level or degree of fusion activity of the native F protein, for example, at least 50% or at least about 50% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 55% or at least about 55% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 60% or at least about 60% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 65% or at least about 65% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 70% or at least about 70% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 75% or at least about 75% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 80% or at least about 80% of the level or degree of fusion activity of the corresponding wild-type F protein, for example, at least 85% or at least about 85% of the level or degree of fusion activity of the corresponding wild-type F protein, for example,This includes activity (in combination with a Henipavirus G protein) that is at least 90% or at least about 90% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 95% or at least about 95% of the level or degree of fusion activity of the corresponding wild-type F protein, e.g., at least 100% or at least about 100% of the level or degree of fusion activity of the corresponding wild-type F protein, or e.g., at least 120% or at least about 120% of the level or degree of fusion activity of the corresponding wild-type F protein.

[0399] In some embodiments, the F protein is a mutant F protein, which is a functionally active fragment or biologically active portion thereof, comprising one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations relative to a reference F protein sequence. In some embodiments, the reference F protein sequence is a wild-type sequence of an F protein or a biologically active portion thereof. In some embodiments, the mutant F protein or a biologically active portion thereof is a mutant of a wild-type Hendra (Hev) virus F protein, Nipah (NiV) virus F protein, Cedar (CedPV) virus F protein, Mojiang virus F protein, or bat paramyxovirus F protein. In some embodiments, the wild-type F protein is encoded by a sequence of nucleotides encoding any one of SEQ ID NOs: 3-7.

[0400] In some embodiments, the Henipavirus F protein molecules described herein comprise an amino acid sequence in Table 4 (e.g., any of SEQ ID NOS:3-7), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., a 100, 200, 300, 400, 500, or 600 amino acid long portion thereof, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. For example, in some embodiments, the Henipavirus F protein molecules described herein comprise an amino acid sequence having at least 80% identity to any of the amino acid sequences in Table 4. In some embodiments, the Henipavirus F protein molecules described herein comprise an amino acid sequence having at least 80% identity to SEQ ID NO:3. In some embodiments, the Henipavirus F protein molecules described herein comprise an amino acid sequence having at least 80% identity to SEQ ID NO:4. In some embodiments, the henipavirus F protein molecules described herein comprise an amino acid sequence at least 80% identical to SEQ ID NO: 5. In some embodiments, the henipavirus F protein molecules described herein comprise an amino acid sequence at least 80% identical to SEQ ID NO: 6. In some embodiments, the henipavirus F protein molecules described herein comprise an amino acid sequence at least 80% identical to SEQ ID NO: 7. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence in Table 4, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, e.g., a 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acid long portion thereof. [Table 5-1] [Table 5-2] [Table 5-3]

[0401] In some embodiments, the mutant F protein is a biologically active portion of a wild-type F protein that is an N-terminally and / or C-terminally truncated fragment. In some embodiments, the mutant F protein, or a biologically active portion thereof, of the wild-type F protein comprises one or more amino acid substitutions. In some embodiments, the mutations described herein may improve transduction efficiency. In some embodiments, the mutations described herein may increase fusion capability. Exemplary mutations include any of those described, see, e.g., Khetawat and Broder 2010 Virology Journal 7:312; Witting et al. 2013 Gene Therapy 20:997-1005; and published international patent application WO / 2013 / 148327.

[0402] In some embodiments, the mutant F protein is truncated and is a biologically active portion that lacks up to 20 contiguous amino acid residues at or near the C-terminus of a wild-type F protein, such as a wild-type F protein encoded by a sequence of nucleotides encoding the F protein set forth in any one of SEQ ID NOs: 3 to 7. In some embodiments, the mutant F protein is truncated and lacks up to 19 contiguous amino acids, e.g., up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 contiguous amino acid at the C-terminus of the wild-type F protein.

[0403] In some embodiments, the F protein, or functionally active variant or biologically active portion thereof, comprises an F1 subunit or fusogenic portion thereof. In some embodiments, the F1 subunit is a proteolytically cleaved portion of an F0 precursor. In some embodiments, the F0 precursor is inactive. In some embodiments, cleavage of the F0 precursor forms a disulfide-linked F1+F2 heterodimer. In some embodiments, the cleavage exposes a fusion peptide and produces a mature F protein. In some embodiments, the cleavage occurs at or around a single basic residue. In some embodiments, the cleavage occurs at arginine 109 of the NiV-F protein. In some embodiments, the cleavage occurs at lysine 109 of the Hendra virus F protein.

[0404] In some embodiments, the F protein is a wild-type Nipah virus F (NiV-F) protein, or a functionally active variant or biologically active portion thereof. In some embodiments, the F precursor is encoded by a sequence of nucleotides that encodes the sequence set forth in SEQ ID NO:7. The encoding nucleic acid may encode a signal peptide sequence having the sequence MVVILDKRCY CNLLILILMI SECSVG (SEQ ID NO:16). In some embodiments, the F protein has the sequence set forth in SEQ ID NO:13. In some examples, the F protein is cleaved into an F1 subunit comprising the sequence set forth in SEQ ID NO:15 and an F2 subunit comprising the sequence set forth in SEQ ID NO:14.

[0405] In some embodiments, the F protein or functionally active variant or biologically active portion thereof includes an Fl subunit having a sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.

[0406] In some embodiments, the F protein or functionally active variant or biologically active portion thereof includes an F2 subunit having a sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least or about 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, or at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14.

[0407] In some embodiments, the F protein is a mutant NiV-F protein that is a biologically active portion thereof comprising a 22 amino acid truncation at or near the C-terminus of a wild-type NiV-F protein (SEQ ID NO: 7 or 13). In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO: 17. In some embodiments, the NiV-F protein is encoded by a nucleotide sequence that encodes a sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 17. In some embodiments, the NiV-F protein has the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the NiV-F protein has an amino acid sequence that has at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 17.

[0408] In some embodiments, the G protein is a Henipavirus G protein or a biologically active portion thereof. In some embodiments, the Henipavirus G protein is a Hendra (HeV) virus G protein, a Nipah (NiV) virus G protein (NiV-G), a Cedar (CedPV) virus G protein, a Mojiang virus G protein, a bat paramyxovirus G protein, or a biologically active portion thereof. Table 5 provides non-limiting examples of G proteins.

[0409] This G-binding protein is a type II transmembrane glycoprotein comprising an N-terminal cytoplasmic tail (e.g., corresponding to amino acids 1-49 of SEQ ID NO: 9), a transmembrane domain (e.g., corresponding to amino acids 50-70 of SEQ ID NO: 9), an extracellular domain including an extracellular stalk (e.g., corresponding to amino acids 71-187 of SEQ ID NO: 9), and a globular head (e.g., corresponding to amino acids 188-602 of SEQ ID NO: 9). The N-terminal cytoplasmic domain is located within the inner lumen of the lipid bilayer, and the C-terminal portion is the extracellular domain exposed outside the lipid bilayer. The C-terminal stalk region (e.g., corresponding to amino acids 159-167 of NiV-G) has been shown to be involved in interaction with the F protein and inducing F protein fusion (Liu et al. 2015 J of Virology 89:1838). In wild-type G proteins, the globular head mediates receptor binding to the henipavirus entry receptors ephrinB2 and ephrinB3 but is dispensable for membrane fusion (Brandel-Tretheway et al. Journal of Virology. 2019. 93(13)e00577-19). In certain embodiments herein, the G protein's tropism is altered by linking the G protein or a biologically active fragment thereof (e.g., cytoplasmic cleavage) to an sdAb variable domain. Binding of the G protein to a binding partner can trigger fusion mediated by a compatible F protein or a biologically active portion thereof. The G protein sequences disclosed herein are primarily disclosed as expressed sequences containing an N-terminal methionine required for translation initiation. Because such N-terminal methionine is generally cleaved co- or post-translationally, the mature protein sequences for all G protein sequences disclosed herein are also contemplated as lacking the N-terminal methionine.

[0410] The G glycoprotein is highly conserved among henipavirus species. For example, the G proteins of NiV and HeV viruses share 79% amino acid identity. Studies have shown a high degree of identity among G proteins with the F proteins of different species, as demonstrated by heterotypic fusion activation (Brandel-Tretheway et al. Journal of of Virology. 2019). As described further below, the retargeting lipid particles can include heterologous G and F proteins from different species.

[0411] In some embodiments, the henipavirus G protein molecules described herein comprise an amino acid sequence in Table 5 (e.g., any of SEQ ID NOS:8-12), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., a 100, 200, 300, 400, 500, or 600 amino acid long portion thereof, having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. For example, in some embodiments, the henipavirus G protein molecules described herein comprise an amino acid sequence having at least 80% identity to any of the amino acid sequences in Table 5. In some embodiments, the henipavirus G protein molecules described herein comprise an amino acid sequence having at least 80% identity to SEQ ID NO:8. In some embodiments, the henipavirus G protein molecules described herein comprise an amino acid sequence having at least 80% identity to SEQ ID NO:9. In some embodiments, a henipavirus G protein molecule described herein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 10. In some embodiments, a henipavirus G protein molecule described herein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 11. In some embodiments, a henipavirus G protein molecule described herein comprises an amino acid sequence at least 80% identical to SEQ ID NO: 12. In some embodiments, a nucleic acid sequence described herein encodes an amino acid sequence in Table 5, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, e.g., a 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acid long portion thereof.

[0412] In certain embodiments, the G protein or functionally active variant or biologically active portion thereof is a protein that retains fusion activity in conjunction with a henipavirus F protein, e.g., an F protein shown in Table 4 (e.g., NiV-F or HeV-F). Fusion activity includes the activity of the G protein in conjunction with the henipavirus F protein to promote or facilitate fusion of two membrane spaces, e.g., the space of a targeted lipid particle having the henipavirus F and G proteins embedded in its lipid bilayer, and the cytoplasm of a target cell, e.g., a cell containing a surface receptor or molecule recognized or bound by the targeting envelope protein. In some embodiments, the F protein and G protein are from the same henipavirus species (e.g., NiV-G and NiV-F). In some embodiments, the F protein and G protein are from different henipavirus species (e.g., NiV-G and HeV-F).

[0413] Reference to retaining fusion activity includes between or exceeding 10% or about 10% and 150% or about 150% of the level or degree of binding of the corresponding wild-type G protein as set forth in SEQ ID NOs: 8-12, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 35% or at least about 35% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 40% or at least about 40% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 10% or at least about 10% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 15% or at least about 15% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 20% or at least about 20% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 25% or at least about 25% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least 30% or at least about 30% of the level or degree of fusion activity of the corresponding wild-type G protein, e at least 45% or at least about 45% of the level or degree of fusion activity of the native G protein, for example, at least 50% or at least about 50% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 55% or at least about 55% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 60% or at least about 60% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 65% or at least about 65% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 70% or at least about 70% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 75% or at least about 75% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 80% or at least about 80% of the level or degree of fusion activity of the corresponding wild-type G protein, for example, at least 85% or at least about 85% of the level or degree of fusion activity of the corresponding wild-type G protein, for example,This includes activity (in combination with a Henipavirus F protein) that is at least or at least about 90% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least or at least about 95% of the level or degree of fusion activity of the corresponding wild-type G protein, e.g., at least or at least about 100% of the level or degree of fusion activity of the corresponding wild-type G protein, or e.g., at least or at least about 120% of the level or degree of fusion activity of the corresponding wild-type G protein. [Table 6-1] [Table 6-2] [Table 6-3]

[0414] In some embodiments, the G protein is a mutant G protein, which is a functionally active variant or biologically active portion thereof comprising one or more amino acid mutations, e.g., one or more amino acid insertions, deletions, substitutions, or truncations. In some embodiments, the mutations described herein relate to amino acid insertions, deletions, substitutions, or truncations relative to a reference G protein sequence. In some embodiments, the reference G protein sequence is the wild-type sequence of a G protein or biologically active portion thereof. In some embodiments, the functionally active variant or biologically active portion thereof is a mutant of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, the wild-type bat paramyxovirus G protein, or a biologically active portion thereof. In some embodiments, the wild-type G protein has a sequence set forth in any one of SEQ ID NOs: 9-12.

[0415] In some embodiments, the G protein is a mutant G protein that is a biologically active portion that is an N-terminally and / or C-terminally truncated fragment of the wild-type Hendra (HeV) virus G protein, the wild-type Nipah (NiV) virus G protein (NiV-G), the wild-type Cedar (CedPV) virus G protein, the wild-type Mojiang virus G protein, or the wild-type bat paramyxovirus G protein. In certain embodiments, the truncation is an N-terminal truncation of all or part of the cytoplasmic domain. In some embodiments, the mutant G protein is a truncated and biologically active portion that lacks up to 49 consecutive amino acid residues at or near the N-terminus of a wild-type G protein, such as the wild-type G protein set forth in any one of SEQ ID NOs: 9-12. In some embodiments, the mutant F protein is truncated and lacks up to 49 consecutive amino acids at the N-terminus of the wild-type G protein, for example, up to 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 30, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 consecutive amino acid(s).

[0416] In some embodiments, the G protein is NiV-G or a functionally active variant or biologically active portion thereof, and binds to ephrin B2 or ephrin B3. In some aspects, the NiV-G has the amino acid sequence set forth in any of SEQ ID NOs: 9-12, or is a functionally active variant or biologically active portion thereof capable of binding to ephrin B2 or ephrin B3. In some embodiments, the functionally active variant or biologically active portion has an amino acid sequence having at least about 80%, at least about 85%, at least or about 90%, at least or about 90%, at least or about 91%, at least 92%, at least or about 92%, at least 93%, at least or about 93%, at least 94%, at least or about 94%, at least 95%, at least or about 95%, 96%, at least 97%, at least 98%, or at least 99% or about 99% sequence identity to SEQ ID NOs: 9-12 and retains binding to ephrin B2 or B3. Exemplary biologically active portions include N-terminally truncated variants lacking all or a portion of the cytoplasmic domain, e.g., one or more, e.g., 1 to 49 consecutive N-terminal amino acid residues. Reference to retaining binding to ephrin B2 or B3 includes, for example, binding that is at least 5% or at least about 5% of the level or extent of binding of the corresponding wild-type NiV-G set forth in SEQ ID NOs: 9-12.

[0417] In some embodiments, the G protein or biologically active portion thereof is a mutant G protein that exhibits reduced binding to a natural binding partner of the wild-type G protein. In some embodiments, the mutant G protein or biologically active portion thereof is a mutant of wild-type Niv-G and exhibits reduced binding to one or both of the natural binding partners, ephrin B2 or ephrin B3. In some embodiments, the mutant G protein or biologically active portion thereof, e.g., mutant NiV-G protein, exhibits reduced binding to a natural binding partner. In some embodiments, the reduced binding to EphrinB2 or EphrinB3 is at or about 5%, 10% or about 10%, 15% or about 15%, 20% or about 20%, 25% or about 25%, 30% or about 30%, 40% or about 40%, 50% or about 50%, 60% or about 60%, 70% or about 70%, 80% or about 80%, 90% or about 90%, or 100% or more than a reduction of at or about 100%.

[0418] In some embodiments, the mutations described herein can improve transduction efficiency. In some embodiments, the mutations described herein enable specific targeting of other desired cell types other than EphrinB2 or EphrinB3. In some embodiments, the mutations described herein at least partially disable binding to at least one native receptor, e.g., reduce binding to at least one of EphrinB2 or EphrinB3. In some embodiments, the mutations described herein interfere with native receptor recognition.

[0419] In some embodiments, the G protein comprises one or more amino acid substitutions at residues involved in interaction with one or both of EphrinB2 and EphrinB3, hi some embodiments, the amino acid substitutions correspond to the mutations E501A, W504A, Q530A, and E533A, with reference to the numbering shown in SEQ ID NO:9.

[0420] In some embodiments, the G protein is a mutant G protein comprising one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A, with reference to the numbering set forth in SEQ ID NO: 9. In some embodiments, the G protein is a mutant G protein comprising one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A, with reference to SEQ ID NO: 9, and a biologically active portion thereof comprising an N-terminal truncation.

[0421] In some embodiments, the G protein is a mutant G protein comprising one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A, with reference to the numbering set forth in SEQ ID NO: 9. In some embodiments, the G protein is a mutant G protein comprising one or more amino acid substitutions selected from the group consisting of E501A, W504A, Q530A, and E533A, with reference to SEQ ID NO: 9, and a biologically active portion thereof comprising an N-terminal truncation. In some embodiments, the mutant NiV-G protein or biologically active portion thereof is truncated and includes up to 5 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 6 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 7 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 8 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 9 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 10 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 11 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 12 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 13 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 14 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 15 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 16 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 17 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 18 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9),19 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 20 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 21 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 22 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 23 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 24 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 25 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 26 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 27 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 28 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 29 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 30 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 31 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 32 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), 33 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), wild-type 34 consecutive amino acid residues at or near the N-terminus of the NiV-G protein (SEQ ID NO: 9), 35 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 36 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 37 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), up to 38 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9),It lacks up to 39 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9), or up to 40 consecutive amino acid residues at or near the N-terminus of the wild-type NiV-G protein (SEQ ID NO: 9).

[0422] In some embodiments, the NiV-G protein is encoded by a nucleotide sequence that encodes the sequence set forth in SEQ ID NO: 18. In some embodiments, the NiV-G protein is encoded by a nucleotide sequence that encodes a sequence having at least or about 90%, at least or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 18. In some embodiments, the mutant NiV-G protein has the amino acid sequence set forth in SEQ ID NO: 18 or an amino acid sequence having at least or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 18. In certain embodiments, the G protein has the amino acid sequence set forth in SEQ ID NO: 18.

[0423] In some embodiments, the NiV-F protein has an amino acid sequence having at least or about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95%, 96%, at least 97%, at least 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 17, and the NiV-G protein has an amino acid sequence having at least or about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or about 99% sequence identity to SEQ ID NO: 18. In some embodiments, the NiV-F protein has the amino acid sequence set forth in SEQ ID NO:17, and the NiV-G protein has the amino acid sequence set forth in SEQ ID NO:18.

[0424] Other proteins In some embodiments, the fusogen may include a pH-dependent protein, a homolog thereof, a fragment thereof, or a protein fusion comprising one or more proteins or fragments thereof. The fusogen may mediate membrane fusion at the cell surface or in an endosome or another membrane-bound space.

[0425] In some embodiments, the fusogens include EFF-1, AFF-1, gap junction proteins such as connexins (e.g., Cn43, GAP43, CX43) (DOI: 10.1021 / jacs.6b05191), other tumor junction proteins, homologs thereof, fragments thereof, variants thereof, and protein fusions comprising one or more proteins or fragments thereof.

[0426] Modification to protein fusogens Protein fusogens or viral envelope proteins (e.g., henipavirus G protein molecules) can be retargeted by mutating amino acid residues in the fusion protein or targeting protein (e.g., hemagglutinin protein). In some embodiments, the fusogens are randomly mutated. In some embodiments, the fusogens are rationally mutated. In some embodiments, the fusogens are subjected to directed evolution. In some embodiments, the fusogens are truncated and only a subset of the peptides are used in the retroviral vector or fusosome. For example, amino acid residues in the measles hemagglutinin protein may be mutated to alter the binding properties of the protein and redirect fusions (doi:10.1038 / nbt942, Molecular Therapy vol.16 no.8,1427-1436 Aug.2008, doi:10.1038 / nbt1060, DOI:10.1128 / JVI.76.7.3558-3563.2002, DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1073pnas.0604993103).

[0427] A protein fusogen (e.g., a henipavirus G protein molecule) can be retargeted by covalently conjugating a targeting moiety to the fusion protein or targeting protein (e.g., the hemagglutinin protein). For example, a G protein can be linked to a targeting moiety (e.g., an antibody or antigen-binding fragment). In some embodiments, the fusogen and targeting moiety are covalently conjugated by expression of a chimeric protein comprising the fusogen linked to the targeting moiety. Targets include any peptide (e.g., a receptor) displayed on target cells. In some examples, the target is expressed at a higher level on target cells than on non-target cells. For example, single-chain variable fragments (scFv) can be conjugated to fusogens to redirect fusion activity to cells that present the scFv-binding target (doi:10.1038 / nbt1060, DOI 10.1182 / blood-2012-11-468579, doi:10.1038 / nmeth.1514, doi:10.1006 / mthe.2002.0550, HUMAN GENE THERAPY 11:817-826, doi:10.1038 / nbt942, doi:10.1371 / journal.pone.0026381, DOI 10.1186 / s12896-015-0142-z). For example, engineered ankyrin repeat proteins (DARPins) can be conjugated to fusogens to redirect fusion activity to cells presenting DARPin-binding targets (doi:10.1038 / mt.2013.16, doi:10.1038 / mt.2010.298, doi:10.4049 / jimmunol.1500956), and combinations of different DARPins (doi:10.1038 / mto.2016.3). For example, receptor ligands and antigens can be conjugated to fusogens to redirect fusion activity to cells presenting the target receptor (DOI:10.1089 / hgtb.2012.054, DOI:10.1128 / JVI.76.7.3558-3563.2002).Targeting proteins can also include, for example, antibodies or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) scaffolds, e.g., fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T cell receptors (TCRs). Protein fusogens can be retargeted by non-covalently conjugating a targeting moiety to the fusion protein or targeting protein (e.g., the hemagglutinin protein). For example, the fusion protein can be engineered to bind to the Fc region of an antibody that targets an antigen on a target cell, redirecting fusion activity to cells that present the antibody's target (DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1038 / nm1192). Modified and unmodified fusogens can be presented in the same retroviral vector or fusosome (doi:10.1016 / j.biomaterials.2014.01.051).

[0428] Targeting moieties can be humanized antibody molecules, intact IgA, IgG, IgE or IgM antibodies, bi- or multispecific antibodies (e.g., Zybodies®, etc.), antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies, e.g., IgNAR or fragments thereof), camelid antibodies, masked antibodies (e.g., Probodies®), Small Modular Antibodies, ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAbs®), VHHs, Anticalins®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, microproteins, Fynomers®, Centyrin®, and KALBITOR®. Targeting moieties can also include antibodies or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single-domain antibodies such as sdAbs (either VL or VH), nanobodies, or camelid VHH domains), antigen-binding fibronectin type III (Fn3) scaffolds, e.g., fibronectin polypeptide minibodies, ligands, cytokines, chemokines, or T-cell receptors (TCRs).

[0429] In some embodiments, the single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. In some embodiments, the single domain antibody is an antibody variable domain of only a heavy chain. In some embodiments, the single domain antibody does not include a light chain.

[0430] In some embodiments, the heavy chain antibody lacking the light chain is referred to as a VHH. In some embodiments, the single domain antibody has a molecular weight of 12-15 kDa. In some embodiments, the single domain antibody includes a camelid antibody or a shark antibody. In some embodiments, the single domain antibody molecule is derived from antibodies established in camelid species, such as camel, llama, dromedary, alpaca, vicuna, and guanaco. In some embodiments, the single domain antibody is referred to as an immunoglobulin new antigen receptor (IgNAR) and is derived from cartilaginous fish. In some embodiments, the single domain antibody is generated by splitting the dimeric variable domain of human or mouse IgG into monomers and camelizing key residues.

[0431] In some embodiments, the single domain antibodies may be generated from a phage display library. In some embodiments, the phage display library is generated from the VHH repertoire of camelids immunized with various antigens, as described in Arbabi et al., FEBS Letters, 414, 521-526 (1997); Lauwereys et al., EMBO J., 17, 3512-3520 (1998); Decaniere et al., Structure, 7, 361-370 (1999). In some embodiments, the phage display library is generated including antibody fragments from non-immunized camelids. In some embodiments, a single domain antibody library of human single domain antibodies is synthetically generated by introducing diversity into one or more scaffolds.

[0432] In some embodiments, the C-terminus of the single domain antibody is linked to the C-terminus of a G protein or a biologically active portion thereof. In some embodiments, the N-terminus of the single domain antibody is exposed to the outer surface of the lipid bilayer. In some embodiments, the N-terminus of the single domain antibody binds to a cell surface molecule of a target cell. In some embodiments, the single domain antibody specifically binds to a cell surface molecule present on a target cell. In some embodiments, the cell surface molecule is a protein, a glycan, a lipid, or a low-molecular-weight molecule.

[0433] In embodiments, the retargeted fusogen binds to a cell surface marker on the target cell, such as a protein, glycoprotein, receptor, cell surface ligand, agonist, lipid, sugar, class I transmembrane protein, class II transmembrane protein, or class III transmembrane protein.

[0434] Retroviral vectors or fusosomes may present targeting moieties that are not conjugated to protein fusogens, redirecting the fusion activity or affecting homing to cells bound by the targeting moiety.

[0435] The targeting moiety attached to the retroviral vector or fusosome can be adjusted to have different binding strengths. For example, scFvs and antibodies with different binding strengths can be used to change the fusion activity of the retroviral vector or fusosome with cells that present a large or small amount of the target antigen (doi:10.1128 / JVI.01415-07, doi:10.1038 / cgt.2014.25, doi:10.1002 / jgm.1151). For example, DARPins with different affinities can be used to change the fusion activity of the retroviral vector or fusosome with cells that present a large or small amount of the target antigen (doi:10.1038 / mt.2010.298). The targeting moiety can also be adjusted to target different regions on the target ligand, which affects the fusion rate with cells that present the target (doi:10.1093 / protein / gzv005).

[0436] In some embodiments, the cell surface molecule of the target cell is an antigen or a portion thereof. In some embodiments, the single domain antibody or portion thereof is an antibody having a single monomer domain antigen binding / recognition domain that can selectively bind to a specific antigen. In some embodiments, the single domain antibody binds to an antigen present on the target cell.

[0437] Exemplary cells include polymorphonuclear cells (also known as PMN, PML, PMNL, or granulocytes), stem cells, embryonic stem cells, neural stem cells, mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), human myogenic stem cells, muscle-derived stem cells (MuStem), embryonic stem cells (ES or ESC), limbal epithelial stem cells, cardiomyogenic stem cells, cardiomyocytes, progenitor cells, immune effector cells, lymphocytes, macrophages, dendritic cells, natural killer cells, T cells, cytotoxic T lymphocytes, allogeneic cells, resident cardiac cells, induced pluripotent stem cells (iPS), adipose tissue-derived or phenotypically modified stem or progenitor cells, CD133+ cells, aldehyde dehydrogenase positive cells (ALDH+), umbilical cord blood (UCB) cells, peripheral blood stem cells (PBSC), neurons, neural progenitor cells, pancreatic beta cells, glial cells, or hepatocytes.

[0438] In some embodiments, the target cell is a cell of a target tissue, which may include the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testes, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye.

[0439] In some embodiments, the target cell is a muscle cell (e.g., a skeletal muscle cell), a kidney cell, a liver cell (e.g., a hepatocyte), or a cardiac cell (e.g., a cardiomyocyte). In some embodiments, the target cell is a cardiac cell, such as a cardiomyocyte (e.g., a quiescent cardiomyocyte), a hepatoblast (e.g., a biliary hepatoblast), an epithelial cell, a T cell (e.g., a naive T cell), a macrophage (e.g., a tumor-infiltrating macrophage), or a fibroblast (e.g., a cardiac fibroblast).

[0440] In some embodiments, the target cell is a tumor-infiltrating lymphocyte, a T cell, a neoplasm or tumor cell, a virus-infected cell, a stem cell, a central nervous system (CNS) cell, a hematopoietic stem cell (HSC), a liver cell, or a fully differentiated cell. In some embodiments, the target cell is a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, a liver cell, 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, a CD133+ cancer cell, an EpCAM+ cancer cell, a CD19+ cancer cell, a Her2 / Neu+ cancer cell, a GluA2+ neuron, a GluA4+ neuron, an NKG2D+ natural killer cell, a SLC1A3+ astrocyte, a SLC7A10+ adipocyte, or a CD30+ lung epithelial cell.

[0441] In some embodiments, the target cell is an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, an atypical antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancer cell.

[0442] In some embodiments, the cell surface molecule is any one of CD8, CD4, asialoglycoprotein receptor 2 (ASGR2), transmembrane 4 L6 family member 5 (TM4SF5), low density lipoprotein receptor (LDLR), or asialoglycoprotein 1 (ASGR1).

[0443] In some embodiments, the G protein or functionally active variant or biologically active portion thereof is directly linked to an sdAb variable domain, hi some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-(C'-G protein-N').

[0444] In some embodiments, the G protein or functionally active variant or biologically active portion thereof is indirectly linked to the sdAb variable domain via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker.

[0445] In some embodiments, the linker is a peptide linker and the targeting envelope protein is a fusion protein comprising a G protein or a functionally active variant or biologically active portion thereof linked to an sdAb variable domain via a peptide linker, hi some embodiments, the targeting envelope protein is a fusion protein having the following structure: (N'-single domain antibody-C')-linker-(C'-G protein-N').

[0446] In some embodiments, the peptide linker is up to 65 amino acids in length. In some embodiments, the peptide linker is 2 or about 2 to 65 amino acids, 2 to 60 amino acids, 2 to 56 amino acids, 2 to 52 amino acids, 2 to 48 amino acids, 2 to 44 amino acids, 2 to 40 amino acids, 2 to 36 amino acids, 2 to 32 amino acids, 2 to 28 amino acids, 2 to 24 amino acids, 2 to 20 amino acids, 2 to 18 amino acids, 2 to 14 amino acids, 2 to 12 amino acids, 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 6 to 65 amino acids, 6 to 60 amino acids, 6 to 56 amino acids, 6 to 52 amino acids, 6 to 48 amino acids, 6 to 44 amino acids, Amino acids, 6-40 amino acids, 6-36 amino acids, 6-32 amino acids, 6-28 amino acids, 6-24 amino acids, 6-20 amino acids, 6-18 amino acids, 6-14 amino acids, 6-12 amino acids, 6-10 amino acids, 6-8 amino acids, 8-65 amino acids, 8-60 amino acids, 8-56 amino acids, 8-52 amino acids, 8-48 amino acids, 8-44 amino acids, 8-40 amino acids, 8-36 amino acids, 8-32 amino acids, 8-28 amino acids, 8-24 amino acids, 8-20 amino acids, 8-18 amino acids, 8-14 amino acids, 8-12 amino acids, 8 ~10 amino acids, 10~65 amino acids, 10~60 amino acids, 10~56 amino acids, 10~52 amino acids, 10~48 amino acids, 10~44 amino acids, 10~40 amino acids, 10~36 amino acids, 10~32 amino acids, 10~28 amino acids, 10~24 amino acids, 10~20 amino acids, 10~18 amino acids, 10~14 amino acids, 10~12 amino acids, 12~65 amino acids, 12~60 amino acids, 12~56 amino acids, 12~52 amino acids, 12~48 amino acids, 12~44 amino acids, 12~40 amino acids, 12~36 amino acids, 2-32 amino acids, 12-28 amino acids, 12-24 amino acids, 12-20 amino acids, 12-18 amino acids, 12-14 amino acids, 14-65 amino acids, 14-60 amino acids, 14-56 amino acids, 14-52 amino acids, 14-48 amino acids, 14-44 amino acids, 14-40 amino acids, 14-36 amino acids, 14-32 amino acids, 14-28 amino acids, 14-24 amino acids, 14-20 amino acids, 14-18 amino acids, 18-65 amino acids, 18-60 amino acids, 18-56 amino acids, 18-52 amino acids, 18-48 amino acids,18-44 amino acids, 18-40 amino acids, 18-36 amino acids, 18-32 amino acids, 18-28 amino acids, 18-24 amino acids, 18-20 amino acids, 20-65 amino acids, 20-60 amino acids, 20-56 amino acids, 20-52 amino acids, 20-48 amino acids, 20-44 amino acids, 20-40 amino acids, 20-36 amino acids, 20-32 amino acids, 20-28 amino acids, 20-26 amino acids, 20-24 amino acids, 24-65 amino acids, 24-6 0 amino acids, 24-56 amino acids, 24-52 amino acids, 24-48 amino acids, 24-44 amino acids, 24-40 amino acids, 24-36 amino acids, 24-32 amino acids, 24-30 amino acids, 24-28 amino acids, 28-65 amino acids, 28-60 amino acids, 28-56 amino acids, 28-52 amino acids, 28-48 amino acids, 28-44 amino acids, 28-40 amino acids, 28-36 amino acids, 28-34 amino acids, 28-32 amino acids, 32-65 amino acids Acid, 32-60 amino acids, 32-56 amino acids, 32-52 amino acids, 32-48 amino acids, 32-44 amino acids, 32-40 amino acids, 32-38 amino acids, 32-36 amino acids, 36-65 amino acids, 36-60 amino acids, 36-56 amino acids, 36-52 amino acids, 36-48 amino acids, 36-44 amino acids, 36-40 amino acids, 40-65 amino acids, 40-60 amino acids, 40-56 amino acids, 40-52 amino acids, 40-48 amino acids, 40 Contains up to 44 amino acids, 44 to 65 amino acids, 44 to 60 amino acids, 44 to 56 amino acids, 44 to 52 amino acids, 44 to 48 amino acids, 48 ​​to 65 amino acids, 48 ​​to 60 amino acids, 48 ​​to 56 amino acids, 48 ​​to 52 amino acids, 50 to 65 amino acids, 50 to 60 amino acids, 50 to 56 amino acids, 50 to 52 amino acids, 54 to 65 amino acids, 54 to 60 amino acids, 54 to 56 amino acids, 58 to 65 amino acids, 58 to 60 amino acids, or 60 to 65 amino acids. In some embodiments, the peptide linker is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,It is a polypeptide that is 59, 60, 61, 62, 63, 64, or 65 amino acids in length.

[0447] In certain embodiments, the linker is a flexible peptide linker. In some such embodiments, the linker is 1 to 20 amino acids, e.g., 1 to 20 amino acids composed primarily of glycine. In some embodiments, the linker is 1 to 20 amino acids, e.g., 1 to 20 amino acids composed primarily of glycine and serine. In some embodiments, the linker is a flexible peptide linker comprising the amino acids glycine and serine, referred to as a GS-linker. In some embodiments, the peptide linker includes the sequence GS, GGS, GGGGS (SEQ ID NO: 19), GGGGGS (SEQ ID NO: 20), or a combination thereof. In some embodiments, the polypeptide linker has the sequence (GGS)n, where n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGS)n (SEQ ID NO: 21), where n is 1 to 10. In some embodiments, the polypeptide linker has the sequence (GGGGGS)n (SEQ ID NO: 22), where n is 1 to 6.

[0448] In some embodiments, protein fusogens can be modified to reduce immune reactivity, e.g., as described herein. For example, protein fusogens can be decorated with molecules that reduce immune interactions, e.g., PEG (DOI: 10.1128 / JVI.78.2.912-921.2004). Thus, in some embodiments, the fusogen comprises PEG, e.g., a pegylated polypeptide. Amino acid residues of fusogens targeted by the immune system can be altered to prevent immune system recognition (doi: 10.1016 / j.virol.2014.01.027, doi: 10.1371 / journal.pone.0046667). In some embodiments, the protein sequence of the fusogen is altered to resemble an amino acid sequence found in humans (humanization). In some embodiments, the protein sequence of the fusogen is altered to a protein sequence that weakens binding to MHC complexes. In some embodiments, the protein fusogen is derived from a virus or organism that does not infect humans (and against which humans have not been vaccinated), increasing the likelihood that the patient's immune system is naive to the protein fusogen (e.g., humoral or cellular adaptive immune responses to the fusogen are negligible) (doi:10.1006 / mthe.2002.0550, doi:10.1371 / journal.ppat.1005641, doi:10.1038 / gt.2011.209, DOI 10.1182 / blood-2014-02-558163). In some embodiments, the glycosylation of the fusogen may be altered to alter immune interactions or reduce immune reactivity. Without wishing to be bound by theory, in some embodiments, a protein fusogen derived from a virus or organism that does not infect humans has no natural fusion targets in the patient and therefore has high specificity.

[0449] Lipid fusogens In some embodiments, the retroviral vector or fusosome may include, for example, one or more fusogenic lipids, such as saturated fatty acids, in addition to the F and G proteins described herein. In some embodiments, the saturated fatty acids have 10-14 carbons. In some embodiments, the saturated fatty acids have longer chain carboxylic acids. In some embodiments, the saturated fatty acids are monoesters.

[0450] In some embodiments, the retroviral vector or fusosome can comprise one or more unsaturated fatty acids. In some embodiments, the unsaturated fatty acids are C16-C18 unsaturated fatty acids. In some embodiments, the unsaturated fatty acids include oleic acid, glycerol monooleate, glycerides, diacylglycerols, modified unsaturated fatty acids, and any combination thereof.

[0451] Without wishing to be bound by theory, in some embodiments, negative curvature lipids promote membrane fusion. In some embodiments, the retroviral vector or fusosome contains one or more negative curvature lipids in the membrane, for example, exogenous negative curvature lipids. In embodiments, the negative curvature lipid or its precursor is added to a medium containing the origin cell, retroviral vector, or fusosome. In embodiments, the origin cell is engineered to express or overexpress one or more lipid synthesis genes. The negative curvature lipid can be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acid (FA).

[0452] Without wishing to be bound by theory, in some embodiments, positive curvature lipids inhibit membrane fusion. In some embodiments, the retroviral vector or fusosome contains a reduced level of one or more positive curvature lipids in the membrane, for example, exogenous positive curvature lipids. In some embodiments, the level is reduced by inhibiting the synthesis of the lipid in the origin cell, for example, by knocking out or knocking down lipid synthesis genes. The positive curvature lipid can be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

[0453] Chemical Fusogens In some embodiments, the retroviral vector or fusosome may be treated with a fusogenic chemical, hi some embodiments, the fusogenic chemical is polyethylene glycol (PEG) or a derivative thereof.

[0454] In some embodiments, the chemical fusogen induces local dehydration between two membranes, which results in unfavorable molecular packing of the bilayer. In some embodiments, the chemical fusogen induces dehydration in the region near the lipid bilayer, causing the movement of aqueous molecules between the two membranes and allowing interaction between the two membranes.

[0455] In some embodiments, the chemical fusogen is a positive cation. Some non-limiting examples of positive cations include Ca, Mg, Mn, Zn, La, Sr, and H.

[0456] In some embodiments, the chemical fusogens bind to target membranes by modifying their surface polarity, which alters hydration-dependent intermembrane repulsion.

[0457] In some embodiments, the chemical fusogen is lipid soluble. Some non-limiting examples include oleoylglycerol, dioleoylglycerol, trioleoylglycerol, and variants and derivatives thereof.

[0458] In some embodiments, the chemical fusogen is a water-soluble chemical, some non-limiting examples of which include polyethylene glycol, dimethyl sulfoxide, and variants and derivatives thereof.

[0459] In some embodiments, the chemical fusogen is a small organic molecule. A non-limiting example is n-hexyl bromide.

[0460] In some embodiments, the chemical fusogen does not alter the composition, cell viability, or ion transport properties of the fusogen or target membrane.

[0461] In some embodiments, the chemical fusogen is a hormone or vitamin, some non-limiting examples of which include abscisic acid, retinol (vitamin A1), tocopherol (vitamin E), and variants and derivatives thereof.

[0462] In some embodiments, the retroviral vector or fusosome comprises an agent that stabilizes actin and polymerized actin. Without wishing to be bound by theory, stabilized actin within the retroviral vector or fusosome may facilitate fusion with target cells. In embodiments, the agent that stabilizes polymerized actin is selected from actin, myosin, biotin-streptavidin, ATP, neuronal Wiskott-Aldrich syndrome protein (N-WASP), or formin. See, e.g., Langmuir. 2011 Aug 16;27(16):10061-71 and Wen et al., Nat Commun. 2016 Aug 31;7. In embodiments, the retroviral vector or fusosome comprises exogenous actin, e.g., wild-type actin or actin containing a mutation that promotes polymerization. In embodiments, the retroviral vector or fusosome comprises ATP or phosphocreatine, e.g., exogenous ATP or phosphocreatine.

[0463] Low molecular weight fusogens In some embodiments, the retroviral vector or fusosome may be treated with a fusogenic small molecule, some non-limiting examples of which include halothane, nonsteroidal anti-inflammatory drugs (NSAIDs) such as meloxicam, piroxicam, tenoxicam, and chlorpromazine.

[0464] In some embodiments, the small molecule fusogen may be present in micelle-like aggregates or may be aggregate-free.

[0465] Positive target cell-specific regulatory elements In some embodiments, the fusosomal nucleic acids described herein comprise a positive target cell-specific regulatory element, such as a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific splice site, a tissue-specific site that extends the half-life of an RNA or protein, a tissue-specific mRNA export-promoting site, a tissue-specific translation-enhancing site, or a tissue-specific post-translational modification site. Additional positive target cell-specific regulatory elements are described, for example, in International Application No. WO 2019 / 222403, which is incorporated herein by reference in its entirety.

[0466] In certain embodiments, the fusosomal nucleic acids described herein comprise regulatory elements capable of, for example, directing, increasing, modulating, or controlling the transcription or expression of an operably linked polynucleotide in a cell-specific manner. In certain embodiments, the fusosomal nucleic acids comprise one or more expression control sequences specific for a particular cell, cell type, or cell lineage, e.g., a target cell. That is, expression of a polynucleotide operably linked to an expression control sequence specific for a particular cell, cell type, or cell lineage is expressed in the target cell and not expressed (or expressed at a lower level) in non-target cells. In certain embodiments, the fusosomal nucleic acids may comprise exogenous, endogenous, or heterologous regulatory sequences, e.g., promoters and / or enhancers.

[0467] In certain embodiments, promoters that function in mammalian cells include an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins, and / or another sequence found approximately 70-80 bases upstream from the transcription start site, i.e., a CNCAAT region, where N can be any nucleotide. In embodiments, an enhancer comprises a segment of DNA containing a sequence capable of enhancing transcription and, in some cases, can function independently of its orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. In some embodiments, a promoter / enhancer segment of DNA comprises a sequence capable of providing both promoter and enhancer function. In some embodiments, the regulatory sequence is a ubiquitous expression control sequence.

[0468] In some embodiments, the promoter is a tissue-specific promoter, e.g., a promoter that drives expression in liver cells, e.g., hepatocytes, liver sinusoidal endothelial cells, cholangiocytes, stellate cells, liver-resident antigen-presenting cells (e.g., Kupffer cells), liver-resident immune lymphocytes (e.g., T cells, B cells, or NK cells), or portal vein fibroblasts.

[0469] Non-target cell-specific regulatory elements In some embodiments, the non-target cell-specific regulatory element comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site. Additional non-target cell-specific regulatory elements are described, for example, in International Application No. WO2019 / 222403, which is incorporated herein by reference in its entirety. In some embodiments, the non-target cell comprises an endogenous miRNA. The fusomal nucleic acid (e.g., the gene encoding the exogenous agent) can comprise the recognition sequence of the miRNA. Thus, when the fusomal nucleic acid enters the non-target cell, the miRNA can downregulate the expression of the exogenous agent. This can further enhance the specificity of the target cell relative to the non-target cell.

[0470] In some embodiments, the miRNA is a 20-22 nucleotide small non-coding RNA, typically excised from a foldback RNA precursor structure of approximately 70 nucleotides known as a pre-miRNA. miRNAs (e.g., naturally occurring or artificially designed) can specifically target any mRNA sequence. In one embodiment, those skilled in the art can design a short hairpin RNA construct that is expressed as a primary transcript of a human miRNA (e.g., miR-30 or miR-21). This design adds a Drosha processing site to the hairpin construct, which has been shown to significantly increase knockdown efficiency (Pusch et al., 2004). The stem of the hairpin consists of a 22 nt dsRNA (e.g., antisense strand perfectly complementary to the desired target) and a 15-19 nt loop from a human miR.

[0471] Hundreds of different miRNA genes are differentially expressed during development and between tissue types. Molecular analysis has revealed that miRNAs have distinct expression profiles in different tissues. Using computational methods, the expression of approximately 7,000 predicted human miRNA targets has been analyzed. These data suggest that miRNA expression broadly contributes to the tissue specificity of mRNA expression in many human tissues. (Sood et al. 2006) See PNAS USA 103(8):2746-51.)

[0472] Thus, miRNA-based approaches can be used to restrict expression of exogenous agents to target cell populations by silencing expression of the exogenous agents in non-target cell types by using endogenous microRNA species. In some embodiments, the fusomal nucleic acids comprise one or more (e.g., multiple) tissue-specific miRNA recognition sequences. In some embodiments, the tissue-specific miRNA recognition sequences are approximately 20-25, 21-24, or 23 nucleotides in length. In embodiments, the tissue-specific miRNA recognition sequences are fully complementary to miRNAs present in non-target cells. In some embodiments, the exogenous agent does not comprise GFP, e.g., does not comprise a fluorescent protein, e.g., does not comprise a reporter protein. In some embodiments, the off-target cells are not hematopoietic cells and / or the miRNA is not present in hematopoietic cells.

[0473] In some embodiments, the methods herein include tissue-specific expression of an exogenous agent in a target cell, which includes contacting a plurality of cells, including target cells and non-target cells, with fusomal nucleic acids comprising nucleotides encoding the exogenous agent and at least one tissue-specific microRNA (miRNA) target sequence, such that the exogenous agent is selectively expressed in the target cell, e.g., restricted to the target cell. In embodiments, the fusomal nucleic acids comprise at least one miRNA recognition sequence operably linked to a nucleotide sequence having a corresponding miRNA in a non-target cell, e.g., a hematopoietic progenitor cell (HSPC) or hematopoietic stem cell (HSC), thereby preventing or reducing expression of the nucleotide sequence in the non-target cell but not in a target cell, e.g., a differentiated cell. In some embodiments, the fusomal nucleic acids comprise at least one miRNA sequence targeting an miRNA present in an effective amount in the non-target cell (e.g., the concentration of the endogenous miRNA is sufficient to reduce or prevent expression of the transgene), and comprise a transgene. In embodiments, the miRNAs used in this system are strongly expressed in non-target cells, e.g., HSPCs and HSCs, but not in differentiated progeny, e.g., myeloid and lymphoid, preventing or reducing transgene expression in susceptible stem cell populations while maintaining expression and therapeutic effect in target cells.

[0474] immunomodulation In some embodiments, the retroviral vectors or fusosomes described herein comprise elevated CD47. See, e.g., U.S. Patent No. 9,050,269, incorporated herein by reference in its entirety. In some embodiments, the retroviral vectors or fusosomes described herein comprise elevated complement regulatory proteins. See, e.g., ES2627445T3 and US6790641, each incorporated herein by reference in its entirety. In some embodiments, the retroviral vectors or fusosomes described herein lack or comprise reduced levels of MHC proteins, e.g., MHC-1 class 1 or class II. See, e.g., US20170165348, incorporated herein by reference in its entirety.

[0475] Sometimes, retroviral vectors or fusosomes can be recognized by the immune system of the target.In the case of enveloped virus vector particles (for example, retroviral vector particles), the membrane-associated protein displayed on the surface of the virus envelope can be recognized, and the virus particle itself can be neutralized.Furthermore, after infecting target cells, the virus envelope can be integrated with the cell membrane, and as a result, the virus envelope protein can be displayed on the surface of the cell, or can remain closely associated with the surface of the cell.Therefore, the immune system can also target the cells that the virus vector particles infect.Both effects can reduce the effectiveness of the delivery of exogenous substances by virus vectors.

[0476] The viral particle envelope is usually derived from the membrane of its cell of origin, and therefore membrane proteins expressed on the cell membrane from which the viral particle buds may be incorporated into the viral envelope.

[0477] immunoregulatory protein CD47 The internalization of extracellular substances into cells is generally carried out by a process called endocytosis (Rabinovitch, 1995, Trends Cell Biol. 5(3):85-7; Silverstein, 1995, Trends Cell Biol. 5(3):141-2; Swanson et al., 1995, Trends Cell Biol. 5(3):89-93; Allen et al., 1996, J. Exp. Med. 184(2):627-37). Endocytosis can be divided into two general categories: phagocytosis, which involves the uptake of particles, and pinocytosis, which involves the uptake of fluids and solutes.

[0478] Professional phagocytes have been shown to distinguish self from non-self based on studies in knockout mice lacking the membrane receptor CD47 (Oldenborg et al., 2000, Science 288(5473):2051-4). CD47 is a ubiquitous member of the Ig superfamily that interacts with the immunosuppressive receptor SIRPα (signal regulatory protein α) found on macrophages (Fujioka et al., 1996, Mol. Cell. Biol. 16(12):6887-99, Veillette et al., 2000). (Jiang et al., 1999, J. Biol. Chem. 274(2):559-62). Although CD47-SIRPα interactions appear to inactivate autologous macrophages in mice, a significant reduction (perhaps 90%) in CD47 expression has been found on human blood cells of several Rh genotypes that show little or no evidence of anemia (Mouro-Chanteloup et al., 2003, Blood 101(1):338-344) and little or no evidence of increased cell interactions with phagocytic monocytes (Arndt et al., 2004, Br. J. Haematol. 125(3):412-4).

[0479] In some embodiments, retroviral vectors or fusosomes (e.g., viral particles having a radius of less than about 1 μm, less than about 400 nm, or less than about 150 nm) comprise at least a biologically active portion of CD47, e.g., on an exposed surface of the retroviral vector or fusosome. In some embodiments, the retroviral vector (e.g., lentivirus) or fusosome comprises a lipid coat. In embodiments, the amount of biologically active CD47 in the retroviral vector or fusosome is about 20-250, 20-50, 50-100, 100-150, 150-200, or 200-250 molecules / μm 2 In some embodiments, the CD47 is human CD47.

[0480] The method described herein can comprise avoiding particle phagocytosis by phagocytes.The method can comprise expressing at least one peptide comprising at least a biologically active part of CD47 in retroviral vector or fusosome, so that when the retroviral vector or fusosome comprising CD47 is exposed to phagocytes, the viral particle avoids phagocyte phagocytosis by the phagocytes or shows reduced phagocytosis compared with the same retroviral vector or fusosome except unmodified.In some embodiments, the half-life of retroviral vector or fusosome in subjects is prolonged compared with the same retroviral vector or fusosome except unmodified.

[0481] MHC deletion Major histocompatibility complex class I (MHC-I) is a host cell membrane protein that can be incorporated into the viral envelope and is highly polymorphic in nature, making it a major target of the body's immune response (McDevitt HO (2000) Annu. Rev. Immunol. 18:1-17). MHC-I molecules exposed on the plasma membrane of the cell of origin are incorporated into the viral particle envelope during the vector budding process. These MHC-I molecules derived from the cell of origin and incorporated into the viral particle can then be transferred to the plasma membrane of the target cell. Alternatively, the MHC-I molecules may remain closely associated with the target cell membrane as a result of the tendency of viral particles to absorb and remain bound to the target cell membrane.

[0482] The presence of exogenous MHC-I molecules on or near the plasma membrane of transduced cells can induce alloreactive immune responses in subjects.This can lead to the immune-mediated killing or phagocytosis of transduced cells, either during ex vivo gene transfer after transduced cells are administered to subjects, or during direct in vivo administration of the viral particles.Furthermore, when MHC-I carrying viral particles are administered in vivo into bloodstream, the viral particles can be neutralized by existing MHC-I specific antibodies before reaching their target cells.

[0483] Thus, in some embodiments, the source cells are modified (e.g., genetically engineered) to reduce expression of MHC-I on the surface of the cells. In embodiments, the source comprises a genetically engineered disruption of the gene encoding β2-microglobulin (β2M). In embodiments, the source cells comprise a genetically engineered disruption of one or more genes encoding the α chain of MHC-I. The cells may comprise a genetically engineered disruption in all copies of the gene encoding β2-microglobulin. The cells may comprise a genetically engineered disruption in all copies of the gene encoding the α chain of MHC-I. The cells may comprise both a genetically engineered disruption of the gene encoding β2-microglobulin and a genetically engineered disruption of the gene encoding the α chain of MHC-I. In some embodiments, the retroviral vector or fusosome comprises a reduced number of surface-exposed MHC-I molecules. The number of surface-exposed MHC-I molecules may be reduced so that the immune response to the MHC-I is reduced to a therapeutically relevant extent. In some embodiments, the enveloped viral vector particles are substantially devoid of surface-exposed MHC-I molecules.

[0484] HLA-G / E overexpression In some embodiments, the retroviral vector or fusosome displays on its envelope a tolerogenic protein, e.g., an ILT-2 or ILT-4 agonist, e.g., HLA-E or HLA-G or any other ILT-2 or ILT-4 agonist. In some embodiments, the retroviral vector or fusosome has increased expression of HLA-E, HLA-G, ILT-2, or ILT-4 compared to a reference retrovirus, e.g., an unmodified but similar retrovirus.

[0485] In some embodiments, the retroviral composition has reduced MHC class I compared to unmodified retrovirus and increased HLA-G compared to unmodified retrovirus.

[0486] In some embodiments, the retroviral vector or fusosome has increased expression of HLA-G or HLA-E, e.g., a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more increase in HLA-G or HLA-E expression, compared to a reference retrovirus, e.g., a retrovirus similar to the retrovirus but unmodified, wherein HLA-G or HLA-E expression is assayed in vitro using flow cytometry, e.g., FACS.

[0487] In some embodiments, the retroviruses with increased HLA-G expression exhibit reduced immunogenicity in a teratoma formation assay, for example, as measured by reduced immune cell infiltration.

[0488] Complement regulatory proteins Complement levels are normally controlled by several complement regulatory proteins (CRPs). These proteins prevent misdirected inflammation and host tissue damage. One group of proteins, including CD55 / decay-accelerating factor (DAF) and CD46 / membrane cofactor protein (MCP), inhibits the C3 / C5 convertase enzymes of the classical and alternative pathways. Another set of proteins, including CD59, regulates MAC assembly. CRPs have been used to prevent rejection of xenotransplant tissues and have also been shown to protect viruses and viral vectors from complement inactivation.

[0489] Membrane-resident complement regulators include, for example, decay-accelerating factor (DAF) or CD55, factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), complement receptor 1 (CD35), membrane cofactor protein (MCP) or CD46, and CD59 (protectin) (e.g., to prevent membrane attack complex (MAC) formation and protect cells from lysis).

[0490] Albumin-binding proteins In some embodiments, the lentivirus binds to albumin. In some embodiments, the lentivirus comprises an albumin-binding protein on its surface. In some embodiments, the lentivirus comprises an albumin-binding protein on its surface. In some embodiments, the albumin-binding protein is a streptococcal albumin-binding protein. In some embodiments, the albumin-binding protein is a streptococcal albumin-binding domain.

[0491] Expression of non-fusogenic proteins on lentiviral envelopes In some embodiments, the lentivirus is engineered to include one or more proteins on its surface. In some embodiments, the protein affects immune interaction with a subject. In some embodiments, the protein affects the pharmacology of the lentivirus in the subject. In some embodiments, the protein is a receptor. In some embodiments, the protein is an agonist. In some embodiments, the protein is a signaling molecule. In some embodiments, the protein on the lentivirus surface includes an anti-CD3 antibody (e.g., OKT3) or IL7.

[0492] In some embodiments, a mitogenic transmembrane protein and / or a cytokine-based transmembrane protein may be present in the cell of origin and incorporated into the retrovirus as it buds from the membrane of the cell of origin, and the mitogenic transmembrane protein and / or cytokine-based transmembrane protein may be expressed as a separate cell surface molecule of the cell of origin rather than as part of the viral envelope glycoprotein.

[0493] In some embodiments of any of the aspects described herein, the retroviral vector, fusosome, or pharmaceutical composition is substantially non-immunogenic. Immunogenicity can be quantified, for example, as described herein.

[0494] In some embodiments, the retroviral vector or fusosome fuses with the target cell to produce a recipient cell. In some embodiments, recipient cells fused with one or more retroviral vectors or fusosomes are assessed for immunogenicity. In embodiments, the recipient cells are analyzed for the presence of cell surface antibodies, for example, by staining with anti-IgM antibodies. In other embodiments, immunogenicity is assessed by a PBMC cytolytic assay. In embodiments, the recipient cells are incubated with peripheral blood mononuclear cells (PBMCs) and then assessed for lysis of the cells by PBMCs. In other embodiments, immunogenicity is assessed by a natural killer (NK) cytolytic assay. In embodiments, the recipient cells are incubated with NK cells and then assessed for lysis of the cells by NK cells. In other embodiments, immunogenicity is assessed by a CD8+ T cell lytic assay. In embodiments, the recipient cells are incubated with CD8+ T cells and then assessed for lysis of the cells by CD8+ T cells.

[0495] In some embodiments, the retroviral vector or fusosome comprises an elevated level of an immunosuppressant (e.g., an immunosuppressant protein) compared to a reference retroviral vector or fusosome, e.g., one produced from a similar but otherwise unmodified cell of origin, or HEK293 cells. In some embodiments, the elevated level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold. In some embodiments, the retroviral vector or fusosome comprises an immunosuppressant that is not present in the reference cell. In some embodiments, the retroviral vector or fusosome comprises a reduced level of an immunostimulatory substance (e.g., an immunostimulatory protein) compared to a reference retroviral vector or fusosome, e.g., one produced from a similar but otherwise unmodified cell of origin, or HEK293 cells. In some embodiments, the reduction in level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% compared to the reference retroviral vector or fusosome, hi some embodiments, the immunostimulatory agent is substantially absent from the retroviral vector or fusosome.

[0496] In some embodiments, the retroviral vector or fusosome, or the cell of origin from which the retroviral vector or fusosome is derived, has one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more of the following characteristics: a. Expresses less than or equal to 50%, 40%, 30%, 20%, 15%, 10%, or 5% of MHC class I or MHC class II compared to a reference retroviral vector or fusosome, e.g., an unmodified retroviral vector or fusosome derived from an otherwise similar cell of origin, or a HeLa cell, or a HEK293 cell. b. Expression of one or more costimulatory proteins, including but not limited to, LAG3, ICOS-L, ICOS, Ox40L, OX40, CD28, B7, CD30, CD30L 4-1BB, 4-1BBL, SLAM, CD27, CD70, HVEM, LIGHT, B7-H3, or B7-H4, is less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% less than that of a reference retroviral vector or fusosome, e.g., an unmodified retroviral vector or fusosome derived from a cell otherwise similar to the cell of origin, or an HEK cell, or a reference cell described herein. c. Expression of a surface protein that inhibits phagocytosis by macrophages, e.g., CD47, as detected by the methods described herein, e.g., greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, compared to a reference retroviral vector or fusosome, e.g., an unmodified retroviral vector or fusosome derived from a cell otherwise similar to the cell of origin, Jurkat cells, or HEK293 cells. d. Expression of a soluble immunosuppressive cytokine, e.g., IL-10, e.g., greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, of a soluble immunosuppressive cytokine, e.g., IL-10, as detectable by the methods described herein, compared to a reference retroviral vector or fusosome, e.g., an unmodified retroviral vector or fusosome derived from a cell otherwise similar to the cell of origin, or HEK293 cells. e. Expression of a soluble immunoinhibitory protein, e.g., PD-L1, e.g., greater than 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, compared to a reference retroviral vector or fusosome, e.g., an unmodified retroviral vector or fusosome derived from a cell otherwise similar to the cell of origin, or HEK293 cells, e.g., expression d...

Claims

[Claim 1] The invention described in this specification.

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