Fusosome composition for T cell delivery

Abstract

Providing a fusosome composition for T cell delivery. [Solution] This disclosure provides, at least in part, methods and compositions for in vivo fusosome delivery. In some embodiments, the fusosome comprises a combination of elements that promote specificity to target cells, e.g., one or more fusogens, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome composition comprises one or more modifications that reduce the immune response to the fusosome.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 767,320, entitled "FUSOSOME COMPOSITIONS FOR T CELL DELIVERY," filed on 14 November 2018, and U.S. Patent Application No. 62 / 900,053, entitled "FUSOSOME COMPOSITIONS FOR T CELL DELIVERY," filed on 13 September 2019, the contents of which are incorporated in their entirety by reference for all purposes.

[0002] Reference to sequence listings This application is submitted with an electronic sequence listing. The sequence listing is provided as a file titled 186152003240SeqList.TXT, created on November 14, 2019, and is 627 kilobytes in size. The electronic information of the sequence listing is incorporated in its entirety by reference. [Background technology]

[0003] Complex biologics are promising therapeutic candidates for a variety of diseases. However, because the plasma membrane acts as a barrier between the cell and the extracellular space, delivering large biological factors to cells is difficult. In this field, there is a need for new methods to deliver complex biologics to the cells of test subjects. [Overview of the project] [Means for solving the problem]

[0004] This disclosure provides, at least in part, fusosome methods and compositions for in vivo delivery. In some embodiments, the fusosome comprises a combination of elements that promote specificity to target cells, e.g., one or more fusogens, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome composition comprises one or more modifications that reduce the immune response to the fusosome.

[0005] List of embodiments 1. The following: a) Lipid bilayer containing Fusogen; and b) Below: (i) Payload genes encoding exogenous active substances, for example, payload genes encoding exogenous active substances in Table 5, (ii) A fusosome comprising a nucleic acid comprising a positive target cell-specific regulatory element (e.g., a target cell-specific promoter) operably linked to a payload gene, wherein the positive target cell-specific regulatory element increases the expression of the payload gene in a target cell compared to a similar fusosome except that lacking the positive target cell-specific regulatory element, and the target cell is a T cell.

[0006] 2. A fusosome of Embodiment 1, wherein the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE) (e.g., a non-target cell-specific miRNA recognition sequence) operably linked to the payload gene, the NTCSRE reducing the expression of the payload gene in non-target cells compared to a similar fusosome except that lacking the NTCSRE, optionally, the target cell is a first type T cell and the non-target cell is a second different type T cell or a non-T cell, optionally, the target cell is a Treg cell and the non-target cell is a conventional CD4+ T cell.

[0007] 3. Below: a) Lipid bilayer containing Fusogen; and b) Below: (i) Exogenous active substances, such as payload genes encoding the exogenous active substances in Table 5, and (ii) A fusosome comprising a nucleic acid comprising a promoter operably ligated to a payload gene, wherein the promoter comprises, for example, the sequence of the promoters in Table 3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, selected from the IL2RA, LRRC32, FOXP3, or IKZF2 promoter.

[0008] 4. Below: a) Lipid bilayer containing Fusogen; and b) Below: (i) Payload genes encoding exogenous active substances, for example, payload genes encoding exogenous active substances in Table 5, (ii) Fusosomes comprising a non-target cell-specific regulatory element (NTCSRE) (e.g., a non-target cell-specific miRNA recognition sequence), i.e., a non-cell-specific regulatory element operably linked to a payload gene, wherein the NTCSRE is a nucleic acid comprising a non-target cell-specific regulatory element operably linked to a payload gene that reduces the expression of the payload gene in non-target cells or tissues compared to fusosomes otherwise identical except for the lack of the NTCSRE.

[0009] 5. Below: a) Lipid bilayer containing Fusogen; and b) Below: (i) Payload genes encoding exogenous active substances, for example, payload genes encoding exogenous active substances in Table 5, (ii) A fusosome comprising a nucleic acid containing a negative target cell-specific regulatory element (negative TCSRE) (e.g., a tissue-specific miRNA recognition sequence) which is a non-T cell-specific regulatory element operably linked to a payload gene, wherein the negative TCSRE reduces the expression of exogenous activators in non-target cells or tissues compared to nucleic acids that are otherwise similar but lacking the negative TCSRE.

[0010] 6. A fusosome of either Embodiment 4 or 5, wherein the nucleic acid further comprises a positive target cell-specific regulatory element (e.g., a target cell-specific promoter) operably linked to the payload gene, the positive target cell-specific regulatory element increasing the expression of the payload gene in the target cell compared to a similar fusosome except lacking the positive target cell-specific regulatory element, the target cell being a first type T cell and optionally the non-target cell being a second different type T cell or a non-T cell, and optionally the target cell being a Treg cell and the non-target cell being a conventional CD4+ T cell.

[0011] 7. Below: a) A lipid bilayer containing Fusogen, b) A payload gene encoding an exogenous active substance, for example, a nucleic acid containing a payload gene encoding an exogenous active substance as shown in Table 5, c) One or both of the following: (i) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer; or (ii) Fusosomes comprising a first immunostimulatory protein present at reduced levels (e.g., reduced to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes generated from otherwise similar unmodified source cells.

[0012] 8. Fusosomes of any of the embodiments described above, one or more of the following: i) Fusosomes fuse with target cells at a higher rate than non-target cells, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x; ii) Fusosomes fuse with target cells at a higher rate than other fusosomes, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x; iii) Fusosomes fuse with target cells at a rate such that the active substance within 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; iv) Fusosomes deliver nucleic acids, such as retroviral nucleic acids, to target cells at a higher rate than to non-target cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 50 times, or 100 times; v) Fusosomes deliver nucleic acids, such as retroviral nucleic acids, to target cells at a higher rate than other fusosomes, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, twice, three times, four times, five times, ten times, twenty times, fifty times, or 100 times; or vi) Fusosomes deliver nucleic acids, such as retroviral nucleic acids, to target cells in such a proportion that the active ingredient 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.

[0013] 9. A fusosome of any of the above embodiments, wherein one or more (e.g., two or all three) of the following is applied: the fusosome is a retroviral vector, the lipid bilayer is composed of an envelope, e.g., a viral envelope, and the nucleic acid is a retroviral nucleic acid.

[0014] 10. A fusosome of any of the above embodiments, wherein the nucleic acid comprises one or more (e.g., all) of the following nucleic acid sequences: a 5'LTR (e.g., containing U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tube (cPPT) promoter operably linked to the payload gene, the payload gene (optionally containing an intron before the open reading frame), a poly-A tail sequence, a WPRE, and a 3'LTR (e.g., lacking U5 and functional U3).

[0015] 11. A fusosome according to any of the above embodiments, comprising one or more (e.g., all) of the following: polymerase (e.g., reverse transcriptase, e.g., pol or a part thereof), integrase (e.g., pol or a part thereof, e.g., functional or non-functional variant), matrix protein (e.g., gag or a part thereof), capsid protein (e.g., gag or a part thereof), nucleocapsid protein (e.g., gag or a part thereof), and protease (e.g., pro).

[0016] 12. A fusosome of Embodiment 7, comprising (i) and (ii).

[0017] 13. A fusosome according to any of Embodiments 7 to 12, further comprising a second exogenous or overexpressed immunosuppressive protein on a lipid bilayer.

[0018] 14. A fusosome of any of Embodiments 7 to 13, comprising a second immunostimulatory protein that is absent or present at a reduced level, wherein the reduced level is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to fusosomes produced from otherwise similar unmodified source cells.

[0019] 15. A fusosome of any embodiment 7-14, wherein the nucleic acid, for example, a retroviral vector, further comprises a positive target cell-specific regulatory element (e.g., a target cell-specific promoter) operably linked to the payload gene, the positive target cell-specific regulatory element increasing the expression of the payload gene in a target cell compared to a similar fusosome except lacking the positive target cell-specific regulatory element, and the target cell is a T cell.

[0020] 16. A fusosome of any embodiment 7 to 15, comprising a nucleic acid, e.g., retroviral nucleic acid, operably linked to a payload gene, a non-target cell-specific regulatory element (NTCSRE) (e.g., a non-target cell-specific miRNA recognition sequence), wherein the NTCSRE reduces the expression of the payload gene in non-target cells or tissues compared to a similar fusosome except that lacking the NTCSRE, optionally, the target cell is a first type T cell and the non-target cell is a second different type T cell or a non-T cell, optionally, the target cell is a Treg cell and the non-target cell is a conventional CD4+ T cell.

[0021] 17. A fusosome according to any of Embodiments 7 to 15, wherein the nucleic acid, for example, retroviral nucleic acid, comprises a negative target cell-specific regulatory element (negative TCSRE) (e.g., tissue-specific miRNA recognition sequence) operably linked to a payload gene, the negative TCSRE reducing the expression of exogenous activators in non-target cells or tissues compared to a similar nucleic acid, for example, retroviral nucleic acid, that lacks the negative TCSRE.

[0022] 18. When administered to a subject (e.g., a human subject or a mouse), the fusosome of any of Embodiments 7 to 17 is one or more of the following: i) Fusosomes do not produce a detectable antibody response (e.g., after a single or multiple dose), or antibodies against fusosomes are present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of the background level, according to a FACS antibody detection assay, e.g., the assay of Example 13 or Example 14; ii) The fusosomes do not produce a detectable cellular immune response (e.g., T cell response, NK cell vesicle response, or macrophage response), or the cellular immune response to the fusosomes is present at background levels of less than 10%, 5%, 4%, 3%, 2%, or greater than 1%, according to, for example, a PBMC lysis assay (e.g., the assay of Example 5), an NK cell lysis assay (e.g., the assay of Example 6), a CD8 killer T cell lysis assay (e.g., the assay of Example 7), or a macrophage phagocytosis assay (e.g., the assay of Example 8); iii) The fusosomes do not produce a detectable innate immune response, e.g., complement activation (e.g., after a single or multiple dose), or, e.g., according to a complement activity assay (e.g., the assay of Example 9), an innate immune response to the fusosomes is present at a background level of less than 10%, 5%, 4%, 3%, 2%, or greater than 1%; iv) For example, according to a serum inactivation assay, such as the assay of Example 11 or Example 12, 10%, 5%, 4%, 3%, 2%, or less than 1% of fusosomes are inactivated by serum; v) Target cells that have received an exogenous activator from fusosomes do not produce a detectable antibody response (e.g., after a single or multiple dose), or, for example, according to a FACS antibody detection assay, e.g., the assay of Example 15, antibodies against the target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of the background level; or vi) Target cells that have received an exogenous activator from fusosomes do not produce a detectable cellular immune response (e.g., a T cell response, an NK cell vesicle response, or a macrophage response), or a cellular response to target cells is present at a level of less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of the background level, according to, for example, a macrophage phagocytic assay (e.g., the assay of Example 16), a PBMC lysis assay (e.g., the assay of Example 17), an NK cell lysis assay (e.g., the assay of Example 18), or a CD8 killer T cell lysis assay (e.g., the assay of Example 19).

[0023] 19. Fusosomes of Embodiment 18, wherein the background level is equivalent to that in the same subject before administration of fusosomes.

[0024] 20. A fusosome according to any of embodiments 7 to 19, wherein the immunosuppressive protein (e.g., the first immunosuppressive protein or the second immunosuppressive protein) is a complement regulatory protein or CD47.

[0025] 21. A fusosome according to any of Embodiments 7 to 20, wherein the immunostimulatory protein (e.g., the first immunostimulatory protein or the second immunostimulatory protein) is an MHC I (e.g., HLA-A, HLA-B, HLA-C, HLA-E, or HLA-G) or an MHC II (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR) protein.

[0026] 22. Exogenous active substances include hair follicle keratinocyte antigen, melanocyte antigen, CXCL8, CXCL9, CXCL10, CXCL11, CCL2, CCL5, intercellular adhesion molecule 1, gluten, PDGF, bacterial flagellin, FAM84A, alpha(2)-Heremans Schmidt glycoprotein (alpha(2)-HSG), transferrin, carbonate anhydrase, food-based antigens, thyroid-stimulating hormone receptor (TSHR), thyroid peroxidase, thyroglobulin, TSH receptor, alpha-enolase, alpha-β-crystallin, beta-arrestin, S100-beta, aquaporin-4, MOG, PLP, MBP, nAChR, MuSK, NC16A-terminal BPAG1 / 2, desmoglein 1, desmoglein 3, BPAG1, plectin, desmoplakin I, desmoplakin II, and A fusosome of any of the above embodiments, which is a binding portion (e.g., a CAR molecule) that binds to an antigen selected from boplakin, periplakin, alpha-2-macroglobulin-like-1, citrullinated peptide, non-self antigen, dsDNA, nucleosome histone, telomere, Sm core protein, hsp60, 437-460 (p277), PPIns / Pins, anti-insulin antigen, glutamate decarboxylase (GAD), carboxypeptidase H, insulinoma antigen-2, IA-2β, or an antigen in the cytoplasm of a neutrophil granulocyte.

[0027] 23. A fusosome of any of the above embodiments, wherein the fusogen contains VSV-G.

[0028] 24. A fusosome of Embodiments 1, 2, 6, 15, 22, or 23, wherein the positive target cell-specific regulatory element includes a T cell-specific promoter, a T cell-specific enhancer, a T cell-specific splice site, a T cell-specific site for extending the half-life of RNA or protein, a T cell-specific mRNA export-promoting site, a T cell-specific translation-enhancing site, or a T cell-specific post-translational modification site.

[0029] 25. Fusosomes of Embodiments 1, 2, 6, 15, or 22-24, wherein a positive target cell-specific regulatory element comprises a T cell-specific promoter.

[0030] 26. A fusosome of Embodiment 25, wherein the T cell-specific promoter includes the motifs shown in Table 3.

[0031] 27. A fusosome of Embodiment 25 or 26, wherein the positive T cell-specific regulatory element comprises a promoter selected from the IL2RA, LRRC32, FOXP3, or IKZF2 promoter.

[0032] 28. A fusosome according to any of Embodiments 4-6 or 16-21, wherein the negative TCSRE or NTCSRE includes a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site.

[0033] 29. A fusosome according to any of Embodiments 4-6, 16-21, or 28, wherein the negative TCSRE or NTCSRE includes 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.

[0034] 30. A fusosome according to any of Embodiments 4-6, 16-21, 28, or 29, wherein the negative TCSRE or NTCSRE includes a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site.

[0035] 31. A fusosome of any of Embodiments 4-6, 16-21, or 28-30, comprising a non-target cell-specific miRNA recognition sequence to which a negative TCSRE or NTCSRE is bound by the miRNAs of Table 4, for example, one or more (e.g., two or more) miR31, miR363, or miR29c.

[0036] 32. A fusosome of any of Embodiments 28 to 31, wherein a negative TCSRE or NTCSRE is located or encoded within a transcription region (e.g., a transcription region encoding an exogenous active agent) such that the RNA produced by the transcription region contains a miRNA recognition sequence within the UTR or coding region.

[0037] 33. A fusosome of any of the above embodiments, wherein the nucleic acid, for example, retroviral nucleic acid, comprises one or more insulator sequences.

[0038] 34. A fusosome of Embodiment 33, wherein the nucleic acid, for example, retroviral nucleic acid, comprises two insulator sequences, for example, a first insulator sequence upstream of the payload gene and a second insulator sequence downstream of the payload gene, and for example, the first insulator sequence and the second insulator sequence comprise the same or different sequences.

[0039] 35. A fusosome of any of the above embodiments that is not genotoxic or does not increase the rate of tumorigenesis in target cells.

[0040] 36. A fusosome of any of the above embodiments, in which nucleic acids, such as retroviral nucleic acids, can be integrated into the genome of a target cell.

[0041] 37. A fusosome of Embodiment 36, wherein the nucleic acid, for example, retroviral nucleic acid, is a lentivirus with integration capability or a lentivirus without integration capability.

[0042] 38. A fusosome according to any of the above embodiments, wherein the target cell is selected from T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, naive T cells, effector T cells, cytotoxic T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymic regulatory T cells, peripheral regulatory T cells, CD4+Foxp3+ regulatory T cells, or CD4+FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T(MAIT) cells.

[0043] 39. One or more of the following fusosomes from embodiments 4-6 and 9-38: i) 10%, 5%, 4%, 3%, 2%, or less than 1% of the exogenous active ingredients detectable in the subject are present in non-target cells; ii) At least 90%, 95%, 96%, 97%, 98%, or 99% of the subject's cells containing the exogenous active ingredient detectably are target cells (e.g., single-cell type cells); iii) Of the cells of the subject that contain detectable exogenous active ingredients, fewer than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 cells are non-target cells; iv) The mean level of exogenous active substances in all target cells of the subject is at least 100 times, 200 times, 500 times, or 1,000 times higher than the mean level of exogenous active substances in all non-target cells of the subject; or v) The exogenous active ingredient is not detected in the non-target cells of the subject.

[0044] 40. A fusosome of any of the above embodiments, wherein nucleic acids, such as retroviral nucleic acids, encode positive TCSRE and / or NTCSRE or negative TCSRE.

[0045] 41. A fusosome of any of the above embodiments, wherein the nucleic acid, for example, retroviral nucleic acid, comprises a complement of positive TCSRE and / or NTCSRE or negative TCSRE.

[0046] 42. A fusosome of either Embodiment 40 or 41, comprising a target cell-specific promoter in which a positive TCSRE has at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, or 1000% activity in target cells compared to non-target cells.

[0047] 43. A fusosome of any of Embodiments 40 to 42, wherein a negative TCSRE or NTCSRE includes a miRNA recognition sequence that reduces gene expression in non-target cells by at least 10%, 25%, 50%, 75%, or 100% compared to target cells.

[0048] 44. A fusosome of any of the above embodiments that does not deliver nucleic acids, such as retroviral nucleic acids, to non-target cells, such as conventional CD4+ T cells, 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, spleen cells, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0049] 45. For example, using quantitative PCR, for example using the assay of Example 1, 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, 0.000001%, or less than 0.000001% of non-target cell types (e.g., one or more of conventional CD4+ T cells, 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) are fusosomes of any of the above embodiments, comprising nucleic acids, such as retroviral nucleic acids.

[0050] 46. ​​Fusosomes of any of the above embodiments, wherein the target cell contains 0.00001-10, 0.0001-10, 0.001-10, 0.01-10, 0.1-10, 0.5-5, 1-4, 1-3, or 1-2 copies of nucleic acid per host genome, for example, retroviral nucleic acid or a portion thereof, and for example, the copy number of the nucleic acid, for example, the retroviral nucleic acid is evaluated after in vivo administration.

[0051] 47. A fusosome of any of the embodiments described above, wherein: Less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, or 0.01% of non-target cells (e.g., conventional CD4+ T cells, 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, spleen cells, B cells, hepatocytes, endothelial cells, or non-cancerous cells) contain exogenous active ingredients; or A fusosome of any of the above embodiments, wherein the exogenous active substance (e.g., protein) is undetectably absent in non-target cells, such as conventional CD4+ T cells, 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, spleen cells, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0052] 48. A fusosome of any of the above embodiments, wherein the fusosome delivers nucleic acids, such as retroviral nucleic acids, to target cells, such as T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, naive T cells, effector T cells, cytotoxic T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymic regulatory T cells, peripheral regulatory T cells, CD4+Foxp3+ regulatory T cells, or CD4+FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T(MAIT) cells.

[0053] 49. For example, using quantitative PCR, for example using the assay of Example 3, at least 0.00001%, 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells (e.g., T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, naive T cells, effector T cells, cytotoxic T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymus-derived regulatory T cells, peripheral-derived regulatory T cells) A fusosome according to any of the above embodiments, wherein one or more T cells (CD4+Foxp3+ regulatory T cells, or CD4+FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosal-associated invariant T(MAIT) cells) comprises nucleic acid, such as retroviral nucleic acid.

[0054] 50. At least 0.00001%, 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of target cells (e.g., T cells, CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, naive T cells, effector T cells, cytotoxic T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymus-derived regulatory T cells, peripheral-derived regulatory T cells, CD4+ Fox A fusosome according to any of the above embodiments, wherein the fusosome contains an exogenous active agent and comprises p3+ regulatory T cells (or CD4+ FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T(MAIT) cells).

[0055] 51. Fusosomes of any of the above embodiments, wherein, at the time of administration, the ratio of target cells containing nucleic acids, e.g., retroviral nucleic acids, to non-target cells containing nucleic acids, e.g., retroviral nucleic acids is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, according to a quantitative PCR assay, for example, using the assays of Example 1 and Example 3.

[0056] 52. A fusosome of any of the above embodiments, wherein the ratio of the average copy number of nucleic acid, e.g., retroviral nucleic acid or a portion thereof, in target cells to the average copy number of nucleic acid, e.g., retroviral nucleic acid or a portion thereof, in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, as determined by quantitative PCR assay, for example, using the assays of Example 1 and Example 3.

[0057] 53. Fusosomes of any of the above embodiments, wherein the ratio of the median copy number of nucleic acid, e.g., retroviral nucleic acid or a portion thereof, in target cells to the median copy number of nucleic acid, e.g., retroviral nucleic acid or a portion thereof, in non-target cells is, for example, 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, as determined by a quantitative PCR assay, for example, using the assays of Example 1 and Example 3.

[0058] 54. A fusosome of any of the above embodiments, wherein the ratio of target cells containing the exogenous RNA activator to non-target cells containing the exogenous RNA activator is, for example, 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, as determined by a reverse transcription quantitative PCR assay.

[0059] 55. A fusosome of any of the above embodiments, wherein the ratio of the average exogenous RNA activity level of target cells to the average exogenous RNA activity level of non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, as determined by a reverse transcription quantitative PCR assay.

[0060] 56. A fusosome of any of the above embodiments, wherein the ratio of the median exogenous RNA activator level of target cells to the median mean exogenous RNA activator level of non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, as determined by a reverse transcription quantitative PCR assay.

[0061] 57. Fusosomes of any of the above embodiments, wherein the ratio of target cells containing an exogenous proteoactive agent to non-target cells containing an exogenous proteoactive agent is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, according to a FACS assay, for example, using the assays of Example 2 and Example 4.

[0062] 58. Fusosomes of any of the above embodiments, wherein the ratio of the average exogenous proteoactive substance level of target cells to the average exogenous proteoactive substance level of non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, according to a FACS assay, for example, using the assays of Example 2 and Example 4.

[0063] 59. Fusosomes of any of the above embodiments, wherein the ratio of the median level of exogenous proteoactive material in target cells to the median level of exogenous proteoactive material in non-target cells is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, or 10,000, according to a FACS assay, for example, using the assays of Example 2 and Example 4.

[0064] 60. A fusosome of any of the embodiments described above, comprising one or both of the following: i) Exogenous or overexpressed immunosuppressive proteins on the lipid bilayer, e.g., envelopes; and (ii) Immunostimulatory proteins that are absent or present at reduced levels (e.g., reduced to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes produced from otherwise similar, unmodified source cells.

[0065] 61. Fusosomes of any of the embodiments described above, including one or more of the following: i) A lipid bilayer, e.g., a first exogenous or overexpressed immunosuppressive protein on the envelope, and a lipid bilayer, e.g., a second exogenous or overexpressed immunosuppressive protein on the envelope; ii) A first exogenous or overexpressed immunosuppressive protein on the lipid bilayer, e.g., the envelope, and a second immunostimulatory protein present in a reduced level (e.g., reduced to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes generated from otherwise similarly unmodified source cells; or iii) A first immunostimulatory protein present in a reduced level (e.g., reduced to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes produced from otherwise similar, unmodified source cells, and a second immunostimulatory protein present in a reduced level (e.g., reduced to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes produced from otherwise similar, unmodified source cells.

[0066] 62. Fusosomes of any of the above embodiments, wherein the fusosomes circulate for at least 0.5, 1, 2, 3, 4, 6, 12, 18, 24, 36, or 48 hours after administration to a subject.

[0067] 63. Fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes are circulating for 30 minutes after administration.

[0068] 64. Fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes are circulating for 1 hour after administration.

[0069] 65. Fusosomes of any of the above 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 circulating for 2 hours after administration.

[0070] 66. Fusosomes of any of the above 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 circulating for 4 hours after administration.

[0071] 67. Fusosomes of any of the above 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 circulating for 8 hours after administration.

[0072] 68. Fusosomes of any of the above 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 circulating for 12 hours after administration.

[0073] 69. Fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes are circulating for 18 hours after administration.

[0074] 70. Fusosomes of any of the above embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes are circulating for 24 hours after administration.

[0075] 71. Fusosomes of any of the above 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 circulating for 36 hours after administration.

[0076] 72. Fusosomes of any of the above 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 circulating for 48 hours after administration.

[0077] 73. A fusosome of any of the above embodiments having reduced immunogenicity, as measured by a decrease in humoral response after one or more administrations of the fusosome to a suitable animal model, e.g., an animal model described herein, compared to an unmodified fusosome otherwise similar to a fusosome.

[0078] 74. Fusosomes of Embodiment 73, in which a decrease in humoral response is measured in a serum sample by anti-cell antibody titer, for example, antiretroviral antibody titer, for example, by ELISA.

[0079] 75. Fusosomes of any of the above embodiments, wherein serum samples from animals administered with fusosomes exhibit a reduction in anti-fusosome antibody titer of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater, compared to serum samples from subjects administered with unmodified cells.

[0080] 76. A serum sample from a subject administered with fusosomes has an increased anti-cell antibody titer, for example, increased by 1%, 2%, 5%, 10%, 20%, 30%, or 40% from baseline, for example, the fusosomes of any of the embodiments described above, where baseline refers to a serum sample from the same subject before fusosome administration.

[0081] 77. A fusosome of any of the embodiments described above: A subject administered with fusosomes or a pharmaceutical composition containing fusosomes has, is known to have, or is tested for, pre-existing antibodies (e.g., IgG or IgM) that are reactive with fusosomes; The subjects receiving fusosomes do not have pre-existing antibodies that are detectable to be reactive with fusosomes; A subject who has received fusosomes or a pharmaceutical composition containing fusosomes has, is known to have, or is tested for antibodies (e.g., IgG or IgM) that are reactive with fusosomes; Subjects administered fusosomes or a pharmaceutical composition containing fusosomes (e.g., at least one, two, three, four, five, or more times) do not possess detectable levels of antibodies that are reactive with fusosomes; or The antibody level does not increase by more than 1%, 2%, 5%, 10%, 20%, or 50% between the two time points, with the first time point being before the first administration of fusosomes and the second time point being after one or more administrations of fusosomes.

[0082] 78. Fusosomes of any of the above embodiments, wherein the fusosomes are produced from cells transfected with, for example, HLA-G or HLA-E cDNA by the method of Example 5, 6, or 7.

[0083] 79. Fusosomes of any of the above embodiments, wherein the rate of lysis, e.g., PBMC-mediated lysis, NK cell vesicle-mediated lysis, and / or CD8+ T cell-mediated lysis, is reduced at a particular time point compared to fusosomes generated from NMC or NMC-empty vectors.

[0084] 80. A fusosome according to any of the above embodiments, wherein the modified fusosome evades phagocytosis by macrophages.

[0085] 81. Fusosomes of any of the above embodiments, wherein the fusosomes are produced, for example, from cells transfected with CD47 cDNA by the method of Example 8.

[0086] 82. Fusosomes of any of the above embodiments, wherein macrophages exhibit a reduced phagocytic index when incubated with a retroviral vector derived from NMC-CD47 compared to a vector derived from NMC or an empty vector from NMC.

[0087] 83. Fusosomes of any of the above embodiments, which, compared to reference fusosomes, for example unmodified fusosomes otherwise similar to fusosomes, exhibit a reduction in macrophage phagocytosis of, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, and in which case the reduction in macrophage phagocytosis is determined by assaying the in vitro phagocytosis index, for example, as described in Example 8.

[0088] 84. Fusosomes of any of the above embodiments, having a phagocytic index of 0, 1, 10, 100, or higher, as measured by the assay of Example 8, for example, when the fusosome composition is incubated with macrophages in an in vitro assay of macrophage phagocytosis.

[0089] 85. A fusosome of any of the above embodiments, which has been modified and has reduced complement activity compared to an unmodified fusosome.

[0090] 86. Fusosomes of any of the above embodiments, produced by the method of Example 9, for example, from cells transfected with a complement regulatory protein, such as a cDNA encoding DAF.

[0091] A fusosome of any of the above embodiments, wherein the dose of fusosomes containing 87,200 pg / ml of C3a is greater in the case of a modified fusosome (e.g., HEK293-DAF) incubated with the corresponding mouse serum (e.g., HEK293 DAF mouse serum) than in the case of a reference fusosome (e.g., HEK293 retroviral vector) incubated with the corresponding mouse serum (e.g., HEK293 mouse serum).

[0092] A fusosome of any of the above embodiments, wherein the dose of fusosomes containing 88,200 pg / ml of C3a is greater in the case of modified fusosomes incubated with naive mouse serum (e.g., HEK293-DAF) than in the case of reference fusosomes incubated with naive mouse serum (e.g., HEK293 retroviral vector).

[0093] 89. Fusosomes of any of the above embodiments, wherein the fusosomes are resistant to complement-mediated inactivation in patient serum 30 minutes after administration, according to the assay of Example 9.

[0094] 90. Fusosomes of any of the above 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.

[0095] 91. A fusosome of any of Embodiments 86 to 90, wherein the complement regulatory protein comprises one or more proteins that bind to complement-aggregating factors (DAF, CD55), such as factor H (FH)-like protein-1 (FHL-1), such as C4b-binding protein (C4BP), such as complement receptor 1 (CD35), such as membrane cofactor proteins (MCP, CD46), such as protectin (CD59), such as proteins that inhibit classical and alternative complement pathway CD / C5 convertase enzymes, such as proteins that regulate MAC assembly.

[0096] 92. For example, fusosomes of any of the above embodiments produced by the method of Example 10 from cells transfected with DNA encoding shRNA targeting MHC class I, wherein, for example, the retroviral vector derived from NMC-shMHC class I has lower MHC class I expression compared to NMC and NMC vector controls.

[0097] 93. Fusosomes of any of the above embodiments, wherein the measure of immunogenicity to fusosomes is serum inactivation, and is measured, for example, as described herein, as described in Example 11.

[0098] 94. Fusosomes of any of the above embodiments, wherein the percentage of cells receiving exogenous agents does not differ between fusosome samples incubated with serum from fusosome-naive mice and thermo-inactivated serum.

[0099] 95. Fusosomes of any embodiment in which the percentage of cells receiving an exogenous agent does not differ between a fusosome sample incubated with serum from a fusosome-naive mouse and a serum-free control incubation.

[0100] 96. Fusosomes of any of the above embodiments, wherein the percentage of cells receiving the exogenous agent is lower in fusosome samples incubated with positive control serum than in fusosome samples incubated with serum from fusosome-naive mice.

[0101] 97. Fusosomes of any of the above embodiments, modified, for example, those modified by the method described herein, which have a reduction in serum inactivation (for example, a reduction compared to administration of unmodified fusosomes) following multiple administrations (e.g., more than one, e.g., two or more) of the modified fusosomes.

[0102] 98. Fusosomes of any of the above embodiments, wherein the fusosomes described herein are not inactivated by serum after multiple administrations.

[0103] 99. Fusosomes of any of the above embodiments, wherein the measure of immunogenicity of the fusosome is, for example, serum inactivation after multiple doses, for example, serum inactivation after multiple doses as measured as described herein, for example, as described in Example 12.

[0104] 100. Fusosomes of any of the above embodiments, wherein the percentage of cells receiving the exogenous agent does not differ between serum from mice treated with modified (e.g., HEK293-HLA-G) fusosomes and fusosome samples incubated with thermo-inactivated serum.

[0105] 101. Fusosomes of any of the above embodiments, wherein the percentage of cells receiving the exogenous agent does not differ between serum from mice treated 1, 2, 3, 5, or 10 times with modified (e.g., HEK293-HLA-G) fusosomes and fusosome samples incubated with thermo-inactivated serum.

[0106] 102. Fusosomes of any of the above embodiments, wherein the percentage of cells receiving the exogenous agent does not differ between fusosome samples incubated with serum from vehicles-treated mice and from mice treated with modified (e.g., HEK293-HLA-G) fusosomes.

[0107] 103. Fusosomes of any of the above embodiments, wherein the percentage of cells receiving the exogenous agent is lower for fusosomes derived from a reference cell (e.g., HEK293) than for fusosomes derived from a modified cell (e.g., HEK293-HLA-G).

[0108] 104. A fusosome of any of the above embodiments, wherein the measure of immunogenicity against the fusosome is an antibody response.

[0109] 105. A fusosome of any of the above embodiments, wherein the subject receiving the fusosome as described herein has an existing antibody that binds to and recognizes the fusosome, measured as described herein, for example, as described in Example 13.

[0110] 106. Fusosomes of any of the above embodiments, wherein serum from a fusosome-naive mouse shows more signal (e.g., fluorescence) than serum from a negative control, e.g., a mouse depleted of IgM and IgG, indicating, for example, that immunogenicity has occurred.

[0111] 107. Fusosomes of any of the above embodiments, wherein serum from fusosome-naive mice exhibits a similar signal (e.g., fluorescence) compared to a negative control, indicating, for example, that no detectable immunogenicity occurred.

[0112] 108. A fusosome of any of the embodiments described above, which is a modified fusosome, for example, modified by the method described herein, and which, as measured for example as described herein, exhibits a reduced humoral response after multiple administrations (e.g., more than one, e.g., two or more) of the modified fusosome (e.g., reduced compared to administration of unmodified fusosomes).

[0113] 109. Fusosomes of any of the above embodiments, wherein the fusosomes are produced from cells transfected with, for example, HLA-G or HLA-E cDNA by the method of Example 5, 6, 7, or 14.

[0114] 110. A fusosome of any of the above embodiments, wherein the humoral response is evaluated by determining the level values ​​of anti-fusosome antibodies (e.g., IgM, IgG1, and / or IgG2 antibodies).

[0115] 111. Fusosomes of any of the above embodiments, wherein the modified (e.g., NMC-HLA-G) fusosomes exhibit reduced antiviral IgM or IgG1 / 2 antibody titers (e.g., measured by fluorescence intensity in FACS) after injection, compared to a control, e.g., NMC fusosomes or NMC-empty fusosomes.

[0116] 112. Fusosomes of any of the above embodiments in which recipient cells are not targeted by an antibody response, or in which the antibody response is below a reference level when measured, for example, as described herein, for example, as described in Example 15.

[0117] 113. Fusosomes of any of the above embodiments, wherein the signal (e.g., mean fluorescence intensity) is similar in recipient cells from mice treated with fusosomes and mice treated with PBS.

[0118] 114. A fusosome of any of the above embodiments, wherein the measure of recipient cell immunogenicity is the macrophage response.

[0119] 115. A fusosome of any of the above embodiments, wherein the recipient cell is not targeted by the macrophage, or is targeted below the reference level.

[0120] 116. Fusosomes of any of the above embodiments, for example, in which the phagocytic index measured as described herein, for example as described in Example 16, is similar in recipient cells from mice treated with fusosomes and mice treated with PBS.

[0121] 117. A fusosome of any of the above embodiments, wherein the measure of recipient cell immunogenicity is the PBMC response.

[0122] 118. A fusosome of any of the above embodiments, wherein the recipient cell does not induce a PBMC response.

[0123] 119. Fusosomes of any of the aforementioned embodiments, where the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated mice and PBS-treated mice, for example, when measured as described herein, for example, as described in Example 17.

[0124] 120. A fusosome of any of the above embodiments, wherein the measure of recipient cell immunogenicity is a natural killer cell response.

[0125] 121. A fusosome of any of the above embodiments, wherein the recipient cell does not induce a natural killer cell response, or induces a natural killer cell response lower than, for example, a reference value.

[0126] 122. Fusosomes of any of the aforementioned embodiments, where the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated mice and PBS-treated mice, for example, when measured as described herein, for example, as described in Example 18.

[0127] 123. A fusosome of any of the above embodiments, wherein the measure of recipient cell immunogenicity is a CD8+ T cell response.

[0128] 124. A fusosome of any of the above embodiments, wherein the recipient cell does not induce a CD8+ T cell response, or induces a CD8+ T cell response lower than, for example, a reference value.

[0129] 125. Fusosomes of any of the aforementioned embodiments, where the percentage of CD3+ / CMG+ cells is similar in recipient cells derived from fusosome-treated mice and PBS-treated mice, for example, when measured as described herein, for example, as described in Example 19.

[0130] 126. A fusosome of any of the above embodiments, wherein the fusogen is a retargeted fusogen.

[0131] 127. A fusosome of any of the embodiments described above, comprising (i) a positive target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous active agent, or (ii) a nucleic acid encoding one or both of a non-target cell-specific regulatory element or a negative TCSRE operably linked to a nucleic acid encoding an exogenous active agent, such as a retroviral nucleic acid.

[0132] 128. A pharmaceutical composition comprising a fusosome of any of the embodiments described above and a pharmaceutically acceptable carrier, diluent, or excipient.

[0133] 129. A method for delivering an exogenous active substance to a subject (e.g., a human subject), comprising administering a fusosome of any of Embodiments 1 to 127 or a pharmaceutical composition described in Embodiment 128 to the subject, thereby delivering the exogenous active substance to the subject.

[0134] 130. A method for modulating function in a subject (e.g., a human subject), a target tissue, or a target cell (e.g., a T cell, e.g., a Treg cell), comprising contacting the subject, target tissue, or target cell with a fusosome of any of Embodiments 1 to 127, or with a pharmaceutical composition of Embodiment 128, for example, administering it thereto.

[0135] 131. The method of Embodiment 130, wherein the target tissue or target cells are present in the subject.

[0136] 132. A method for treating a disease or disorder in a subject (e.g., a human subject), comprising administering to the subject a fusosome of any of Embodiments 1 to 127, or a pharmaceutical composition of Embodiment 128.

[0137] 133. The method of Embodiment 132, wherein the disease or disorder is one of the diseases or disorders listed in Table 5.

[0138] 134. The method of Embodiment 132 or 133, wherein the disease or disorder is a disease or disorder that can be treated by a payload gene encoding an exogenous active agent.

[0139] 135. Any method of Embodiments 132 to 134, wherein the disease or disorder is selected from acute GvHD, alopecia areata, autoimmune uveitis, celiac disease, chronic GvHD, Crohn's disease, endometriosis, eosinophilic esophagitis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, bullous pemphigoid, pemphigus, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, type 1 diabetes mellitus, and ulcerative colitis.

[0140] 136. Fusosomes of any of Embodiments 1 to 127 or a pharmaceutical composition of Embodiment 128 for use in treating subjects (e.g., human subjects) having a disease or disorder.

[0141] 137. Use of fusosomes of any of Embodiments 1 to 127 or the pharmaceutical composition of Embodiment 128 for the production of an active substance for use in treating a subject with a disease or disorder (e.g., a human subject).

[0142] 138. Fusosomes or pharmaceutical compositions for use in Embodiment 137, or use in Embodiment 138, wherein the fusosomes contain a payload gene encoding an exogenous active substance for treating a disease or disorder.

[0143] 139. Fusosomes or pharmaceutical compositions for use in Embodiment 136 or 138, or use in Embodiment 137 or 138, for a disease or disorder selected from acute GvHD, alopecia areata, autoimmune uveitis, celiac disease, chronic GvHD, Crohn's disease, endometriosis, eosinophilic esophagitis, Graves' disease, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, bullous pemphigoid, pemphigus, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, type 1 diabetes mellitus, or ulcerative colitis.

[0144] 140. A method for producing fusosomes according to any of Embodiments 1 to 127, including the following: a) To provide cells containing nucleic acids, such as retroviral nucleic acids, and fusogens; b) Culturing the above cells under conditions that enable the production of fusosomes, and c) Isolating, concentrating, or purifying fusosomes from the above-mentioned cells, and thereby producing fusosomes.

[0145] Other features, purposes, and advantages of the present invention will become apparent from the description and drawings, as well as from the claims.

[0146] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referenced herein, for example, in any of the tables, are incorporated by reference. Unless otherwise specified, sequence accession numbers specified herein, including in the tables herein, refer to database entries as of May 15, 2018. If a gene or protein references multiple sequence accession numbers, all sequence variants are included. In addition, materials, methods, and examples are illustrative and not intended to limit.

[0147] The following detailed description of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, specific embodiments described herein are illustrated herein. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings. [Brief explanation of the drawing]

[0148] [Figure 1] Figure 1 quantifies the staining of fusosomes with F-actin dye. [Figure 2]Figure 2 is a graph showing the ability of fusosomes and parent cells to polymerize actin over periods of 3, 5, and 24 hours. [Figure 3] Figure 3 is a table showing the size distribution statistics of fusosomes and parent cells as measured by NTA and microscopy. [Figure 4] Figure 4 is a table showing the average size and volume of fusosomes and parent cells. [Figure 5] Figure 5 is a series of diagrams showing the soluble:insoluble ratios observed for fusosomes or cell preparations. [Figure 6] Figure 6 is a series of diagrams showing the absolute amounts of MvH(CD8)+F fusosome fusion to target cells or non-target cells, and target fusions. [Figure 7] Figure 7 shows the average fluorescence intensity of 2-NBDG in VSV-G fusosomes. [Figure 8] Figure 8 shows the esterase activity in the cytosol of VSV-G fusosomes. [Figure 9-1] Figures 9A–9B are a series of images showing Cre recombinase delivery by fusosomes as detected by bioluminescence imaging in mice. (A) Ventral images of the exposed liver and spleen of IV fusosome-treated mice (1x and 3x concentrations) with luminescence signal overlays. The lower part shows only the luminescence signal. (B) Total luminosity signals of the spleen and liver targeting fusosomes; the γ-scale is on a log10 scale. Mice treated with a 3x fusosome treatment concentration showed a significantly larger signal in the spleen than the background 72 hours post-treatment (p=0.0004). [Figure 9-2]Figures 9A–9B are a series of images showing Cre recombinase delivery by fusosomes as detected by bioluminescence imaging in mice. (A) Ventral images of the exposed liver and spleen of IV fusosome-treated mice (1x and 3x concentrations) with luminescence signal overlays. The lower part shows only the luminescence signal. (B) Total luminosity signals of the spleen and liver targeting fusosomes; the γ-scale is on a log10 scale. Mice treated with a 3x fusosome treatment concentration showed a significantly larger signal in the spleen than the background 72 hours post-treatment (p=0.0004). [Figure 10-1] Figures 10A–10B are a series of images showing Cre recombinase mediated by fusosomes in mouse liver and spleen, as detected by bioluminescence imaging. (A) From left to right; dorsal images and luminescence signal overlays of resected liver, heart, lung, kidney, small intestine, pancreas, and spleen, collected and imaged within 5 minutes of euthanasia. The lower part shows only the luminescence signal. (B) Total luminescence signals of spleen and liver, as well as other tissues, targeted by fusosomes; the γ-scale is on a log10 scale. Mice treated with 3x higher fusosome concentrations showed significantly stronger signals in the spleen compared to the tissue with the lowest signal (heart) (p<0.0001). [Figure 10-2] Figures 10A–10B are a series of images showing Cre recombinase mediated by fusosomes in mouse liver and spleen, as detected by bioluminescence imaging. (A) From left to right; dorsal images and luminescence signal overlays of resected liver, heart, lung, kidney, small intestine, pancreas, and spleen, collected and imaged within 5 minutes of euthanasia. The lower part shows only the luminescence signal. (B) Total luminescence signals of spleen and liver, as well as other tissues, targeted by fusosomes; the γ-scale is on a log10 scale. Mice treated with 3x higher fusosome concentrations showed significantly stronger signals in the spleen compared to the tissue with the lowest signal (heart) (p<0.0001). [Figure 11] Figure 11 is a table showing the delivery of Cre cargo by NivG+F fusosomes via a non-endocytosis pathway. [Figure 12]Figure 12 is a graph showing the GAPDH:total protein ratio measured by the bicinchoninate assay in fusosomes and parental cells. [Figure 13] Figure 13 is a graph showing the lipid:protein ratio measured by the bicinchoninate assay in fusosomes and parental cells. [Figure 14] Figure 14 is a graph showing the protein:DNA ratio measured by the bicinchoninate assay in fusosomes and parental cells. [Figure 15] Figure 15 is a graph showing the lipid:DNA ratio measured by the bicinchoninate assay in fusosomes and parental cells. [Figure 16] Figure 16 is a graph showing the protein levels of the exosome marker CD63 in exosomes and fusosomes. [Figure 17] Figure 17 is a graph showing the intensity of the calnexin signal detected in fusosomes and parental cells. [Figure 18] Figure 18 is a graph showing the lipid-to-DNA ratio determined for fusosomes and parental cells. [Figure 19-1] Figures 19A-19B are a series of graphs showing the proportion of lipid species as a percentage of total lipids in parent cells, exosomes, and fusosomes. [Figure 19-2] Figures 19A-19B are a series of graphs showing the proportion of lipid species as a percentage of total lipids in parent cells, exosomes, and fusosomes. [Figure 20] Figure 20 is a series of graphs showing the protein content of parent cells, exosomes, and fusosomes with respect to proteins associated with specific compartments, as shown. [Figure 21] Figure 21 is a series of graphs showing the levels of ARRDC1 (left panel) or TSG101 (right panel) as a percentage of total protein content in parenteral cells, exosomes, and fusosomes. [Modes for carrying out the invention]

[0149] This disclosure provides, at least in part, fusosome methods and compositions for in vivo delivery. In some embodiments, the fusosome comprises a combination of elements that promote specificity to target cells, e.g., one or more retargeted fusogens, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome composition comprises one or more modifications that reduce the immune response to the fusosome.

[0150] I. Definition Unless otherwise specified, terms used in the claims and specification are defined as follows:

[0151] As used herein, the term “antigen-binding domain” refers to that portion of an antibody or chimeric antigen receptor that binds to an antigen. In some embodiments, the antigen-binding domain binds to a cell surface antigen of a cell. In some embodiments, the antigen-binding domain binds to an antigen characteristic of cancer, for example, a tumor-associated antigen in neoplastic cells. In some embodiments, the antigen-binding domain binds to an antigen characteristic of infectious diseases, for example, a virus-associated antigen in virus-infected cells. In some embodiments, the antigen-binding domain binds to an antigen characteristic of a cell targeted by the immune system in question, in an autoimmune disease, for example, an autoantigen. In some embodiments, the antigen-binding domain is an antibody or its antigen-binding portion, or includes them. In some embodiments, the antigen-binding domain is an scFv or Fab, or includes them.

[0152] The terms “cancer,” “malignant tumor,” “neoplasm,” “tumor,” and “carcinoma” are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormal proliferation phenotype characterized by a significant loss of control over cell proliferation. In some embodiments, tumors may be or include cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic. This disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the cancers in question may be characterized by solid tumors. In some embodiments, tumors may be dispersed tumors or liquid tumors. In some embodiments, the cancers in question may be characterized by hematological tumors. Generally, examples of different types of cancer known in the art include, for example, leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma and myeloproliferative disorders, sarcoma, melanoma, adenoma, solid tissue carcinoma, squamous cell carcinoma of the mouth, throat, larynx, and lung, genitourinary cancers such as liver cancer, prostate, cervix, bladder, endometrial and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, melanoma of the skin or eye, endocrine cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancer, benign lesions such as papillomas, etc.

[0153] As used herein, “CDR” refers to a complementarity-determining region that can be located, for example, within an antibody variable region. Each variable region of the heavy and light chains has three CDRs, referred to as CDR1, CDR2, and CDR3 for each variable region. A “set of CDRs” or “CDR set” refers to a group of three or six CDRs that occur in a single variable region capable of binding to an antigen or in any of the congeneral heavy and light chain variable regions capable of binding to an antigen. Specific systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.), and those skilled in the art can understand the differences between and within these systems and understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0154] As used herein, "detectably present" means that the exogenous active substance itself is detectably present when used in the context of an exogenous active substance being detectably present. For example, if the exogenous active substance is a protein, an exogenous proteoactive substance can be detectably present regardless of whether the nucleic acid encoding it is detectably present.

[0155] As used herein, “fusosome” refers to an amphiphilic lipid bilayer surrounding a lumen or cavity, and fusogens that interact with the amphiphilic lipid bilayer. In embodiments, fusosomes contain nucleic acids. In some embodiments, fusosomes are membrane-bound preparations. In some embodiments, fusosomes are derived from source cells.

[0156] As used herein, “fusosome composition” refers to a composition comprising one or more fusosomes.

[0157] As used herein, “fusogen” refers to an active substance or molecule that creates an interaction between two membrane-enclosed lumens. In some embodiments, the fusogen promotes membrane fusion. In other embodiments, the fusogen creates a connection, e.g., a pore, between two lumens (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., none of which possess fusion activity alone. In some embodiments, the fusogen includes a targeting domain.

[0158] As used herein, “insulator sequence” refers to a nucleotide sequence that blocks an enhancer or prevents the diffusion of heterochromatin. The insulator sequence may be wild-type or a variant.

[0159] As used herein, the term “effective dose” means an amount of a pharmaceutical composition sufficient to significantly and positively modify (e.g., provide a positive clinical response) the symptom and / or condition being treated. The effective dose of an active ingredient for use in a pharmaceutical composition varies depending on the specific condition being treated, the severity of the condition, the duration of treatment, the nature of the combination therapy, the specific active ingredient(s) used, the specific pharmaceutically acceptable excipients and / or carriers utilized, and similar factors based on the knowledge and expertise of the attending physician.

[0160] As used herein with respect to viruses, VLPs, or fusosomes, “exogenous active material” refers to an active material that is not contained in and does not encode the fusogen produced from the corresponding wild-type virus or the corresponding wild-type source cell. In some embodiments, the exogenous active material is not naturally occurring, such as a protein or nucleic acid having a sequence that is modified (e.g., by insertion, deletion, or substitution) compared to a naturally occurring protein. In some embodiments, the exogenous active material is not naturally present in the source cell. In some embodiments, the exogenous active material is naturally present in the source cell but is exogenous with respect to the virus. In some embodiments, the exogenous active material is not naturally present in the recipient cell. In some embodiments, the exogenous active material is naturally present in the recipient cell but is not present at a desired level or for a desired time. In some embodiments, the exogenous active material includes RNA or a protein.

[0161] As used herein, the term “pharmaceutically acceptable” means an excipient, composition and / or dosage form suitable for use in contact with human and animal tissues, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, and with a reasonable benefit-to-risk ratio.

[0162] As used herein, “promoter” refers to a cis-regulatory DNA sequence that, when operably ligated to a gene coding sequence, drives the transcription of a gene. A promoter may include a transcription factor binding site. In some embodiments, a promoter functions in conjunction with one or more enhancers located distal to the gene.

[0163] As used herein, “positive target cell-specific regulatory element” (or positive TCSRE) refers to a nucleic acid sequence that increases the level of an exogenous activator in target cells compared to non-target cells, wherein the nucleic acid encoding the exogenous activator is operably ligated to the positive TCSRE. In some embodiments, the positive TCSRE is a functional nucleic acid sequence, for example, the positive TCSRE may include a promoter or enhancer. In some embodiments, the positive TCSRE encodes a functional RNA sequence, for example, the positive TCSRE may encode a splice site that promotes correct splicing of RNA in target cells. In some embodiments, the positive TCSRE encodes a functional protein sequence, or the positive TCSRE may 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 down-regulator or inhibitor of an exogenous activator.

[0164] As used herein, “negative target cell-specific regulatory element” (or negative TCSRE) refers to a nucleic acid sequence that reduces the level of an exogenous activator in non-target cells compared to target cells, where the nucleic acid encoding the exogenous activator is operably linked to the negative TCSRE. In some embodiments, the negative TCSRE is a functional nucleic acid sequence, e.g., a miRNA recognition site that causes degradation or inhibition of retroviral nucleic acids in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, e.g., the nucleic acid encodes a miRNA sequence present in mRNA encoding an exogenous proteoactive agent, resulting in the degradation or inhibition of the mRNA in non-target cells. In some embodiments, the negative TCSRE increases the level or activity of a down-regulator or inhibitor of an exogenous activator.

[0165] As used herein, “Non-Target Cell Specific Regulatory Elements” (or NTCSRE) refers to a nucleic acid sequence that reduces the level of an exogenous activator in non-target cells compared to target cells, where the nucleic acid encoding the exogenous activator is operably linked to the NTCSRE. In some embodiments, the NTCSRE is a functional nucleic acid sequence, for example, a miRNA recognition site that causes degradation or inhibition of retroviral nucleic acids in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, for example, the nucleic acid encodes a miRNA sequence present in mRNA encoding an exogenous proteoactive agent, resulting in the degradation or inhibition of the mRNA in non-target cells. In some embodiments, the NTCSRE increases the level or activity of a down-regulator or inhibitor of an exogenous activator. The terms “negative TCSRE” and “NTCSRE” are used herein to mean the same thing.

[0166] As used herein, “non-T cell-specific regulatory element” refers to a non-target cell-specific regulatory element (NTCSRE), where the target cell is a T cell. Thus, a non-T cell-specific regulatory element refers to a nucleic acid sequence that reduces the level of exogenous activators in non-T cells compared to T cells, where the nucleic acid encoding the exogenous activator is operably linked to the non-T cell-specific regulatory element.

[0167] As used herein, “retargeted fusogen” refers to fusogen that includes a targeting moiety having sequences not part of the naturally occurring form of fusogen. In embodiments, the fusogen includes a different targeting moiety compared to the targeting moiety of the naturally occurring form of fusogen. In embodiments, the naturally occurring form of fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting moiety not present in the naturally occurring form of fusogen. In embodiments, the fusogen is modified to include a targeting moiety. In embodiments, the fusogen includes, for example, one or more sequence changes outside the targeting moiety in a transmembrane domain, a fusogenically active domain, or a cytoplasmic domain compared to the naturally occurring form of fusogen.

[0168] As used herein, “retroviral nucleic acid” refers to a nucleic acid that, alone or in combination with a helper cell, helper virus, or helper plasmid, comprises at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector. In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous activator, a positive target cell-specific regulatory element, a non-target cell-specific regulatory element, or a negative TCSRE. In some embodiments, the retroviral nucleic acid comprises one or more of the following: 5'LTR (e.g., to promote integration), U3 (e.g., to activate viral genomic RNA transcription), R (e.g., a Tat-binding region), U5, 3'LTR (e.g., to promote integration), a packaging site (e.g., psi(Ψ)), and RRE (e.g., to bind to Rev and promote nuclear export). The retroviral nucleic acid may comprise RNA (e.g., if it is part of a virion) or DNA (e.g., if it is introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, retroviral nucleic acids are packaged using helper cells, helper viruses, or helper plasmids containing one or more (e.g., all) of gag, pol, and env.

[0169] As used herein, “target cells” refers to the type of cells to which it is desired that fusosomes (e.g., lentiviral vectors) deliver exogenous activators. In embodiments, target cells are cells of a specific tissue type or class, e.g., T cells, e.g., Treg cells. In some embodiments, target cells are disease cells, e.g., cancer cells. In some embodiments, fusogens, e.g., retargeted fusogens (alone, or in combination with positive TCSRE, NTCSRE, negative TCSRE, or any combination thereof) lead to preferential delivery of exogenous activators to target cells compared to non-target cells.

[0170] As used herein, “non-target cells” refers to a type of cell to which it is undesirable for a lentiviral vector to deliver an exogenous activator. In some embodiments, non-target cells are cells of a particular tissue type or class. In some embodiments, non-target cells are non-disease cells, e.g., non-cancerous cells. In some embodiments, fusogens, e.g., retargeted fusogens (alone, or in combination with positive TCSRE, NTCSRE, negative TCSRE, or any combination thereof), lead to lower delivery of the exogenous activator to non-target cells compared to target cells.

[0171] As used herein, the terms “to treat / administer,” “to treat / administer,” or “treatment / administer” mean improving a disease or disorder, for example, at least one of its underlying causes or clinical symptoms, for example, delaying, preventing, or reducing the onset of the disease or disorder.

[0172] As used herein, “cell biological material” refers to a portion of a cell, including its lumen and cell membrane, or a cell having partial or complete nuclear inactivation. In some embodiments, the cell biological material comprises one or more cytoskeletal components, organelles, and ribosomes. In embodiments, the cell biological material is an enucleated cell, microvesicle, or cellular ghost.

[0173] II. Fusosomes, e.g., cell-derived fusosomes Fusosomes can take various forms. For example, in some embodiments, the fusosomes described herein are derived from source cells. Fusosomes may be, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, apoptotic bodies (derived from apoptotic cells), microparticles (e.g., can be derived from platelets), ectosomes (e.g., can be derived from neutrophils and monocytes in serum), prostatosomes (obtained from prostate cancer cells), cardiosomes (obtained from cardiac cells), or any combination thereof, or may include them. In some embodiments, fusosomes are spontaneously released from source cells, and in some embodiments, source cells are treated to enhance fusosome formation. In some embodiments, fusosomes have a diameter of about 10 to 10,000 nm, for example, a diameter of about 30 to 100 nm. In some embodiments, fusosomes contain one or more synthetic lipids.

[0174] In some embodiments, the fusosome is or contains a virus, such as a retrovirus or a lentivirus. For example, in some embodiments, the amphiphilic lipid bilayer of the fusosome is or contains a viral envelope. The viral envelope may contain a fusogen, such as a fusogen endogenous to the virus or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome contains viral nucleic acid, such as a retroviral nucleic acid or a lentiviral nucleic acid. The viral nucleic acid may be a viral genome. In some embodiments, the fusosome further contains, for example, one or more non-structural viral proteins in its cavity or lumen.

[0175] Fusosomes may possess various properties that facilitate the delivery of payloads to target cells. For example, in some embodiments, both the fusosome and the source cell contain sufficient nucleic acids (which may be more) to form particles that can fuse with the target cell. In embodiments, these nucleic acids (which may be more) encode proteins having one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0176] Fusosomes may also contain various structures that facilitate the delivery of payloads to target cells. For example, in some embodiments, both the fusosome and the source cell contain enough nucleic acids (which may be more) to form particles that can fuse with the target cell. In embodiments, these nucleic acids (which may be more) encode proteins having one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0177] Fusosomes may also include various structures that facilitate the delivery of payloads to target cells. For example, in some embodiments, a fusosome (e.g., a virus, e.g., a retrovirus, e.g., a lentivirus) comprises 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 comprises rev. In some embodiments, one or more of the aforementioned proteins are encoded in a retroviral genome, and in some embodiments, one or more of the aforementioned proteins are provided trans by, for example, a helper cell, a helper virus, or a 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: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine tube (cPPT) promoter operably linked to the payload gene, payload gene (optionally containing an intron before the open reading frame), poly-A tail sequence, WPRE, and 3'LTR (e.g., lacking U5 and 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, one or more miRNA recognition sites are located downstream of the poly-A tail sequence, for example, between the poly-A tail sequence and the WPRE.

[0178] In some embodiments, the fusosomes provided herein are administered to a subject, for example, a mammal, for example, a human. In such embodiments, the subject is at risk of, symptoms of, or can be diagnosed with, or can be identified as, a particular disease or condition (for example, a disease or condition described herein). In one embodiment, the subject has a disease or disorder such as those listed in Table 5. In some embodiments, the fusosomes include a nucleic acid sequence encoding an exogenous active agent for treating a disease or condition, such as for treating the disease or disorder.

[0179] A. Fusosomes generated from a virus. For example, in some embodiments, a fusosome (e.g., a virus, e.g., a retrovirus, e.g., a lentivirus) comprises 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 comprises rev. In some embodiments, one or more of the aforementioned proteins are encoded in a retroviral genome, and in some embodiments, one or more of the aforementioned proteins are provided trans by, for example, a helper cell, a helper virus, or a 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: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), central polypurine tube (cPPT) promoter operably linked to the payload gene, payload gene (optionally containing an intron before the open reading frame), poly-A tail sequence, WPRE, and 3'LTR (e.g., lacking U5 and 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, one or more miRNA recognition sites are located downstream of the poly-A tail sequence, for example, between the poly-A tail sequence and the WPRE.

[0180] i) Lentiviral components and helper cells In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5' promoter (e.g., for controlling the expression of the entire packaged RNA), a 5'LTR (e.g., U5 including R (a polyadenylated tail signal) and / or a primer activation signal), a primer binding site, a psi packaging signal, an RRE element for nuclear export, a promoter immediately upstream of the transgene for controlling the expression of the transgene, the transgene (or other exogenous activator element), a polyprint lacte, and a 3'LTR (e.g., including mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more cPPT, WPRE, and / or insulator sequences.

[0181] Retroviruses typically replicate by reverse transcription of their genomic RNA into a linear double-stranded DNA copy, followed by covalent integration of the genomic DNA into the host genome. Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney's mouse leukemia virus (M-MuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary cancer virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), Supumavirus, Friend's mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV), as well as lentiviruses.

[0182] 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. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spuma retrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0183] Examples of lentiviruses, though not limited to these, include: HIV (human immunodeficiency virus; including HIV types 1 and HIV 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); and equine infectious anemia virus (EIAV). Anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, an HIV-based vector skeleton (i.e., an HIV cis-acting sequence element) is used.

[0184] In some embodiments, the vectors described herein are nucleic acid molecules capable of importing or transporting another nucleic acid molecule. The imported nucleic acid is generally ligated, for example, inserted, into the vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication within a cell, or sequences sufficient to enable integration into host cell DNA. Useful vectors include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include replication-defective retroviruses and lentiviruses.

[0185] Viral vectors may include, for example, nucleic acid molecules (e.g., transfer plasmids) that typically contain virus-derived nucleic acid elements that facilitate the transfer of nucleic acid molecules or integration into viral particles that mediate the transfer of a cell's genome or nucleic acid. Viral particles typically contain various viral components in addition to nucleic acids (which may be multiple), and sometimes host cell components. Viral vectors may include, for example, viruses or viral particles that can transfer nucleic acids into cells or transfer them to transferred nucleic acids (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 include viral vectors or plasmids that primarily contain structural and functional genetic elements, or parts thereof, primarily derived from retroviruses. Lentiviral vectors may include viral vectors or plasmids that primarily contain structural and functional genetic elements, or parts thereof, including LTRs, primarily derived from lentiviruses.

[0186] In embodiments, a lentiviral vector (e.g., a lentiviral expression vector) may 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, and heterologous nucleic acids, it should be understood that the sequences of these elements may exist in the form of RNA in lentiviral particles and in the form of DNA in DNA plasmids.

[0187] 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. This can lead to defects in viral vector replication. In some embodiments, the vector can transduce a target non-dividing host cell and / or integrate its genome into the host genome.

[0188] The structure of a wild-type retroviral genome often includes a 5' terminal repeat (LTR) and a 3' LTR, between or within it, the gag, pol, and env genes encoding packaging signals that enable genome packaging, primer binding sites, integration sites for integration into the host cell genome, and packaging components that facilitate the assembly of viral particles. More complex retroviruses have additional functions, such as the rev and RRE sequences in HIV, which allow for the efficient export of the integrated proviral RNA transcript from the nucleus to the cytoplasm of the infected target cell. In proviruses, regions called terminal repeats (LTRs) are adjacent to both ends of the viral gene. LTRs are involved in proviral integration and transcription. LTRs also function as enhancer-promoter sequences, regulating the expression of viral genes. Capsid formation of retroviral RNA occurs via the psi sequence located at the 5' end of the viral genome.

[0189] The LTR itself is usually a similar (e.g., identical) sequence and can be divided into three elements called U3, R, and U5. U3 originates from a sequence specific to the 3' end of the RNA. R originates from a sequence repeated at both ends of the RNA, and U5 originates from a sequence specific to the 5' end of the RNA. The sizes of the three elements vary considerably among retroviruses.

[0190] In viral genomes, the transcription start site is typically located at the U3-R boundary of one LTR, and the poly(a) addition (termination) site is located at the R-U5 boundary of the other LTR. U3 contains most of the proviral transcriptional regulatory elements, including promoters and multiple enhancer sequences that respond to cellular, and sometimes viral, transcription-activating proteins. Some retroviruses contain one or more of the following genes that encode proteins involved in regulating gene expression: tot, rev, tax, and rex.

[0191] Regarding the structural genes gag, pol, and env themselves, gag encodes the internal structural proteins of the virus. The gag protein is processed by proteolysis into mature proteins MA (matrix), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), including DNA polymerase, associated RNase H, and integrase (IN), which mediate genome replication. The env gene encodes virion surface (SU) glycoprotein and transmembrane (TM) proteins that form a complex that specifically interacts with cell receptor proteins. This interaction facilitates infection, for example, by fusion of the viral membrane and the cell membrane.

[0192] In replication-deficient retroviral vector genomes (gag), pol and env may be absent or non-functional. The R regions at both ends of the RNA are typically repetitive sequences. U5 and U3 represent unique sequences located at the 5' and 3' ends of the RNA genome, respectively.

[0193] Retroviruses may contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev, and nef (in HIV). EIAV has (among other things) an additional gene, S2. The proteins encoded by these additional genes perform a variety of functions, some of which may overlap with functions provided by cellular proteins. For example, in EIAV, tat functions as a transcriptional activator of the viral LTR (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). Tat binds to a stable stem-loop RNA secondary structure called the TAR. Rev regulates and modulates viral gene expression via the Rev response element (RRE) (Martarano et al. 1994 J.Virol. 68:3102-11). The mechanisms of action of these two proteins are thought to be largely the same as those of primate viruses. In addition, the EIAV protein Ttm has been identified, which is encoded by the first exon of tat, spliced ​​into the env coding sequence at the initiation of the transmembrane protein.

[0194] In addition to proteases, reverse transcriptases, and integrases, non-primate lentiviruses contain a fourth pol gene product that encodes dUTPase. This may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

[0195] In embodiments, a recombinant lentiviral vector (RLV) is a vector containing sufficient retroviral genetic information to enable the packaging of an RNA genome into viral particles capable of infecting target cells in the presence of a packaging component. Infection of target cells may involve reverse transcription and integration into the target cell genome. RLVs typically possess nonviral coding sequences that are delivered to target cells by the vector. In embodiments, RLVs are unable to independently replicate to produce infectious retroviral particles within target cells. Typically, RLVs lack functional gag-pol and / or env genes and / or other genes involved in replication. The vector may be configured as a split-intron vector, for example, as described in International Publication 99 / 15683 of a PCT patent application, which is incorporated entirely herein by reference.

[0196] In some embodiments, the lentiviral vector comprises a minimal viral genome, and, for example, as described in International Publication No. 98 / 17815, the entirety of which is incorporated herein by reference, the viral vector is manipulated to remove non-essential elements and retain essential elements in order to provide the necessary functions for infection, transduction, and delivery of the desired nucleotide sequence to a target host cell.

[0197] The smallest lentiviral genome may, for example, contain (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, plasmid vectors used to produce lentiviral genomes in source cells may also contain transcriptional regulatory sequences operably linked to the lentiviral genome to direct the transcription of the genome in the source cells. These regulatory sequences may contain the transcribed retroviral sequence, e.g., a native sequence related to the 5'U3 region, or a different viral promoter, e.g., a heterologous promoter such as the CMV promoter. Some lentiviral genomes contain additional sequences to facilitate efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively or in combination, codon optimization can be used; for example, a gene encoding an exogenous active agent may be codon-optimized, as described, for example, in International Publication No. 01 / 79518, which is incorporated in its entirety herein by reference. 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. Known as CTE, it contains RRE-type sequences in the genome that are thought to interact with factors in infected cells. These cellular factors can be considered rev analogs. Therefore, CTE can be used as a substitute for the rev / RRE system. In addition, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Both Rev and Rex have similar effects to IRE-BP.

[0198] In some embodiments, a retroviral nucleic acid (e.g., a lentiviral nucleic acid, e.g., a primate or non-primate lentiviral nucleic acid) comprises a deleted gag gene where (1) the deletion in gag removes one or more nucleotides downstream of about 350 or 354 nucleotides of the gag coding sequence; (2) has one or more accessory genes not present in the retroviral nucleic acid; (3) lacks the tat gene but includes a leader sequence between the end of the 5′ LTR and the ATG of gag; (4) is a combination of (1), (2), and (3). In one embodiment, a lentiviral vector comprises all of features (1), (2), and (3). This strategy is described in more detail in International Publication No. WO 99 / 32646, which is hereby incorporated by reference in its entirety.

[0199] In some embodiments, a primate lentiviral minimal system does not require any of the HIV / SIV accessory genes vif, vpr, vpx, vpu, tat, rev, and nef for either vector production or transduction of dividing and non-dividing cells. In some embodiments, the EIAV minimal vector system does not require S2 for either vector production or transduction of dividing and non-dividing cells.

[0200] Deletion of the additional genes may allow for the generation of vectors without genes associated with diseases of retroviral (e.g., HIV) infections. In particular, tat is associated with disease. Second, deletion of the additional genes may allow the vector to package more heterologous DNA. Third, omitting genes of unknown function such as S2 can reduce the risk of unwanted effects. Examples of minimal lentiviral vectors are disclosed in International Publication No. WO 99 / 32646 and International Publication No. WO 98 / 17815.

[0201] In some embodiments, the retroviral nucleic acid is lacking at least tat and S2 (in the case of the EIAV vector system), and perhaps also vif, vpr, vpx, vpu, and nef. In some embodiments, the retroviral nucleic acid is also lacking rev, RRE, or both.

[0202] In some embodiments, the retroviral nucleic acid includes vpx. The Vpx polypeptide binds to the SAMHD1 restriction factor and induces its degradation, degrading free dNTPs in the cytoplasm. Thus, when Vpx degrades SAMHD1 and reverse transcription activity increases, the concentration of free dNTPs in the cytoplasm increases, promoting reverse transcription of the retroviral genome and integration into the target cell genome.

[0203] If the cells are different, the usage of specific codons is also different. This codon bias corresponds to the bias in the relative abundance of specific tRNAs in the cell type. Expression can be increased by changing the codons within the sequence so that they are adjusted to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by intentionally selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, additional translational control is available. Additional explanations of codon optimization can be found, for example, in WO 99 / 41397, which is hereby incorporated by reference in its entirety.

[0204] Many viruses, including HIV and other lentiviruses, use a number of rare codons, and by changing them to correspond to commonly used mammalian codons, the expression of packaging components in mammalian producer cells can be increased.

[0205] Codon optimization offers many other advantages. Thanks to the alteration of their sequences, the nucleotide sequences encoding the packaging components may have RNA instability sequences (INS) that have been reduced or eliminated from them. At the same time, the amino acid sequence coding sequences of the packaging components are maintained so that the viral component encoded by the sequence remains the same, or at least similar enough that the function of the packaging component is not impaired. In some embodiments, codon optimization also overcomes Rev / RRE requirements for export and makes the optimized sequence Rev-independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within the vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titer and / or improved safety.

[0206] In some embodiments, only codons related to INS are codon-optimized. In other embodiments, the sequence as a whole is codon-optimized, excluding sequences containing gag-pol frameshift regions.

[0207] The gag-pol gene contains two overlapping reading frames encoding the gag-pol protein. The expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of "slippage" of the ribosome during translation. This slippage is thought to be caused at least partially by a secondary structure of RNA that stalls the ribosome. Such a secondary structure is located downstream of the frameshift site of the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 (nucleotide 1 is A in gag ATG) downstream of the beginning of gag to the end of gag (nt 1503). As a result, 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, retaining this fragment would allow for more efficient expression of the gag-pol protein. In the case of EIAV, the beginning of the overlap is at nt 1262 (nucleotide 1 is A in gag ATG). The end of the overlap is at nt 1461. To ensure that the overlap of frameshift regions and gag-pols is reliably maintained, the wild-type sequence can be retained from nt 1156 to 1465.

[0208] For example, it is possible to derive optimal codon usage to address convenient restriction sites and introduce conservative amino acid changes into the gag-pol protein.

[0209] In some embodiments, codon optimization is based on codons with low codon usage frequency in mammalian systems. The third and sometimes the second and third bases may be modified.

[0210] It will be understood that, due to the degrading properties of the genetic code, a large number of gag-pol sequences can be achieved by those skilled in the art. Furthermore, many retroviral variants have been described that can be used as starting points for generating codon-optimized gag-pol sequences. Lentiviral genomes can be quite variable. For example, there are still many functional pseudospecies of HIV-I. This is also true for EIAV. These variants can be used to enhance 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.

[0211] The codon-optimized gag-pol sequence strategy can be used in relation to retroviruses such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I, and HIV-2. Furthermore, this method can be used to increase gene expression from HTLV-I, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERV), MLV, and other retroviruses.

[0212] As described above, the packaging components of a retroviral vector may include expression products of the gag, pol, and env genes. Furthermore, the packaging can utilize short sequences of four stem-loops followed by partial sequences from gag and env as a packaging signal. Thus, the retroviral vector genome can include deleted gag sequences (in addition to the complete gag sequence on the packaging construct). In embodiments, the retroviral vector includes a packaging signal containing 255-360 nucleotides of gag in the vector, which still retains the env sequence, or about 40 nucleotides of gag in a specific combination of gag and env deletions that are splice donor mutations. In some embodiments, the retroviral vector includes a gag sequence containing one or more deletions, for example, the gag sequence containing about 360 nucleotides derived from the N-terminus.

[0213] A retroviral vector, helper cell, helper virus, or helper plasmid may include retroviral structures 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 two or more retroviruses, such as 2, 3, 4, or more retroviruses.

[0214] The gag and pol coding sequences are generally organized as Gag-Pol precursors in natural lentiviruses. The gag sequence encodes a 55kD Gag precursor protein, also known as p55. p55 is cleaved by the virus-encoded protease 4 (a product of the pol gene) during the process of maturing into four small proteins called MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved from Gag by the virus-encoded proteases and further digested to separate the activities of protease (p10), RT (p50), RNase H (p15), and integrase (p31).

[0215] Native Gag-Pol sequences can be used in helper vectors (such as helper plasmids or helper viruses) or modified. These modifications include chimeric Gag-Pol, where the Gag and Pol sequences are obtained from different viruses (e.g., different species, subspecies, strains, clades, etc.) and / or modified to improve transcription and / or translation and / or reduce recombination.

[0216] In various examples, the retroviral nucleic acid contains (i) a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of the gag protein, which includes a mutant INS1 inhibitory sequence that reduces the restriction of RNA export compared to wild-type INS1; (ii) two nucleotide insertions resulting in frameshift and premature termination; and / or (iii) no INS2, INS3, and INS4 inhibitory sequences of the gag.

[0217] In some embodiments, the vectors described herein are hybrid vectors comprising both retroviral (e.g., lentiviral) sequences and non-lentiviral virus sequences. In some embodiments, the hybrid vectors include retroviral, e.g., lentiviral sequences for reverse transcription, replication, integration, and / or packaging.

[0218] According to certain embodiments, most or all of the viral vector backbone sequence 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, or that combined and numerous substitutions and modifications in particular lentiviral sequences can be adapted without impairing the ability of the transfer vector to perform the functions described herein. Various lentiviral vectors are described in Naldini et al., (1996a, 1996b, and 1998), Zufferey et al., (1997), Dull et al., 1998, U.S. Patent No. 6,013,516, and U.S. Patent No. 5,994,136, many of which can be adapted to produce retroviral nucleic acids.

[0219] Proviruses typically contain long terminal repeats (LTRs) at both ends. LTRs usually contain a domain located at the end of the retroviral nucleic acid, which, in the context of its natural sequence, is a direct repeat and includes U3, R, and U5 regions. LTRs generally promote the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs can contain numerous regulatory signals, including transcriptional regulatory elements, polyadenylation signals, and sequences for replication and integration of the viral genome. Viral LTRs are typically divided into three regions called U3, R, and U5. The U3 region usually contains enhancer and promoter elements. The U5 region is typically the sequence between the primer binding site and the R region and can contain polyadenylation sequences. The R (repeat) region can be adjacent to the U3 and U5 regions. LTRs typically consist of the U3, R, and U5 regions and can 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 efficient packaging of viral RNA into particles (Psi site).

[0220] Packaging signals can include sequences located within the retroviral genome that mediate the insertion of 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 minimal packaging signals (psi[Ψ] sequences) for capsid formation of the viral genome.

[0221] In various embodiments, the retroviral nucleic acid comprises modified 5'LTR and / or 3'LTR. Either or both LTRs may comprise one or more modifications, including but not limited to one or more deletions, insertions, or substitutions. Modifications to the 3'LTR are often made to improve the safety of a lentiviral or retroviral system by making the virus replication-defective (e.g., a virus that cannot replicate completely and effectively so as not to produce infectious virions (e.g., a descendant of a replication-defective lentivirus)).

[0222] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-deficient vector, e.g., a retroviral or lentiviral vector, where the right (3')LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because, without the U3 enhancer promoter, viral replication is inhibited, as the right (3')LTR U3 region can be used as a template for the left (5')LTR U3 region during viral replication. In embodiments, the 3'LTR is modified such that the U5 region is removed, altered, or substituted, e.g., with an exogenous poly(A) sequence. The 3'LTR, 5'LTR, or both 3' and 5' LTRs may be modified LTRs.

[0223] In some embodiments, the U3 region of the 5’ LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles. Examples of heterologous promoters that can be used include, for example, 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 additional advantages in controlling how 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 when an inducer is present. Inducers include, but are not limited to, one or more compounds in which the host cells are cultured, or physiological conditions such as temperature or pH.

[0224] In some embodiments, the viral vector includes, for example, a TAR (trans-activation response) element located in the R region of a lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral trans-activator (tat) gene element to enhance viral replication. However, this element is not required, for example, in embodiments where the U3 region of the 5’ LTR has been replaced by a heterologous promoter.

[0225] The R region, for example, the region within a retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends just prior to the start of the polyA tract, can be adjacent to the U3 and U5 regions. The R region plays a role during reverse transcription in the transfer of nascent DNA from one end of the genome to the other.

[0226] Retroviral nucleic acids may also include FLAP elements, for example, nucleic acids whose sequences include the central polyprint lactate and central termination sequences (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173, which are incorporated herein by reference in their entirety. During HIV-1 reverse transcription, central start of the positive-strand DNA at the central polyprint lactate (cPPT) and central termination at the central termination sequence (CTS) may lead to the formation of a triple-stranded DNA structure, i.e., a single HIV-1 central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone includes one or more FLAP elements upstream or downstream of the gene encoding the exogenous active agent. For example, in some embodiments, a transfer plasmid includes FLAP elements, for example, FLAP elements derived from or isolated from HIV-1.

[0227] In embodiments, the retroviral or lentiviral nucleic acid includes one or more export elements, such as cis-acting post-transcriptional regulatory elements that regulate the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements, but not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the hepatitis B virus post-transcriptional regulatory element (HPRE), which are incorporated herein by reference in their entirety. Generally, RNA export elements are located within the 3' UTR of a gene and can be inserted as one or more copies.

[0228] In some embodiments, the expression of heterologous sequences in viral vectors is increased by incorporating one or more post-transcriptional regulatory elements, polyadenylation sites, and transcription termination signals into the vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); and the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864) (Liu et al., 1995, Genes Dev., 9:1766), each of which is incorporated herein by reference in whole. In some embodiments, the retroviral nucleic acids described herein include post-transcriptional regulatory elements such as WPRE or HPRE.

[0229] In some embodiments, the retroviral nucleic acids described herein lack or do not contain post-transcriptional regulatory elements such as WPRE or HPRE.

[0230] Elements that direct the termination and polyadenylation of heterologous nucleic acid transcripts may be included, for example, to increase the expression of exogenous activators. The transcription termination signal may be found downstream of the polyadenylation signal. In some embodiments, the vector includes a polyadenylation sequence at the 3' end of a polynucleotide encoding an exogenous activator. The polyA site may include a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. The polyadenylation sequence enhances mRNA stability by adding a polyA tail to the 3' end of the coding sequence, contributing to improved translation efficiency. Examples of polyA signals that can be used with retroviral nucleic acids include AATAAA, ATTAAA, AGTAAA, the bovine growth hormone polyA sequence (BGHpA), the rabbit β-globin polyA sequence (rβgpA), or another suitable heterologous or endogenous polyA sequence.

[0231] In some embodiments, the retrovirus or lentiviral vector further comprises one or more insulator sequences, for example, the insulator sequences described herein.

[0232] In various embodiments, the vector comprises a promoter operably ligated to a polynucleotide encoding an exogenous active agent. The vector may have one or more LTRs, where any LTR comprises one or more modifications such as one or more nucleotide substitutions, additions, or deletions. The vector may include one or more accessory elements to enhance transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., Psi(Ψ) packaging signal, RRE), and / or other elements to increase exogenous gene expression (e.g., poly(A) sequence), and may optionally include WPRE or HPRE.

[0233] In some embodiments, the lentiviral nucleic acid includes one or more of the following: 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 that drives the expression of an exogenous activator, a gene encoding an exogenous activator, 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), all of which include, for example, 5' to 3'.

[0234] ii) A vector manipulated to remove the splice site Some lentiviral vectors possess potent splicing and polyadenylation signals that can be integrated into active genes, potentially leading to the formation of abnormal, and in some cases, truncated, transcripts.

[0235] The mechanism of proto-oncogene activation may involve the generation of chimeric transcripts resulting from the interaction between promoter elements or splice sites in the genome of the insertion mutagen and the target cellular transcriptional unit for integration (Gabriel et al. 2009. Nat Med 15:1431-1436; Bokhoven, et al. J (Virol 83:283-29). Chimeric fusion transcripts containing vector sequences and cellular mRNA can be generated by read-through transcription, starting from the vector sequence and proceeding to adjacent cellular genes, or vice versa.

[0236] In some embodiments, the lentiviral nucleic acids described herein include a lentiviral skeleton from which at least two splice sites have been removed, for example, to improve the safety profile of the lentiviral vector. The species and methods for identifying such splice sites are described in International Publication No. 2012156839A2, and are all included by reference.

[0237] iii) Methods for producing retroviruses Large-scale production of viral particles is often useful for achieving desired viral titers. Viral particles can be produced by transfecting a transfer vector into a packaging cell line containing viral structures and / or accessory genes, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes, or other retroviral genes.

[0238] In some embodiments, the packaging vector is an expression vector or viral vector lacking a packaging signal and containing a polynucleotide encoding 1, 2, 3, 4, or more viral structures and / or accessory genes. Typically, the packaging vector is contained within a packaging cell and introduced into the cell via transfection, transduction, or infection. A retrovirus, such as a lentivirus transfer vector, can be introduced into a packaging cell line via transfection, transduction, or infection to generate source cells or cell lines. The packaging vector can be introduced into human cells or cell lines by standard methods, such as calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into cells along with a major selectable marker such as neomycin, hygromycin, puromycin, blastosidine, zeosin, thymidine kinase, DHFR, Gln synthase, or ADA, and then selected and isolated clones in the presence of the appropriate drug. The selectable marker gene can be physically ligated by the packaging vector to a gene encoded, for example, by IRES or autocleaved viral peptides.

[0239] Packaging cell lines include cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replication enzymes (e.g., gag, pol, and env) capable of packaging viral particles. Any suitable cell line, e.g., mammalian cells, e.g., human cells, can be used. Suitable usable cell lines 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 this embodiment, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0240] Source cell lines include cell lines capable of producing recombinant retroviral particles, including a packaging cell line and a transfer vector construct containing a packaging signal. Methods for preparing the viral stock solution are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and NRLandau et al. (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference. Infectious viral particles can be collected from packaging cells, for example, by cell lysis or collection of the supernatant of a cell culture. If necessary, the collected viral particles can be concentrated or purified.

[0241] iv) Packaging of plasmids and cell lines In some embodiments, the source cells contain one or more plasmids encoding viral structural proteins and replication enzymes (e.g., gag, pol, and env) capable of packaging viral particles. In some embodiments, sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, sequences encoding gag, pol, and env precursors are on different plasmids. In some embodiments, sequences encoding gag, pol, and env precursors have the same expression signal, e.g., a promoter. In some embodiments, sequences encoding gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, expression of gag, pol, and env precursors is inducible. In some embodiments, the plasmids encoding viral structural proteins and replication enzymes are transfected simultaneously or at different times. In some embodiments, the plasmids encoding viral structural proteins and replication enzymes are transfected from a packaging vector simultaneously or at different times.

[0242] In some embodiments, the source cell line contains one or more stably incorporated viral structural genes. In some embodiments, the expression of the stably incorporated viral structural genes is inducible.

[0243] In some embodiments, the expression of viral structural genes is regulated at the transcriptional level. In some embodiments, the expression of viral structural genes is regulated at the translational level. In some embodiments, the expression of viral structural genes is regulated at the post-translational level.

[0244] In some embodiments, the expression of viral structural genes is regulated by a tetracycline (Tet)-dependent system, where a Tet-regulated transcriptional repressor (Tet-R) binds to a DNA sequence 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, enabling transcription. Several other suitable transcriptional regulatory promoters, transcription factors, and small molecule inducers are also suitable for regulating the transcription of viral structural genes.

[0245] In some embodiments, third-generation lentiviral components, human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and an envelope bound to an antibiotic resistance cassette, under the control of a Tet regulatory promoter, are separately integrated into the source cell genome. In some embodiments, the source cell has only one copy each of Rev and Gag / Pol, and the envelope protein integrated into the genome.

[0246] In some embodiments, nucleic acids encoding exogenous activators (e.g., retroviral nucleic acids encoding exogenous activators) are also incorporated into the source cell genome. In some embodiments, the nucleic acids encoding exogenous activators are maintained episomalally. In some embodiments, the nucleic acids encoding exogenous activators are transfected into source cells having Rev, Gag / Pol, and envelope proteins stably incorporated into the genome. For example, Milani et al., whose entire work is incorporated herein by reference. See al. EMBO Molecular Medicine, 2017.

[0247] In some embodiments, the retroviral nucleic acids described herein are not subject to reverse transcription. Such nucleic acids may, in embodiments, transiently express exogenous active agents. Retroviruses or VLPs may contain or may not contain inactivated reverse transcriptase proteins. In embodiments, retroviral nucleic acids contain inactivated primer-binding sites (PBS) and / or att sites. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, and S2 or their functional equivalents, are inactivated or absent in the retroviral nucleic acids. In embodiments, one or more accessory genes selected from S2, rev, and tat are inactivated or absent in the retroviral nucleic acids.

[0248] v) Strategies for packaging retroviral nucleic acids Typically, modern retroviral vector systems consist of a viral genome having a cis-acting vector sequence for transcription, reverse transcription, integration, translation, and packaging of viral RNA into viral particles, and (2) a producer cell line expressing a trans-acting retroviral gene sequence (e.g., gag, pol, and env) necessary for the production of viral particles. By completely separating the cis-acting and trans-acting vector sequences, the virus cannot maintain replication for more than two cycles of infection. The generation of live viruses can be avoided by several strategies, for example, by minimizing overlap between the cis-acting and trans-acting sequences to avoid recombination.

[0249] Viral vector particles lacking viral RNA or containing sequences lacking viral RNA may be the result of removing or eliminating viral RNA from the sequence. In one embodiment, this can be achieved by using an endogenous packaging signal binding site on the gag, or the endogenous packaging signal binding site is on the pol. In this embodiment, the delivered RNA will contain a homogeneous packaging signal. In another embodiment, packaging of the delivered RNA can be ensured by using a heterogeneous binding domain located on the delivered RNA (heterogeneous to the gag) and a homogeneous binding site located on the gag or pol. The heterogeneous sequence may be nonviral or viral, in which case it may originate from a different virus. Therapeutic RNA can be delivered using vector particles, in which case functional integrase and / or reverse transcriptase are not required. These vector particles can also be used to deliver the therapeutic gene of interest, in which case pol is usually included.

[0250] In one embodiment, the gag-pol is modified, and the packaging signal is replaced with a corresponding packaging signal. In this embodiment, the particle can package RNA with the new packaging signal. An advantage of this approach is that it can package RNA sequences that lack a viral sequence, such as RNAi.

[0251] Another approach relies on the overexpression of the RNA to be packaged. In one embodiment, the RNA to be packaged is overexpressed in the absence of RNA containing a packaging signal. This may result in a significant level of therapeutic RNA being packaged, an amount sufficient to transduce cells and produce a biological effect.

[0252] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a viral gag protein or retroviral gag and pol proteins, the gag protein or pol protein comprising a heterologous RNA-binding domain that can recognize a corresponding sequence in the RNA sequence to facilitate the packaging of the RNA sequence into a viral vector particle.

[0253] In some embodiments, the heterologous RNA-binding domain includes RNA-binding domains derived from bacteriophage coat protein, Rev protein, U1 micronucleus ribonucleoprotein particle protein, Nova protein, TF111A protein, TIS11 protein, trp RNA-binding attenuation protein (TRAP), or pseudouridine synthase.

[0254] In some embodiments, the methods described herein include detecting or confirming the absence of a replicative retrovirus. This method may include evaluating the RNA level of one or more target genes, such as structural or packaging genes, whose gene product is expressed in specific cells infected with a replicative retrovirus, such as a gamma retrovirus or lentivirus, but is not present in the viral vector used to transduce the cells with heterologous nucleic acid, and is present and / or not expressed, or not expected to be present and / or expressed, in cells that do not contain a replicative retrovirus. A replicative retrovirus may be determined to be present if the RNA level of one or more target genes is higher than a reference value that can be measured, for example, directly or indirectly from a positive control sample containing the target genes. For further disclosure, see International Publication No. 2018023094A1.

[0255] vi) Repression of genes encoding exogenous active substances within source cells Proteins (over)expressed in source cells may indirectly or directly affect the assembly and / or infectivity of vector virions. The uptake of exogenous activators into vector virions may also affect the downstream processing of vector particles.

[0256] In some embodiments, tissue-specific promoters are used to restrict the expression of exogenous activators in source cells. In some embodiments, heterologous translation control systems are used in eukaryotic cell cultures to suppress the translation of exogenous activators in source cells. More specifically, retroviral nucleic acids may include a binding site operably linked to a gene encoding an exogenous activator, where the binding site can interact with an RNA-binding protein so that the translation of the exogenous activator is suppressed or prevented in source cells.

[0257] In some embodiments, the RNA-binding protein is a tryptophan RNA-binding attenuated protein (TRAP), such as a bacterial tryptophan RNA-binding attenuated protein. The use of an RNA-binding protein (e.g., a bacterial trp operon regulator protein, tryptophan RNA-binding attenuation protein, TRAP) and the RNA target to which it binds suppresses or prevents the translation of the transgene in the source cell. This system is called transgene repression in vector production cell system or the TRIP system.

[0258] In this embodiment, by positioning the binding site of an RNA-binding protein (e.g., a TRAP binding sequence, tbs) upstream of the NOI translation start codon, specific repression of mRNA translation from the internal expression cassette becomes possible without adversely affecting the production or stability of the vector RNA. The number of nucleotides between the tbs of the gene encoding the exogenous activator and the translation start codon can vary from 0 to 12 nucleotides. The tbs can be positioned downstream of the internal ribosome entry site (IRES) to repress the translation of the gene encoding the exogenous activator of multicistronic mRNA.

[0259] vii) Kill switch system and amplification In some embodiments, polynucleotides or cells possessing genes encoding exogenous activators utilize suicide genes, such as inducible suicide genes, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain embodiments, the suicide genes are not immunogenic to the host cells harboring the exogenous activator. Examples of suicide genes include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 may be activated using certain dimerizing chemical inducers (CIDs).

[0260] In certain embodiments, the vector includes a gene segment that makes 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 the organism's in vivo state. Negatively selectable phenotypes may result from the insertion of a gene that confers sensitivity to an administered active 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 that confers ganciclovir sensitivity (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)).

[0261] In some embodiments, transduced cells, such as immune effector cells like T cells, further contain polynucleotides comprising positive markers that enable the selection of cells with a negatively selectable phenotype in vitro. The positive selection marker may be a gene that, when introduced into target cells, expresses a dominant phenotype that enables positive selection of cells possessing the gene. Examples of this type of gene include, among others, the hygromycin-B phosphotransferase gene (hph) that confers resistance to hygromycin B, the Tn5-derived aminoglycoside phosphotransferase gene (neo or aph) encoding resistance to the antibiotic G418, the dihydrofolate reductase (DHFR), the adenosine deaminase gene (ADA), and multidrug resistance (MDR) genes.

[0262] In some embodiments, positive selectable markers and negative selectable elements are fused such that the loss of the negative selectable element inevitably results in the loss of the positive selectable marker. For example, positive and negative selectable markers can be fused in such a way that the loss of one results in the loss of the other. An example of a fusion polynucleotide that yields a polypeptide as an expression product that gives both the desired positive and negative selective functions is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that gives hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See Lupton SD, et al, Mol. and Cell. Biology 1 1:3374-3378, 1991. Furthermore, in embodiments, the polynucleotide encoding the chimeric receptor is present in a retroviral vector, such as the HyTK retroviral vector described by Lupton SD, et al, (1991), which includes a fusion gene, particularly a fusion gene conferring hygromycin B resistance to positive selection in vitro and ganciclovir sensitivity to negative selection in vivo. See also PCT publications U591 / 08442 and PCT / U594 / 05601, which describe the use of a bifunctional selection fusion gene derived from the fusion of a dominant positive selection marker and a negative selection marker.

[0263] Appropriate positive selection markers are derived from genes selected from the group consisting of hph, nco, and gpt, and appropriate negative selection markers may be derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. Other appropriate 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 gene, or from a selectable marker.

[0264] viii) Strategies for regulating lentiviral integration Retroviral and lentiviral nucleic acids are disclosed in which key proteins / sequences are missing or disabled to prevent the integration of retroviral or lentiviral genomes into target cell genomes. For example, highly conserved DDE motifs of retroviral integrases (Engelman and Craigie (1992) J. Virol. 66:6361-6369; Johnson et al. (1986) Proc. Natl. Acad. Sci. USA 83:7648-7652; Khan et al. al. (1991) Nucleic Acids Res. 19:851-860) enables the production of integrated deficiency retroviral nucleic acids.

[0265] For example, in some embodiments, the retroviral nucleic acids herein include a lentiviral integrase containing a mutation that prevents the integrase from catalyzing the integration of the viral genome into the cellular genome. In some embodiments, the mutation is a type I mutation that directly affects integration, or a type II mutation that induces a pleiotropic defect that affects virion morphogenesis and / or reverse transcription. A non-limiting example of a type I mutation is a mutation that affects any of the three residues involved in the catalytic core domain of the integrase:DX 39-58 DX 35 E (residues D64, D116, and E152 of HIV-1 integrase). In certain embodiments, the mutation that prevents the integrase from catalyzing the integration of the viral genome into the cellular genome is the substitution of one or more amino acid residues in the DDE motif of the catalytic core domain of the integrase, preferably the substitution of the first aspartic acid residue of the DEE motif with an asparagine residue. In some embodiments, the retroviral vector does not contain the integrase protein.

[0266] In some embodiments, the retrovirus is integrated into an active transcription unit. In some embodiments, the retrovirus is not integrated near the transcription start site, the 5' end of the gene, or a DNAse1 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 is integrated into an intron.

[0267] In some embodiments, retroviral nucleic acids are integrated into the genome of target cells at a specific copy number. The average copy number can be determined from a single cell, a population of cells, or individual cell colonies. Exemplary methods for determining the copy number include polymerase chain reaction (PCR) and flow cytometry.

[0268] In some embodiments, DNA encoding exogenous active agents is integrated into the genome. In some embodiments, DNA encoding exogenous active agents is maintained episomalally. In some embodiments, the ratios of DNA integrated into episomal DNA encoding exogenous active agents are at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, and 100.

[0269] In some embodiments, the DNA encoding the exogenous active agent is linear. In some embodiments, the DNA encoding the exogenous active agent is circular. In some embodiments, the ratio of linear copies to circular copies of the DNA encoding the exogenous active agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, or 100.

[0270] In some embodiments, the DNA encoding the exogenous active substance is circular and contains one LTR. In some embodiments, the DNA encoding the exogenous active substance is circular and contains two LTRs. In some embodiments, the ratio of the circular DNA containing one LTR encoding the exogenous active substance to the circular DNA containing two LTRs encoding the exogenous active substance is at least 0.1, 0.5, 1.0, 2, 5, 10, 20, 50, and 100.

[0271] ix) Maintenance of episomal viruses In retroviruses with poor integration, circular cDNA byproducts of reverse transcription (e.g., 1-LTR and 2-LTR) may accumulate in the cell nucleus without being integrated into the host genome (Yanez-Munoz RJ et al., Nat. Med. 2006, 12:348-353). Then, like other exogenous DNAs, these intermediates may be transcribed at the same frequency (e.g., 10 3 ~10 5 It can be integrated into cellular DNA (in cells).

[0272] In some embodiments, episomal retroviral nucleic acids do not replicate. Episomal viral DNA can be modified to be maintained in replicating cells by including matrix-binding regions (S / MARs) for association with eukaryotic replication origins and scaffold / nuclear matrix.

[0273] Accordingly, in some embodiments, the retroviral nucleic acids described herein include eukaryotic origins of replication or variants thereof. Examples of eukaryotic origins of replication in question include the β-globin gene origin described by Aladjem et al (Science, 1995, 270:815-819), consensus sequences derived from autocopulatory sequences associated with alpha-satellite sequences previously isolated from monkey CV-1 cells and human dermal fibroblasts, such as those described by Price et al Journal of Biological Chemistry, 2003, 278(22):19649-59, and the human c-myc promoter region origin described by McWinney and Leffak (McWinney C. and Leffak M., Nucleic Acid Research 1990, 18(5):1233-42). In embodiments, the variant substantially maintains the ability to initiate replication in eukaryotes. The ability of a particular sequence to initiate replication can be determined by any suitable method, for example, by an autonomous replication assay based on bromodeoxyuridine incorporation and density shift (Araujo FD et al., cited above; Frappier L. et al.). al.)

[0274] In some embodiments, retroviral nucleic acids include scaffold / matrix attachment regions (S / MARs) or variants thereof, such as non-consensus-like AT-rich DNA elements of several hundred base pairs in length, which organize nuclear DNA in eukaryotic genomes into chromatin domains by periodic attachment of proteins to the matrix of the cell nucleus. They are typically found in non-coding regions such as adjacent regions, chromatin boundary regions, and introns. An example of an S / MAR region is the human IFN-γ gene (hIFN-γ) described by Bode et al (Bode J. et al., Science, 1992, 255:195-7). large) 1.8kbp S / MAR, the 0.7kbp minimum region of the human IFN-γ gene described by Ramezani (Ramezani A. et al., Blood 2003, 101:4717-24) (hIFN-γ shortThe S / MAR of the human starfish hydrofolate reductase gene (hDHFR), described by Mesner LD et al., Proc Natl Acad Sci USA, 2003, 100:3281-86, is a 0.2 kbp minimal region. In embodiments, a functionally equivalent variant of the S / MAR is a sequence selected based on a set of six rules that have been suggested to contribute to the S / MAR function, either together or individually (Kramer et al (1996) Genomics 33, 305; Singh et al (1997) Nucl. Acids Res 25, 1419). These rules were merged into the MAR-Wiz computer program, which is freely available at genomecluster.secs.oakland.edu / MAR-Wiz. In embodiments, the variant substantially retains the same function of the S / MAR from which it originates, in particular its ability to specifically bind to the nuclear matrix. Those skilled in the art can determine, for example, whether a particular variant can specifically bind to the nuclear matrix by an in vitro or in vivo MAR assay described by Mesner et al. (Mesner LD et al., cited above). In some embodiments, a particular sequence is an S / MAR variant if it exhibits a tendency for DNA strand segregation. This property can be determined using a specific program based on equilibrium statistical mechanics methods. The stress-induced double destabilization (SIDD) analysis method calculates the extent to which a given level of superhelical stress reduces the free energy required to unwind the double helix at each location along the DNA sequence. The results are represented as a SIDD profile, where sites of strong destabilization are indicated as deep minimums, as defined by Bode et al (2005) J.Mol.Biol.358597. The SIDD algorithm and mathematical foundations (Bi and Benham (2004) Bioinformatics 20,1477), as well as the analysis of SIDD profiles, can be performed using internet resources freely available at WebSIDD (www.genomecenter.ucdavis.edu / benham).Therefore, in some embodiments, a polynucleotide is considered a variant of an S / MAR sequence if it exhibits a SIDD profile similar to that of an S / MAR.

[0275] B. Cell-derived fusosomes Fusosome compositions can be generated from cells in culture, such as cultured mammalian cells, such as cultured human cells. The cells may be primordial cells or non-primordial (e.g., differentiated) cells. The cells may be primary cells or cell lines (e.g., mammalian, such as human, or the cell lines described herein). In embodiments, cultured cells may be primordial cells, such as bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPCs), endothelial progenitor cells (EPCs), blasts, intermediate progenitor cells formed in the subventricular zone, neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, progenitor cells, muscle progenitor cells, myoblasts, cardiomyocytes, neural progenitor cells, glial progenitor cells, neural progenitor cells, or hepatocytes.

[0276] In some embodiments, source cells include endothelial cells, fibroblasts, blood cells (e.g., macrophages, neutrophils, granulocytes, leukocytes), stem cells (e.g., mesenchymal stem cells, umbilical cord stem cells, bone marrow stem cells, hematopoietic stem cells, induced pluripotent stem cells, e.g., induced pluripotent stem cells derived from the subject's cells), embryonic stem cells (e.g., embryonic yolk sac, placenta, stem cells from the umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythrocyte-producing tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes), alveolar cells, and neurons (e.g., retinal neurons). These include transcellular cells, progenitor cells (e.g., retinal progenitor cells, myeloblasts, myeloprogenitor cells, thymocytes, meiogenic progenitor cells, megakaryoblasts, premegakaryoblasts, melanin-forming blasts, lymphoblasts, myeloprogenitor cells, normoblasts, or angioblasts), originating cells (e.g., cardiac originating cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic originating cells, endothelial originating cells, blast cells), or immortalized cells (e.g., HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cells).

[0277] Cultured cells may be cells derived from epithelial, connective, muscular, or nerve tissues or cells, or combinations thereof. Fusosomes may be generated from cultured cells derived from any eukaryotic (e.g., mammalian) organ system, such as the cardiovascular system (heart, vascular system); the digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, and anus); the endocrine system (hypothalamus, pituitary gland, pineal gland or pineal gland, thyroid gland, parathyroid gland, adrenal gland); the excretory system (kidney, ureter, bladder); the lymphatic system (lymph, lymph nodes, lymphatic vessels, tonsils, pharyngeal tonsils, thymus, spleen); the cutaneous system (skin, hair, nails); the muscular system (e.g., skeletal muscle); the nervous system (brain, spinal cord, nerves); the reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); the respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); the skeletal system (bone, cartilage), and combinations thereof. In the embodiments, the cells are derived from highly mitotic tissue (e.g., healthy, highly mitotic tissue such as epithelium, embryonic tissue, bone marrow, or intestinal crypts). In the embodiments, the tissue sample is a highly metabolic tissue (e.g., skeletal tissue, nerve tissue, or cardiomyocytes).

[0278] In some embodiments, the cells are derived from young donors, for example, donors aged 25, 20, 18, 16, 12, 10, 8, 5, or 1 year or younger. In some embodiments, the cells are derived from fetal tissue.

[0279] In some embodiments, the cells are derived from a subject and administered to the same subject or a subject having similar genetic characteristics (e.g., MHC compatibility).

[0280] In certain embodiments, cells have telomeres of average size exceeding 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 nucleotides in length (e.g., 4,000–10,000 nucleotides, 6,000–10,000 nucleotides).

[0281] In some embodiments, fusosomes are generated from cell clones identified, sorted, or selected based on a desired phenotype or genotype for use as a source of the fusosome compositions described herein. For example, cell clones are identified, sorted, or selected based on a low mitochondrial mutagenesis, long telomere length, differentiation state, or specific genetic features (e.g., genetic features matching the recipient).

[0282] The fusosome compositions described herein may consist of fusosomes from a single cell or tissue source, or from a combination of sources. For example, a fusosome composition may include fusosomes from a heterogeneous source (e.g., animal, tissue culture of cells of the aforementioned species), allogeneic, autologous, derived from a specific tissue (e.g., liver, skeleton, nerve, adipose tissue) resulting in different protein concentrations and distributions, or derived from cells in different metabolic states (e.g., glycolytic, respiratory). The composition may also include fusosomes in different metabolic states, such as bound or unbound fusosomes, as described elsewhere herein.

[0283] In some embodiments, fusosomes are generated from source cells expressing a fusogen, for example, a fusogen described herein. In some embodiments, the fusogen is located on the membrane of the source cell, for example, a lipid bilayer membrane, for example, a cell surface membrane, or an intracellular membrane (for example, a lysosomal membrane). In some embodiments, fusosomes are generated from source cells having a fusogen located on the cell surface membrane.

[0284] In some embodiments, fusosomes are generated by inducing the budding of exosomes, microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, macroplasma membrane vesicles, apoptotic bodies, mites, pyrenocytes, lysosomes, or other membrane-encapsulated vesicles.

[0285] In some embodiments, fusosomes are produced by inducing cell enucleation. Nucleation can be performed by genetic, chemical (e.g., using actinomycin D, see Bayona-Bafaluy et al., "A chemical enucleation method for transfer of mitochondrial DNA to ρ° cells," Nucleic Acids Res. 2003 Aug 15;31(16):e98), or mechanical methods (e.g., compression or aspiration, see Lee et al.) This can be carried out using assays such as al., "A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods." Anim Biotechnol. 2008;19(2):71-9), or combinations thereof. Enucleation means not only completely removing the nucleus, but also moving the nucleus from its typical location so that the cell contains it but it is non-functional.

[0286] In the embodiment, the production of fusosomes includes producing cell ghosts, giant plasma membrane vesicles, or apoptotic bodies. In the embodiment, the fusosome composition includes one or more cell ghosts, giant plasma membrane vesicles, and apoptotic bodies.

[0287] In some embodiments, fusosomes are produced by inducing cell fragmentation. In some embodiments, cell fragmentation can be carried out using the following methods, which include, but are not limited to, chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation or density centrifugation), freeze-thaw cycles, or sonication), or combinations thereof.

[0288] In some embodiments, fusosomes may be generated from a source cell expressing a fusogen by any one, all, or a combination of the following methods, as described herein. i) Inducing budding of filamentous particles, exosomes, or other membrane-bound vesicles; ii) Inducing nuclear inactivation, e.g., enucleation, by any or a combination of the following methods: a) Genetic methods; b) A chemical method using, for example, actinomycin D; or c) Mechanical methods, e.g., squeezing or suction; or iii) For example, inducing cell fragmentation by one or a combination thereof: a) chemical methods; b) Mechanical methods, such as centrifugation (e.g., ultracentrifugation or density centrifugation); freeze-thaw; or ultrasonic treatment.

[0289] i) Modification of cells before fusosome formation In some embodiments, modifications are made to cells, such as modifying the target, tissue, or cells, before fusosome formation. Such modifications may be effective, for example, in improving fusion, fusogen expression or activity, cargo structure or function, or the structure or function of target cells.

[0290] a) Physical modification In some embodiments, cells are physically modified before generating fusosomes. For example, as described elsewhere herein, fusogens may be ligated to the surface of cells.

[0291] In some embodiments, cells are treated with chemicals before generating fusosomes. For example, cells may be treated with chemical or lipid fusogens, which then interact with the cell surface non-covalently or covalently, or become embedded within the cell surface. In some embodiments, cells are treated with agents to enhance the fusion properties of lipids in the cell membrane.

[0292] In some embodiments, cells are physically modified before generating fusosomes with one or more covalent or non-covalent attachment sites for synthetic or endogenous small molecules or lipids that enhance fusosome targeting to organs, tissues, or cell types.

[0293] In some embodiments, fusosomes include an increase or decrease in the level of endogenous molecules. For example, fusosomes may include endogenous molecules that are naturally present in the source cell but are present in higher or lower levels than fusosomes. In some embodiments, polypeptides are expressed from exogenous nucleic acids in the source cell or fusosomes. In some embodiments, polypeptides are isolated from the source and loaded into or bound to the source cell or fusosomes.

[0294] In some embodiments, cells are treated with chemical agents, such as small molecules, to increase the expression or activity of intracellular endogenous fusogens (e.g., endogenous in some embodiments to source cells and endogenous in some embodiments to target cells) before generating fusosomes. In some embodiments, the small molecule may increase the expression or activity of transcription activators of endogenous fusogens. In some embodiments, the small molecule may decrease the expression or activity of regulatory transcriptional repressors of endogenous fusogens. In some implementations, the small molecule is an epigenetic modifier that increases the expression of endogenous fusogens.

[0295] In some embodiments, fusosomes are generated from cells treated with a fusion-stopping compound, such as lysophosphatidylcholine. In some embodiments, fusosomes are generated from cells treated with a fusogen, such as a non-acutase-cleaving dissociation reagent.

[0296] In some embodiments, source cells are physically modified, for example, with a CRISPR activator, before generating fusosomes to add or increase the concentration of fusogens.

[0297] In some embodiments, cells are physically modified to increase or decrease in quantity, or to enhance the structure or function of organelles, such as mitochondria, Golgi apparatus, endoplasmic reticulum, or intracellular vesicles (lysosomes, autophagosomes, etc.).

[0298] b) Genetic modification In some embodiments, cells are genetically modified before fusosome production to increase the expression of endogenous fusogens within the cell (e.g., endogenous to source cells in some embodiments, and endogenous to target cells in some embodiments). In some embodiments, the genetic modification may increase the expression or activity of a transcription activator of endogenous fusogens. In some embodiments, the genetic modification may decrease the expression or activity of a regulatory transcriptional repressor of endogenous fusogens. In some embodiments, the activator or repressor is a nuclease-inactive Cas9 (dCas9) linked by guide RNA to a transcription activator or repressor targeting endogenous fusogens. In some embodiments, the genetic modification epigenetically modifies the endogenous fusogen gene to increase its expression. In some embodiments, the epigenetic activator is a nuclease-inactive Cas9 (dCas9) linked by guide RNA to an epigenetic modifier targeting endogenous fusogens.

[0299] In some embodiments, cells are genetically modified before fusosome generation to increase the expression of exogenous fusogens within the cell, such as the delivery of transgenes. In some embodiments, nucleic acids, such as DNA, mRNA, or siRNA, are transferred into cells before fusosome generation to increase or decrease the expression of cell surface molecules (proteins, glycans, lipids, or low molecular weight molecules) used, for example, for targeting organs, tissues, or cells. In some embodiments, the nucleic acids target fusogen repressors, such as shRNA or siRNA constructs. In some embodiments, the nucleic acids encode inhibitors of fusogen repressors.

[0300] In some embodiments, the method involves introducing an exogenous nucleic acid into source cells encoding a fusogen. The exogenous nucleic acid may be, for example, DNA or RNA. In some embodiments, the exogenous nucleic acid may be, for example, DNA, gDNA, cDNA, RNA, premRNA, mRNA, miRNA, siRNA, etc. In some embodiments, the exogenous DNA may be linear DNA, circular DNA, or artificial chromosome. In some embodiments, the DNA is maintained episomalally. In some embodiments, the DNA is integrated into the genome. The exogenous RNA may be chemically modified RNA, which may include, for example, one or more skeletal modifications, glycosylation, noncanonical bases, or caps. Skeletal modifications include, for example, phosphorothioate, N3' phosphoramidite, boranophosphate, phosphonoacetate, thio-PACE, morpholinophosphoramidite, or PNA. Sugar modifications include, for example, 2'-O-Me, 2'F, 2'F-ANA, LNA, UNA, and 2'-O-MOE. Non-standard bases include, for example, 5-bromo-U and 5-iodo-U, 2,6-diaminopurine, C-5 propynylpyrimidine, difluorotoluene, difluorobenzene, dichlorobenzene, 2-thiouridine, pseudouridine, and dihydrouridine. Caps include, for example, ARCA. Additional modifications are discussed, for example, in Deleavey et al., "Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing," Chemistry & Biology Volume 19, Issue 8, 24 August 2012, Pages 937-954, which is incorporated herein by reference in its entirety.

[0301] In some embodiments, cells are treated with chemicals, such as small molecules, to increase the expression or activity of exogenous fusogens relative to the intracellular source cells before generating fusosomes. In some embodiments, the small molecule may increase the expression or activity of transcription activators of exogenous fusogens. In some embodiments, the small molecule may decrease the expression or activity of regulatory transcriptional repressors of exogenous fusogens. In some embodiments, the small molecule is an epigenetic modifier that increases the expression of exogenous fusogens.

[0302] In some embodiments, nucleic acids encode modified fusion genes, such as modulogenic fusion genes, e.g., fusion genes having specific cell type, tissue type, or local microenvironment activity. Such modulogenic fusion activity may include activation and / or initiation of fusion activity by exposure to low or high pH, ​​heat, infrared light, extracellular enzyme activity (eukaryotes or prokaryotes), or small molecules, proteins, or lipids. In some embodiments, small molecules, proteins, or lipids are displayed on target cells.

[0303] In some embodiments, cells are genetically modified before fusosome generation to alter (i.e., upregulate or downregulate) the expression of a signaling pathway (e.g., the Wnt / beta-catenin pathway). In some embodiments, cells are genetically modified before fusosome generation to alter (i.e., upregulate or downregulate) the expression of one or more genes of interest. In some embodiments, cells are genetically modified before fusosome generation to alter (i.e., upregulate or downregulate) the expression of a nucleic acid (e.g., miRNA or mRNA) or a nucleic acid of interest. In some embodiments, nucleic acids, e.g., DNA, mRNA or siRNA, are transferred to cells before fusosome generation to increase or decrease the expression of, for example, a signaling pathway, a gene, or a nucleic acid. In some embodiments, the nucleic acid targets a signaling pathway, a gene, or a nucleic acid repressor, or represses a signaling pathway, a gene, or a nucleic acid. In some embodiments, the nucleic acid encodes a transcription factor that upregulates or downregulates a signaling pathway, a gene, or a nucleic acid. In some embodiments, the activator or repressor is a nuclease-inactive Cas9 (dCas9) linked by guide RNA to a transcription activator or repressor targeting a signaling pathway, gene, or nucleic acid. In some embodiments, the gene modification epigenetically alters the expression of an endogenous signaling pathway, gene, or nucleic acid. In some embodiments, the epigenetic activator is a nuclease-inactive Cas9 (dCas9) linked by guide RNA to an epigenetic modifier targeting a signaling pathway, gene, or nucleic acid. In some embodiments, the cellular DNA is edited before fusosome generation to alter the expression of a signaling pathway (e.g., the Wnt / beta-catenin pathway), gene, or nucleic acid (e.g., upregulate or downregulate). In some embodiments, the DNA is edited using guide RNA and CRISPR-Cas9 / Cpf1 or other gene editing techniques.

[0304] Cells can be genetically modified using recombination techniques. Nucleic acid sequences encoding a desired gene can be obtained using recombination techniques, for example, by screening a library from cells expressing the gene, by inducing the gene from a vector known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be generated synthetically rather than by cloning.

[0305] The expression of natural or synthetic nucleic acids is typically achieved by operably ligating the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and incorporation in eukaryotes. A typical cloning vector contains transcription and translation terminators, start sequences, and promoters useful for expressing the desired nucleic acid sequence.

[0306] In some embodiments, cells can be genetically modified with one or more expression regions, for example, genes. In some embodiments, cells can be genetically modified with exogenous genes (e.g., exogenous gene products such as RNA or polypeptide products) and / or exogenous regulatory nucleic acids. In some embodiments, cells can be genetically modified with exogenous sequences encoding exogenous regulatory nucleic acids that can regulate the expression of gene products that are endogenous to target cells and / or endogenous genes. In some embodiments, cells can be genetically modified with exogenous genes and / or regulatory nucleic acids that regulate the expression of exogenous genes. In some embodiments, cells can be genetically modified with exogenous genes and / or regulatory nucleic acids that regulate the expression of endogenous genes. It will be understood by those skilled in the art that the cells described herein can be genetically modified to express, for example, various exogenous genes encoding proteins or regulatory molecules that can act on gene products of the endogenous or exogenous genome of a target cell. In some embodiments, such genes characterize fusosomes and, for example, regulate fusion with target cells. In some embodiments, cells may be genetically modified to express endogenous genes and / or regulatory nucleic acids. In some embodiments, the endogenous genes or regulatory nucleic acids regulate the expression of other endogenous genes. In some embodiments, cells may be genetically modified to express endogenous genes and / or regulatory nucleic acids that are expressed differently from (e.g., inductively, tissue-specifically, constitutively, or at higher or lower levels) than versions of endogenous genes and / or regulatory nucleic acids on other chromosomes.

[0307] Promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located 30–110 bp upstream of the initiation site, although some promoters have recently been shown to contain functional elements downstream of the initiation site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be widened to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0308] One example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably ligated to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the Simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long-terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Rous sarcoma virus promoter, and human gene promoters such as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0309] Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence to which it is operably ligated when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, tissue-specific promoters, metallothione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters. In some embodiments, fusogen expression is upregulated before fusosomes are formed, for example, 3, 6, 9, 12, 24, 26, 48, 60, or 72 hours before fusosome formation.

[0310] The expression vector introduced into the source may also include either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via the viral vector. In other embodiments, the selectable marker may be carried on a separate DNA fragment and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0311] Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is either not present in the recipient source or not expressed by the recipient source, and whose expression is revealed by some readily detectable characteristic, such as enzymatic activity. Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or are commercially available. Generally, a construct with a minimum 5' flanking region exhibiting the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be ligated to the reporter gene and used to evaluate a drug's ability to regulate promoter-driven transcription.

[0312] In some embodiments, cells may be genetically modified to alter the expression of one or more proteins. The expression of one or more proteins may be altered for a specific time, for example, for the developmental or differentiated state of the source. In some embodiments, fusosomes are generated from a source of cells genetically modified to alter the expression of one or more proteins, for example, fusogenic or non-fusogenic proteins that affect fusion activity, structure, or function. The expression of one or more proteins may be restricted to a specific location or spread throughout the entire source.

[0313] In some embodiments, fusosomes are generated from cells having modified fusogen protein expression, for example, an increase or decrease of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or more in fusogen expression.

[0314] In some embodiments, cells may be engineered to express cytosolic enzymes (e.g., proteases, phosphatases, kinases, demethylases, methyltransferases, acetylases) that target fusogen proteins. In some embodiments, the cytosolic enzymes affect one or more fusogens by altering post-translational modifications. Post-translational protein modifications of proteins can affect nutrient availability and responsiveness to redox conditions, as well as protein-protein interactions. In some embodiments, fusosomes contain fusogens having altered post-translational modifications, e.g., an increase or decrease of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, or more.

[0315] Methods for introducing modifications into cells include physical, biological, and chemical methods. For example, Geng. & Lu, Microfluidic electroporation for cellular analysis and delivery. Lab. See a Chip.13(19):3803-21.2013;Sharei, A. et al., A vector-free microfluidic platform for intracellular delivery.PNAS vol.110 no.6.2013;Yin, H. et al., Non-viral vectors for gene-based therapy. Nature Reviews Genetics.15:541-555.2014. Suitable methods for modifying cells for use in the generation of fusosomes as described herein include, for example, diffusion, osmotic pulse, osmotic shock, hypotonic lysis, hypotonic dialysis, ion electrophoresis, electroporation, sonication, microinjection, calcium precipitation, membrane intercalation, lipid-mediated transfection, surfactant treatment, viral infection, receptor-mediated endocytosis, use of protein transduction domains, particle calcination, membrane fusion, freeze-thaw, mechanical disruption, and filtration.

[0316] Various assays are used to confirm the presence of genetic modification. Such assays include, for example, molecular biological assays such as Southern blotting and Northern blotting, RT-PCR and PCR; and biochemical assays such as detecting the presence or absence of a specific peptide by immunological means (ELISA and Western blotting) or assays described herein.

[0317] The present disclosure provides, in some embodiments, fusosomes comprising: (a) a lipid bilayer; (b) a lumen surrounded by the lipid bilayer (e.g., including a cytosol); (c) an exogenous or overexpressed fusogen, for example, a fusogen in which the fusogen is arranged in the lipid bilayer, wherein the fusosome is derived from a source cell; and wherein the fusosome has partial or complete nuclear inactivation (e.g., nuclear removal).

[0318] This disclosure provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer, (d) a nucleic acid, e.g., a nucleic acid containing a payload gene; and wherein the fusosomes do not contain a nucleus; wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; wherein (i) when the plurality of fusosomes come into contact with a cell population including target cells and non-target cells, the cargo is present at least 10 times more in the target cells than in the non-target cells or reference cells, or (ii) A number of fusosomes fuse with target cells at a rate at least 50% higher than with non-target cells or reference cells; where the target cells are selected from alpha-beta T cells, effector T cells, CD8+ cytotoxic T cells, thymic regulatory T cells, peripheral regulatory T cells, CD4+ Foxp3+ regulatory T cells, or CD4+ FoxP3-1 type regulatory T(Tr1) cells, helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T(MAIT) cells.

[0319] In some aspects, this disclosure provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; and (d) a nucleic acid comprising a payload gene encoding an exogenous active agent as shown in Table 5, wherein the fusosomes do not contain a nucleus; and wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0320] This disclosure provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing a cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen positioned in the lipid bilayer; and (d) a nucleic acid comprising a payload gene, wherein the nucleic acid comprises an NTCSRE operably linked to the payload gene, the NTCSRE comprising a non-target cell-specific miRNA recognition sequence bound by a non-target cell-specific miRNA sequence, e.g., a non-target cell-specific miRNA recognition sequence bound by a miRNA present in the non-target cell at a higher level in the target cell than in the target cell, e.g., a non-target cell-specific miRNA recognition sequence bound by a miRNA, e.g., a miRNA listed in Table 4, wherein the target cell is a first type T cell, and optionally, the non-target cell is a second different type T cell or a non-T cell; wherein the fusosome does not contain a nucleus; and wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0321] In some embodiments, miRNA is present in non-target cells (e.g., non-target cells as described herein) at levels at least 10, 100, 1,000, or 10,000 times higher than the levels of miRNA present in target cells (e.g., T cells). In some embodiments, miRNA is undetectably absent in target cells (e.g., T cells, e.g., T cells as described herein). In some embodiments, miRNA is absent in target cells (e.g., T cells, e.g., T cells as described herein).

[0322] This disclosure provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer; (b) a lumen containing a cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; and (d) a nucleic acid comprising a payload gene, comprising a promoter operably linked to the payload gene, wherein the promoter is a T cell-specific promoter, e.g., T cell, CD4+ T cell, CD8+ T cell, alpha-beta T cell, gamma-delta T cell, naive T cell, effector T cell, cytotoxic T cell (e.g., CD8+ cytotoxic T cell), regulatory T cell (e.g., For example, nucleic acids that are promoters specific to thymic regulatory T cells, peripheral regulatory T cells, CD4+Foxp3+ regulatory T cells, or CD4+FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosal-associated invariant T (MAIT) cells; wherein the fusosome does not contain a nucleus; and wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0323] In some aspects, this disclosure provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen positioned in the lipid bilayer; and (d) a nucleic acid comprising a payload gene, comprising a promoter having a sequence of Table 3 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; wherein the fusosomes do not contain a nucleus; and wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0324] This disclosure provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing a cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; and (d) a nucleic acid comprising: (i) a payload gene; and (ii) an NTCSRE operably linked to the payload gene, for example, the NTCSRE comprising a non-target cell-specific miRNA recognition sequence, for example, a non-target cell-specific miRNA recognition sequence linked by the miRNAs of Table 4, and (iii) optionally a positive target cell-specific regulatory element, e.g., a positive target cell-specific regulatory element operably linked to a payload gene (e.g., a target cell-specific promoter), wherein the positive target cell-specific regulatory element increases the expression of the payload gene in the target cell compared to a fusosome that is otherwise identical except for the absence of the positive target cell-specific regulatory element, wherein the target cell is a first type T cell; optionally, the non-target cell is a second different type T cell or a non-T cell, optionally, the target cell is a Treg cell and the non-target cell is a conventional CD4+ T cell; wherein the fusosome does not contain a nucleus; and wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0325] In some embodiments, one or more of the following exist: i) the fusosome contains or is contained within a cell biological substance; ii) the fusosome contains an enucleated cell; iii) the fusosome contains an inactivated nucleus; iv) the fusosome contains a higher proportion of target cells than non-target cells, for example, in the assay of Example 42, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, or (v) Fusosomes fuse with target cells at a higher rate than other fusosomes, for example, in the assay of Example 42, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, or 100x; (vi) Fusosomes fuse with target cells at a higher rate than other fusosomes, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 100x, e.g., in the assay of Example 42, e.g., after 24, 48, or 72 hours, the active substance within the fusosomes fuses with target cells at a rate of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, Alternatively, it fuses with target cells at a rate such that up to 90% is delivered; vii) Fusogen, for example, when measured by the assay of Example 26, contains at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies, or so (viii) Fusosomes contain therapeutic agents in copies of at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or fewer, as measured by the assay of Example 88;ix) The ratio of the copy number of Fusogen to the copy number of the therapeutic agent is as follows: 1,000,000:1~100,000:1, 100,000:1~10,000:1, 10,000:1~1,000:1, 1,000:1~100:1, 100:1~50:1, 50:1~20:1, 20:1~10:1, 10:1~5:1, 5:1~2:1, 2:1~1:1, 1:1~1:2, 1:2~1:5, 1:5~1:10, 1:10~1:20, 1:20~1:50, 1:50~1:100, 1:100~1:1,000, 1:1,000~1:10,000, 1: x) The fusosomes contain a lipid composition substantially similar to that of the source cells, or one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the corresponding lipid levels in the source cells; xi xii) Fusosomes contain a proteomics composition similar to that of the source cells, for example, using the assay of Example 87; xii) Fusosomes contain a ratio of lipids to proteins that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assay of Example 40; xiii) Fusosomes contain nucleic acids (e.g., DNA) that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured, for example, using the assay of Example 41 This includes the ratio of protein to fusosomes; xiv) Fusosomes include the ratio of lipids to nucleic acids (e.g., DNA) which, when measured using the assay of Example 91, are within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in source cells; xv) Fusosomes have half-lives within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the half-life of reference cells, e.g., source cells, in a subject, e.g., mouse, e.g., by the assay of Example 60; xvi) Fusosomes transport at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more (e.g., about 11.6% more) glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane in the absence of glucose, as measured, for example, using the assay of Example 50; xvii) Fusosomes contain esterase activity in the lumen within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the esterase activity in reference cells, e.g., source cells or mouse embryonic fibroblasts, as measured, for example, using the assay of Example 51; xviii) Fusosomes include metabolic activity levels within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the citrate synthase activity in a reference cell, e.g., a source cell, as described, for example, in Example 53; xiv) Fusosomes include metabolic activity levels within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the respiratory level in a reference cell, e.g., a source cell, as described, for example, in Example 54. xx) Fusosomes containing a respiratory level (e.g., oxygen consumption rate) of less than %; for example, using the assay of Example 55, annexin V staining levels of up to 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, or 10,000 MFI, or fusosomes having annexin V staining levels at least 5%, 10%, 20%, 30%, 40%, or 50% lower than annexin V staining levels of otherwise similar fusosomes treated with menadione in the assay of Example 55. xxi) Fusosomes containing annexin V staining levels, or fusosomes containing annexin V staining levels at least 5%, 10%, 20%, 30%, 40%, or 50% lower than the annexin V staining levels of macrophages treated with menadione in the assay of Example 55; xxii) Fusosomes having miRNA content levels at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher than those of source cells, for example, by the assay of Example 33; The soluble:insoluble protein ratio of the soms is within or greater than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of that of the source cells, for example, by the assay in Example 38, or, for example, within or greater than 1%-2%, 2%-3%, 3%-4%, 4%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% of that of the source cells;xxiii) Fusosomes have LPS levels of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, or 0.00001% of the source cell's LPS content, as measured, for example, by mass spectrometry, in the assay of Example 39; xxiv) Fusosomes have LPS levels of 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of a negative control, for example, in the absence of insulin, using the assay of Example 49, compared to otherwise similar fusosomes. , 90%, 100% more, signal transduction, e.g., delivery of extracellular signals in response to insulin, e.g., AKT phosphorylation, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG) in response to insulin;xxv) Fusosomes target tissues such as the liver, lungs, heart, spleen, pancreas, gastrointestinal tract, kidneys, testes, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eyes, and when administered to a subject, e.g., a mouse, e.g., by the assay of Example 64 After 24, 48, or 72 hours, for example, at least 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the administered fusosome population is present in the target tissue; xxvi) Fusosomes are present at least 1%, 2%, 3%, 4%, or 5% higher than the juxtaclin signaling induced by reference cells, e.g., source cells or bone marrow stromal cells (BMSCs), as determined by the assay in Example 56. (xxvii) Fusosomes have paracrine signaling levels that are 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the level of paracrine signaling induced by reference cells, e.g., source cells or macrophages, as determined by the assay in Example 57;xxviii) Fusosomes polymerize actin at levels within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the actin polymerized in reference cells, e.g., source cells or C2C12 cells, as measured by the assay in Example 58; xxix) Fusosomes polymerize actin at levels within 1%, 2%, 3%, 4%, 5%, 10%, or 2% of the membrane potential of reference cells, e.g., source cells or C2C12 cells, as measured by the assay in Example 59. Fusosomes have a membrane potential of 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or fusosomes have a membrane potential of approximately -20 to -150mV, -20 to -50mV, -50 to -100mV, or -100 to -150mV;xxx) Fusosomes are found in blood vessels or in a proportion of source cells or cells of the same type as source cells, for example, at a rate of at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, using the assay of Example 44. Extravasation of these cells is possible, where the source cells are neutrophils, lymphocytes, B cells, macrophages, or NK cells;xxxi) Fusosomes can pass through cell membranes, e.g., endothelial cell membranes or the blood-brain barrier;xxxii) Fusosomes can be detected in proportions greater than, for example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of reference cells, e.g., mouse embryonic fibroblasts, using the assay of Example 48. xxxiii) Fusosomes are capable of secreting proteins; xxxiv) Fusosomes are manufactured in accordance with Good Manufacturing Practice (GMP) standards; xxxv) Fusosomes have pathogen levels below a specified threshold, e.g., substantially free of pathogens; xxxvi) Fusosomes have contaminant levels below a specified threshold, e.g., substantially free of contaminants; xxxvii) Fusosomes have low immunogenicity, e.g., as described herein;xxxviii) Source cells are selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, such as retinal nerve cells; or xxxix) Source cells are other than 293 cells, HEK cells, human endothelial cells, or human epithelial cells, monocytes, macrophages, dendritic cells, or stem cells.

[0326] The disclosure also provides, in some embodiments, fusosomes comprising: a) a lipid bilayer and an aqueous solution, e.g., miscible with water, in which the fusosome has a lumen derived from a source cell; b) exogenous or overexpressed fusogens arranged in the lipid bilayer; and c) organelles arranged in the lumen, e.g., a therapeutically effective number of organelles.

[0327] In some embodiments, one or more of the following are present: i) Source cells are selected from endothelial cells, macrophages, neutrophils, granulocytes, leukocytes, stem cells (e.g., mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal nerve cells; ii) Organelles include Golgi apparatus, lysosomes, endoplasmic reticulum, mitochondria, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxisomes, hydrogenosomes, melanosomes, mitosomes, cnidocytes, peroxisomes, proteasomes, vesicles, and stress cells. iii) Selected from granules; iv) Fusosomes have a size greater than 5 μm, 10 μm, 20 μm, 50 μm, or 100 μm; iv) Fusosomes, or compositions or preparations containing multiple fusosomes, have a density other than 1.08 g / ml to 1.12 g / ml when measured by the assay of Example 30, for example, fusosomes have a density of 1.25 g / ml + / - 0.05; v) Fusosomes are not captured by the circulating scavenger system or Kupffer cells in the hepatic sinus; vi) Source cells are other than 293 cells; vii) Source cells are not transformed or immortalized; viii) Source cells are transformed or immortalized using methods other than adenovirus-mediated immortalization, e.g., by spontaneous mutation or telomerase expression; ix) Fusogens are other than VSVG, SNARE proteins, or secretory granule proteins; x) Fusosomes do not contain Cre or GFP, e.g., EGFP; xi) Fusosomes further contain exogenous proteins other than Cre or GFP, e.g., EGFP; xii) Fusosomes further contain, for example, exogenous nucleic acids (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or exogenous proteins (e.g., antibodies, e.g., antibodies) in their lumen; or xiii) Fusosomes do not contain mitochondria.

[0328] The disclosure also provides, in some embodiments, a fusosome comprising: (a) a lipid bilayer; (b) a lumen surrounded by the lipid bilayer (e.g., including a cytosol); (c) an exogenous or overexpressed fusogen, e.g., the fusogen is located within the lipid bilayer; and (d) a functional nucleus, where the fusosome originates from a source cell.

[0329] In some embodiments, one or more of the following are present: i) the source cell is other than a dendritic cell or tumor cell, for example, the source cell is selected from endothelial cells, macrophages, neutrophils, granulocytes, leukocytes, stem cells (e.g., mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, for example, retinal nerve cells; ii) the fusogen is other than a fusion glycoprotein; iii) the fusogen is a mammalian protein other than fertilin-beta; iv) the fusosome is low immunogenic, for example, as described herein; v) the fusosome is a pharmaceutical or Good Manufacturing Practice (GMP) standard; vi) the fusosome is manufactured in accordance with Good Manufacturing Practice (GMP); vii) the fusosome has a pathogen level below a predetermined threshold, for example, substantially free of pathogens; or viiii) the fusosome has a contaminant level below a predetermined threshold, for example, substantially free of contaminants.

[0330] The disclosure also provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen positioned in the lipid bilayer; and (d) cargo; wherein the fusosomes do not contain a nucleus; wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; wherein the plurality of fusosomes deliver cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population when in contact with a target cell population in the presence of an endocytosis inhibitor, and when in contact with a reference target cell population not treated with an endocytosis inhibitor.

[0331] The disclosure also provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen containing cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed retargeted fusogen arranged in the lipid bilayer; (d) cargo; wherein the fusosomes do not contain a nucleus; wherein the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; wherein (i) when the plurality of fusosomes come into contact with a cell population including target and non-target cells, the cargo is present at least twice, five times, ten times, twenty times, fifty times, or 100 times more in the target cells than in the non-target cells; or (ii) the plurality of fusosomes fuse with the target cells at a rate at least 50% higher than in the non-target cells.

[0332] The disclosure also provides, in some embodiments, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer, (b) a lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen, wherein the fusogen is located within the lipid bilayer; and (d) a cargo, wherein the fusosome does not contain a nucleus; and wherein one or more (e.g., at least two, three, four, or five): i) the fusogen is present in a copy number of at least 1,000 copies; ii) the fusosome contains a copy number of at least 1,000 therapeutic agents; iii) the fusosome contains one or more lipids of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG, which are within 75% of the corresponding lipid levels in the source cell; iv) the fusosome is similar to that of the source cell. The proteomics composition comprises; v) Fusosomes capable of signal transduction, e.g., delivery of extracellular signals in response to insulin, e.g., AKT phosphorylation, or uptake of insulin-responsive glucose (e.g., labeled glucose, e.g., 2-NBDG), in the absence of insulin, e.g., 10% more than negative controls, e.g., otherwise similar fusosomes; vi) Fusosomes target tissues, e.g., liver, lungs, heart, spleen, pancreas, gastrointestinal tract, kidneys, testes, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eyes, and when administered to a subject, e.g., mouse, after 24 hours, e.g., at least 0.1% or 10% of the administered population of fusosomes are present in the target tissue; or source cells are selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal nerve cells.

[0333] In the embodiment, one or more of the following apply: i) the source cells are other than 293 cells; ii) the source cells are not transformed or immortalized; iii) the source cells are transformed or immortalized using a method other than adenovirus-mediated immortalization, e.g., by spontaneous mutation or telomerase expression; iv) the fusogen is other than VSVG, SNARE protein, or secretory granule protein; v) the therapeutic agent is other than Cre or EGFP; vi) the therapeutic agent is, for example, a luminal nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., antibody, e.g., antibody); or vii) the fusosome does not contain mitochondria.

[0334] In the embodiment, one or more of the following apply: i) the source cells are other than 293 cells or HEK cells; ii) the source cells are not transformed or immortalized; iii) the source cells are transformed or immortalized using a method other than adenovirus-mediated immortalization, e.g., by spontaneous mutation or telomerase expression; iv) the fusogen is not a viral fusogen; or v) the fusosomes have a size other than 40-150 nm, e.g., larger than 150 nm, 200 nm, 300 nm, 400 nm, or 500 nm.

[0335] In the embodiments, one or more of the following apply: i) the therapeutic agent is a soluble protein expressed by source cells; ii) the fusogen is other than TAT, TAT-HA2, HA-2, gp41, Alzheimer's disease beta-amyloid peptide, Sendai virus protein, or amphiphilic net-negative peptide (WAE 11); iii) the fusogen is a mammalian fusogen; iv) the fusosome contains a polypeptide selected from enzymes, antibodies, or antiviral polypeptides in its lumen; v) the fusosome does not contain an exogenous therapeutic transmembrane protein; or vi) the fusosome does not contain CD63 or GLUT4, or the fusosome contains 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% or less (e.g., about 0.048% or less) of CD63, as determined according to the method described in Example 89, for example.

[0336] In the embodiments, the fusosomes are: i) virus-free, non-infectious, or do not replicate in host cells; ii) not viral vectors; iii) not VLPs (virus-like particles); iv) not containing viral structural proteins, e.g., proteins derived from gag, e.g., viral capsid proteins, e.g., viral capsule proteins, e.g., viral nucleocapsid proteins, or the amount of viral capsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, e.g., by mass spectrometry using the assay of Example 93; v) not containing viral matrix proteins; vi) not containing viral non-structural proteins, e.g. For example, not containing pol or its fragments or variants, viral reverse transcriptase protein, viral integrase protein, or viral protease protein; vii) not containing viral nucleic acids, such as viral RNA or viral DNA; viii) containing less than 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies per vesicle; or ix) fusosomes are not virososomes.

[0337] In some embodiments, the fusosomes contain (or are identified as containing) less than 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% viral capsid protein (e.g., about 0.05% viral capsid protein). In embodiments, the viral capsid protein is a complex of rabbit endogenous lentivirus (RELIK) capsid and cyclophyllin A. In the embodiment, the viral capsid protein:total protein ratio is (or identified as) approximately 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1.

[0338] In some embodiments, the fusosomes are (or are identified as) not containing gag protein or its fragments or variants, or the amount of gag protein or its fragments or variants is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, for example, by the assay in Example 93.

[0339] In embodiments, the ratio of the number of copies of fusogen to the number of copies of viral structural proteins on fusosomes is at least 1,000,000:1, 100,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, 5:1, or 1:1, or between 100:1 and 50:1, 50:1 and 20:1, 20:1 and 10:1, 10:1 and 5:1, or 1:1. In embodiments, the ratio of the number of copies of fusogen to the number of copies of viral matrix proteins on fusosomes is at least 1,000,000:1, 100,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, 5:1, or 1:1.

[0340] In embodiments, one or more of the following are present: i) the fusosome does not contain water-immiscible droplets; ii) the fusosome includes an aqueous lumen and a hydrophilic outer layer; iii) the fusogen is a protein fusogen; or iv) the organelle is selected from mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxisomes, hydrogenosomes, melanosomes, mitosomes, cnidocytes, peroxisomes, proteasomes, vesicles, and stress granules.

[0341] In the embodiment, one or more of the following apply: i) the fusogen is a mammalian fusogen or a viral fusogen; ii) the fusosome was not produced by loading a therapeutic or diagnostic substance into the fusosome; iii) the source cell was not loaded with a therapeutic or diagnostic substance; iv) the fusosome does not contain doxorubicin, dexamethasone, cyclodextrin, polyethylene glycol, microRNA, e.g., miR125, VEGF receptor, ICAM-1, E-selectin, iron oxide, fluorescent protein, e.g., GFP or RFP, nanoparticles, or RNase, or does not contain any of the above exogenous forms; or v) the fusosome further comprises an exogenous therapeutic substance having one or more posttranslational modifications, e.g., glycosylation.

[0342] In the embodiment, the fusosome is monolayer or multilayer.

[0343] In embodiments, one or more of the following apply: i) the fusosome is not an exosome; ii) the fusosome is a microvesicle; iii) the fusosome contains a non-mammalian fusogen; iv) the fusosome is designed to incorporate a fusogen; v) the fusosome contains an exogenous fusogen; vi) the size of the fusosome is at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the average size of the fusosome population is at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; vi i) A fusosome comprises one or more organelles, such as mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxisomes, hydrogenosomes, melanosomes, mitosomes, cnidocytes, peroxisomes, proteasomes, vesicles, and stress granules; viii) A fusosome comprises the cytoskeleton or its components, such as actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; ix) A composition or preparation comprising a fusosome or multiple fusosomes is, for example, Thery et al., "Isolation and characterization of exosomes from cell culture supernatants and biological fluids." Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22 describes, for example, in a sucrose gradient centrifugation assay, the suspension density should not be 1.08-1.22 g / ml, or at least 1.18-1.25 g / ml, or 1.05-1.x) The lipid bilayer has a density of 12 g / ml; the lipid bilayer is enriched with ceramide or sphingomyelin, or a combination thereof, compared to the source cell, or the lipid bilayer is not enriched with glycolipids, free fatty acids or phosphatidylserine, or a combination thereof, compared to the source cell (e.g., depleted); xi) The fusosomes, as measured by the assay of Example 92, contain phosphatidylserine (PS) or CD40 ligand, or both PS and CD40 ligand; xii) The fusosomes are enriched with PS compared to the source cell, e.g., Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:By assay E1433-E1442, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosome population are positive for PS;xiii) Fusosomes are substantially free of acetylcholinesterase (AChE), or for example, by assay Example 52, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 (xiv) Fusosomes substantially do not contain AChE active units / protein less than ug, or substantially do not contain tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, Actinin-4, Mitophyllin, Syntenin-1, TSG101, ADAM10, EHD4, Syntenin-1, TSG101, EHD1, Flotillin-1, Heat shock 70-kDa proteins (HSC70 / HSP73, HSP70 / HSP72), or any combination thereof, compared to, for example, source cells or those not enriched with any one or more of these proteins by the assay of Example 89, at 0.05%, 0.1%, 0.containing any individual exosome marker protein of less than 5%, 1%, 2%, 3%, 4%, 5%, 5% or 10%, and / or containing any total exosome marker protein of less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or 25%, or not concentrated any one or more of these proteins; xv) the fusosome contains, for example, when using the assay of Example 36, less than 500, 250, 100, 50, 20, 10, 5 or 1 ng GAPDH / μg total protein, or less than the level of GAPDH in the source cells, for example, 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% lower than the level of GAPDH per μg total protein in the source cells in ng / μg units; xvi) the fusosome is enriched with one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasome proteins, or one or more mitochondrial proteins, or any combination thereof. For example, the amount of calnexin is less than 500, 250, 100, 50, 20, 10, 5, or 1 ng calnexin / μg total protein, or the fusosome contains, for example, using the assay of Example 37 or 90, 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin per μg total protein compared to the source cells, or the average fractional content of calnexin in the fusosome is about 1×10. -4 , 1.5×10 -4 , 2×10 -4 , 2.1×10 -4 , 2.2×10 -4 , 2.3×10 -4 , 2.4×10 -4 , 2.43×10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7×10 -4 , 2.8×10 -4 , 2.9×10 -4 , 3×10 -4 , 3.5×10-4 Or 4 x 10 -4 xvii) Fusosomes contain an amount of calnexin per total protein that is less than or more than approximately 70%, 75%, 80%, 85%, 88%, 90%, 95%, or 99% less than that of parent cells; or xviii) Fusosomes contain an exogenous active agent (e.g., exogenous protein, mRNA, or siRNA) as measured, for example, using the assay of Example 34; or xviii) Fusosomes can be immobilized on the surface of mica by atomic force microscopy for at least 30 minutes, for example, by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442.

[0344] In embodiments, one or more of the following apply: i) the fusosome is an exosome; ii) the fusosome is not a microvesicle; iii) the size of the fusosome is less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the average size of the fusosome population is less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; iv) the fusosome does not contain organelles; v) the fusosome does not contain the cytoskeleton or its components, such as actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; vi) the fusosome, or a composition or preparation containing multiple fusosomes, is described, for example, in Thery et al., "Isolation and characterization of exosomes from "Cell culture supernatants and biological fluids." Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22, for example, having a suspension density of 1.08-1.22 g / ml in a sucrose gradient centrifugation assay; vii) The lipid bilayer is not enriched (e.g., depleted) with ceramide or sphingomyelin, or a combination thereof, compared to the source cells, or the lipid bilayer is enriched with glycolipids, free fatty acids or phosphatidylserine, or a combination thereof, compared to the source cells; viiii) The fusosomes, as measured by the assay of Example 92, do not contain phosphatidylserine (PS) or CD40 ligand, or both PS and CD40 ligand, or they are depleted compared to the source cells; ix) The fusosomes are not enriched (e.g., depleted) with PS compared to the source cells, for example, Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. By assay Proc Natl Acad Sci USA 112:E1433-E1442, for example, less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a population of fusosomes are positive for PS; x) Fusosomes are, for example, by assay Example 52, for example, at least 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 xi) Fusosomes contain acetylcholinesterase (AChE) in the form of AChE active units / protein ug; tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, actinin-4, mitophilin, syntenin-1, TSG101, ADAM10, EHD4, syntenin-1, TSG101, EHD1, flutirin-1, heat shock 70-kDa proteins (HSC70 / HSP73, HSP70 / HSP72) or any combination thereof. Including, for example, by the assay of Example 89, the fusosomes contain, for example, more than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5%, or 10% of any individual exosome marker protein compared to the source cells, and / or contain less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% of any of the total exosome marker proteins, or are enriched with one or more of these proteins; xii) Fusosomes, for example, by the assay of Example 36, exceed 500, 250, 100, 50, 20, 10, 5, or 1 ng GAPDH / ug total protein, or are below the level of GAPDH in the source cells, for example, at least 1% higher than the level of GAPDH per ng / ug unit of total protein in the source cells, 2.xiii) Fusosomes containing levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are 5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher; xiii) Fusosomes not enriched (e.g., depleted) one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasome proteins, or one or more mitochondrial proteins, or any combination thereof, e.g., calnexin The amount of calnexin is 500, 250, 100, 50, 20, 10, 5, or 1 ng calnexin / ug total protein, or the fusosomes contain 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin per total protein in ng / ug units compared to the source cells, for example using the assay in Example 90, or the average fractional content of calnexin in the fusosomes is approximately 1 × 10⁻⁶. -4 , 1.5×10 -4 , 2×10 -4 , 2.1 × 10 -4 , 2.2 × 10 -4 , 2.3 × 10 -4 , 2.4×10 -4 , 2.43 × 10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7×10 -4 , 2.8×10 -4 , 2.9×10 -4 , 3 x 10 -4 , 3.5×10 -4 Or 4 x 10 -4The fusosomes contain less than 70%, 75%, 80%, 85%, 88%, 90%, 95%, or 99% less calnexin per total protein than the parent cell; or xiv) the fusosomes cannot be immobilized on the surface of mica by atomic force microscopy for at least 30 minutes, for example, by the assay in Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442.

[0345] In the embodiments, one or more of the following apply: i) the fusosomes are VLP-free; ii) the fusosomes are virus-free; iii) the fusosomes are virus-free; iv) the fusosomes are virus-free, e.g., viral structural proteins, e.g., capsid proteins or viral matrix proteins; v) the fusosomes are virus-free, e.g., capsid proteins or viral matrix proteins; vi) the fusosomes are virus-free, e.g., nucleocapsid proteins; or vii) the fusogen is not a viral fusogen.

[0346] In this embodiment, the fusosome contains cytosol.

[0347] In the embodiment, one or more of the following apply: i) The fusosomes or source cells do not form teratomas when transplanted into a subject, for example, by the assay of Example 65; ii) The fusosomes are capable of chemotaxis of, for example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than reference cells, for example, by the assay of Example 45; iii) The fusosomes can home to the injury site, for example, and the fusosomes or cell biological material are derived from human cells, for example, the source cells being neutrophils, for example, by the assay of Example 46; iv) The fusosomes are capable of phagocytosis, for example, the source cells being macrophages, and phagocytosis by fusosomes can be detected within 0.5, 1, 2, 3, 4, 5, or 6 hours, for example, by the assay of Example 47.

[0348] In the embodiment, the fusosome or fusosome composition retains one, two, three, four, five, six or more of the above characteristics for a period of 5 days or less, for example, 4 days or less, 3 days or less, 2 days or less, or 1 day or less, for example, for about 12 to 72 hours, after administration to a subject, for example, a human subject.

[0349] In embodiments, the fusosome has one or more of the following characteristics: a) contains one or more endogenous proteins from the source cell, e.g., membrane proteins or cytosolic proteins; b) contains at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different proteins; c) contains at least 1, 2, 5, 10, 20, 50, or 100 different glycoproteins; d) contains at least 10%, 20%, 30%, or 4% of the proteins in the fusosome. 0%, 50%, 60%, 70%, 80%, or 90% by mass of proteins of natural origin; e) containing at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different RNAs; or f) containing at least 2, 3, 4, 5, 10, or 20 different lipids, selected from, for example, CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG.

[0350] In the embodiments, the fusosomes are manipulated to have one, two, three, four, five, or more of the following characteristics, or the fusosomes are not of natural origin and the nucleus does not naturally possess one, two, three, four, five, or more of the following characteristics: a) Partial nuclear inactivation results in a reduction of at least 50%, 60%, 70%, 80%, 90% or more of nuclear function, e.g., transcription or DNA replication, or both, where transcription is measured by the assay of Example 24 and DNA replication is measured by the assay of Example 25; b) The fusosomes are incapable of transcription, or e.g., using the assay of Example 24. a) The fusosomes are unable to replicate nuclear DNA, or, for example, using the assay of Example 25, they have less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the transcriptional activity of the reference cells, e.g., the source cells; c) The fusosomes are unable to replicate nuclear DNA, or, for example, using the assay of Example 25, they have less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nuclear DNA replication of the reference cells, e.g., the source cells; d) The fusosomes lack chromatin, or, for example, using the assay of Example 32, they have less than 1%, 2.5%, or 90% of the chromatin content of the reference cells, e.g., the source cells.e) The fusosomes have a chromatin content of less than 5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; f) The fusosomes lack a nuclear membrane or, by assay in Example 31, have a nuclear membrane in an amount less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of that of a reference cell, e.g., a source cell or Jurkat cell; f) The fusosomes lack a functional nuclear pore complex or their nuclear import or export activity is reduced by at least 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% by assay in Example 31, or the fusosomes lack a nuclear pore protein, e.g., NUP98 or importin 7; g) The fusosomes are hist h) The fusosomes are either free of DNA or have histone levels less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the source cell's histone levels (e.g., H1, H2a, H2b, H3, or H4) as determined by the assay in Example 32; i) the fusosomes contain fewer than 20, 10, 5, 4, 3, 2, or 1 chromosome; i) nuclear function is eliminated; j) the fusosomes are enucleated mammalian cells; k) the nucleus is removed or inactivated, for example, by mechanical force, radiation, or chemical ablation; or l) the fusosomes are derived from mammalian cells with DNA that has been completely or partially removed, for example, during interphase or mitosis.

[0351] In the embodiment, the fusosome contains mtDNA or vector DNA. In the embodiment, the fusosome does not contain DNA.

[0352] In the embodiments, the source cells are primary cells, immortalized cells, or cell lines (e.g., myeloblast cell lines, e.g., C2C12). In the embodiments, the fusosomes are derived from source cells having a modified genome with reduced immunogenicity (e.g., by genome editing to remove MHC proteins or MHC complexes). In the embodiments, the source cells are derived from a cell culture treated with an anti-inflammatory signal. In the embodiments, the source cells are derived from a cell culture treated with an immunosuppressant. In the embodiments, the source cells are substantially non-immunogenic, for example, using the assay described herein. In the embodiments, the source cells contain an exogenous agent, e.g., a therapeutic agent. In the embodiments, the source cells are recombinant cells.

[0353] In embodiments, the fusosome further comprises an exogenous active agent, e.g., a therapeutic agent, e.g., a protein or nucleic acid (e.g., DNA, chromosome (e.g., human artificial chromosome), RNA, e.g., mRNA or miRNA). In embodiments, the exogenous active agent is at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 500,000,000, or 1 ,000,000,000 or fewer copies are contained, for example, by fusosomes, or exist at an average level of at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 or fewer copies per fusosome. In embodiments, fusosomes have one or more endogenous molecules, such as proteins or nucleic acids, whose levels have been altered, for example, increased or decreased, by treating mammalian cells with siRNA or gene editing enzymes. In the embodiment, the endogenous active substance is, for example, at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 100 They are present at an average level of 0,000,000 or fewer copies (e.g., contained by fusosomes), or at an average level of at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 or fewer copies per fusosome. In embodiments, endogenous molecules (e.g., RNA or protein) are present at a concentration of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10 3, 5.0×10 3 , 10 4 , 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0 × 10 7 , 5.0×10 7 , or 1.0 × 10 8 It exists in high concentrations.

[0354] In embodiments, the active substance is selected from proteins, protein complexes (e.g., at least 2, 3, 4, 5, 10, 20, or 50 proteins, e.g., at least 2, 3, 4, 5, 10, 20, or 50 different proteins), polypeptides, nucleic acids (e.g., DNA, chromosomes, or RNA, e.g., mRNA, siRNA, or miRNA), or small molecules. In embodiments, the exogenous active substance includes site-specific nucleases, e.g., Cas9 molecules, TALENs, or ZFNs.

[0355] In the embodiment, the fusogen is a viral fusogen, e.g., HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In the embodiment, the fusogen is a mammalian fusogen, e.g., SNARE, Syncytin, myomaker, myomixer, myomerger, or FGFRL1. In the embodiment, the fusogen is active at pH 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In the embodiment, the fusogen is inactive at pH 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In the embodiment, the fusogen fuses to the target cell on its surface. In the embodiment, the fusogen facilitates fusion in a lysosome-independent manner. In the embodiment, the fusogen is a protein fusogen. In the embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glycerides, diacylglycerol, or modified unsaturated fatty acids. In the embodiments, the fusogen is a chemical fusogen, such as PEG. In the embodiments, the fusogen is a small molecule fusogen, such as an NSAID such as halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine. In the embodiments, the fusogen is a recombinant. In the embodiments, the fusogen is biochemically incorporated, for example, the fusogen is provided as a purified protein and contacted with a lipid bilayer under conditions that allow the fusogen to associate with the lipid bilayer. In the embodiments, the fusogen is biosynthetically incorporated, for example, expressed in a source cell under conditions that allow the fusogen to bind to the lipid bilayer.

[0356] In the embodiment, fusosomes bind to target cells. In the embodiment, the target cells are other than HeLa cells, or the target cells are not transformed or immortalized.

[0357] In some embodiments of the fusosome composition, the multiple fusosomes are identical. In some embodiments, the multiple fusosomes are different. In some embodiments, the multiple fusosomes originate from one or more source cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the multiple fusosomes have a diameter within 10%, 20%, 30%, 40%, or 50% of the average diameter of fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the multiple fusosomes have a volume within 10%, 20%, 30%, 40%, or 50% of the average volume of fusosomes in the fusosome composition. In some embodiments, the fusosome composition has a variation in size distribution of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less than 5%, with a variation in the source cell population in size distribution of 10%, 50%, or 90%, as based on Example 28, for example. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have fusogens with a copy number within 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the average fusogen copy number in the fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of a plurality of fusosomes have a number of therapeutic agents that is within 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the average number of therapeutic agents in the fusosomes in the fusosome composition. In some embodiments, the fusosome composition has at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15It contains or more fusosomes. In some embodiments, the fusosome composition is in a volume of at least 1 μl, 2 μl, 5 μl, 10 μl, 20 μl, 50 μl, 100 μl, 200 μl, 500 μl, 1 ml, 2 ml, 5 ml, or 10 ml.

[0358] In some embodiments, the fusosome composition delivers cargo to at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the target cell population compared to a reference target cell population.

[0359] In some embodiments, the fusosome composition delivers at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more cargo to the target cell population compared to a reference target cell population or a non-target cell population.

[0360] In some embodiments, less than 10% of the cargo enters the cell by endocytosis.

[0361] In some embodiments, the endocytosis inhibitor is a lysosomal acidification inhibitor, such as bafilomycin A1. In some embodiments, the endocytosis inhibitor is a dynamin inhibitor, such as dynasoa.

[0362] In some embodiments, the target cell population is at a physiological pH (e.g., 7.3–7.5, e.g., 7.38–7.42).

[0363] In some embodiments, the cargo to be delivered is determined using an endocytosis inhibition assay, for example, the assay of Example 80.

[0364] In some embodiments, the cargo enters the cell via a dynamin-independent or lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., an endocytosis inhibitor, e.g., a macropinocytosis inhibitor at a concentration of, for example, 25 μM, e.g., 5-(N-ethyl-N-isopropyl)amyloride (EIPA)), or an actin-independent pathway (e.g., an endocytosis inhibitor, e.g., an actin polymerization inhibitor at a concentration of, for example, 6 μM, e.g., ratranchlin B).

[0365] In some embodiments, multiple fusosomes further comprise a targeting moiety. In these embodiments, the targeting moiety is comprised by a fusogen or by a separate molecule.

[0366] In some embodiments, when multiple fusosomes come into contact with a cell population including target and non-target cells, cargo is present at least 10 times more in the target cells than in the non-target cells.

[0367] In some embodiments, when multiple fusosomes come into contact with a cell population including target and non-target cells, the cargo is present at least twice, five times, ten times, twenty times, or fifty times more in the target cells than in the non-target cells, and / or the cargo is present at least twice, five times, ten times, twenty times, or fifty times more in the target cells than in the reference cells.

[0368] In some embodiments, multiple fusosomes fuse with target cells at a rate 50% higher than with non-target cells.

[0369] In some embodiments, upon contact with a target cell population, fusosomes deliver cargo to target cell locations other than endosomes or lysosomes, such as the cytosol. In embodiments, 50%, 40%, 30%, 20%, or less than 10% of the cargo is delivered to endosomes or lysosomes.

[0370] In some embodiments, the fusosomes include exosomes, microvesicles, or a combination thereof.

[0371] In some embodiments, the fusosomes have an average size of at least 50 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm. In other embodiments, the fusosomes have an average size of 100 nm, 80 nm, 60 nm, 40 nm, or less than 30 nm.

[0372] In some embodiments, the fusogen (e.g., retargeted fusogen) includes mammalian fusogens. In some embodiments, the fusogen (e.g., retargeted fusogen) includes viral fusogens. In some embodiments, the fusogen (e.g., retargeted fusogen) is a protein fusogen. In some embodiments, the fusogen (e.g., retargeted fusogen) is Nipah virus protein F, measles virus F protein, Tupia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipa virus F protein, Morbillivirus F protein, Respirovirus F protein, and includes a sequence selected from Sendai virus F protein, Rubra virus F protein, or Abura virus F protein, or derivatives thereof.

[0373] In some embodiments, Fusogen (e.g., retargeted Fusogen) is active at pH 4–5, 5–6, 6–7, 7–8, 8–9, or 9–10. In some embodiments, Fusogen (e.g., retargeted Fusogen) is not active at pH 4–5, 5–6, 6–7, 7–8, 8–9, or 9–10.

[0374] In some embodiments, fusogens are present in copy numbers of at least 1, 2, 5, or 10 copies per fusosome.

[0375] In some embodiments, the fusogen (e.g., retargeted fusogen) is Nipah virus protein G, measles protein H, Tupia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, and includes Sendai virus HN protein, Rubra virus HN protein, Abraham virus HN protein, or derivatives thereof. In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupia 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, Henipa virus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubra virus F and HN proteins, or Abura virus F and HN proteins, or derivatives thereof, or any combination thereof.

[0376] In some embodiments, the cargo comprises an exogenous protein or exogenous nucleic acid. In some embodiments, the cargo comprises or encodes a cytosolic protein. In some embodiments, the cargo comprises or encodes a membrane protein. In some embodiments, the cargo comprises a therapeutic agent. In some embodiments, the cargo exists in copy numbers of at least 1, 2, 5, 10, 20, 50, 100, or 200 copies per fusosome (e.g., up to approximately 1,000 copies per fusosome). In some embodiments, the ratio of the number of copies of fusogen (e.g., retargeted fusogen) to the number of copies of cargo is 1000:1 to 1:1, or 500:1 to 1:1, or 250:1 to 1:1, or 150:1 to 1:1, or 100:1 to 1:1, or 75:1 to 1:1, 50:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, or 15:1 to 1:1, or 10:1 to 1:1, or 5:1 to 1:1, or 2:1 to 1:1, or 1:1 to 1:2.

[0377] In some embodiments, the fusosome composition comprises a viral capsid protein or a DNA-integrated polypeptide. In some embodiments, the cargo comprises a viral genome.

[0378] In some embodiments, fusosomes can deliver nucleic acids to target cells, for example, for gene therapy, or for example, to stably modify the genome of target cells.

[0379] In some embodiments, the fusosome composition does not contain viral nucleocapsid protein, and, for example, by mass spectrometry, using the assay of Example 93, for example, the amount of viral nucleocapsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein.

[0380] In one embodiment, the fusosome composition is at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 It contains fusosomes. In embodiments, the fusosome composition contains at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 L, 2 L, 5 L, 10 L, 20 L, or 50 L.

[0381] In the embodiment, the fusosomes are derived from mammalian cells having a genome modified to reduce immunogenicity (for example, by genome editing to remove MHC proteins or MHC complexes). In the embodiment, the source cells are derived from a cell culture treated with an anti-inflammatory signal. In the embodiment, the method further comprises contacting the source cells of step a) with an immunosuppressant or anti-inflammatory signal, for example, before or after enucleation of the cells, such as inactivating the nucleus.

[0382] In one embodiment, the herein provides a fusosome composition comprising a plurality of fusosomes derived from source cells, wherein the plurality of fusosomes comprises: (a) a lipid bilayer; (b) a lumen containing cytosol surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen positioned within the lipid bilayer; and (d) cargo; the fusosomes do not contain a nucleus; the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; and the plurality of fusosomes, when in contact with a target cell population in the presence of an endocytosis inhibitor and when in contact with a reference target cell population not treated with an endocytosis inhibitor, deliver cargo to at least 30% of the cells in the target cell population compared to the reference target cell population.

[0383] In some embodiments, the fusosome composition delivers cargo to at least 40%, 50%, 60%, 70%, or 80% of the cells in the target cell population compared to a reference target cell population or a non-target cell population, or delivers, for example, at least 40%, 50%, 60%, 70%, or 80% of the cargo to the target cell population compared to a reference target cell population or a non-target cell population. In some embodiments, less than 10% of the cargo enters the cells by endocytosis. In some embodiments, the endocytosis inhibitor is a lysosomal acidification inhibitor, such as bafilomycin A1. In some embodiments, the delivered cargo is determined using an endocytosis inhibition assay, such as the assay of Example 80. In the embodiment, the cargo enters the cell via a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., an endocytosis inhibitor, for example, a macropinocytosis inhibitor at a concentration of, for example, 25 μM, e.g., 5-(N-ethyl-N-isopropyl)amyloride (EIPA)), or an actin-independent pathway (e.g., an endocytosis inhibitor, for example, an actin polymerization inhibitor at a concentration of, for example, 6 μM, e.g., ratranchlin B).

[0384] Fusosome compositions can be generated from cells in culture, such as cultured mammalian cells, such as cultured human cells. The cells may be primordial cells or non-primordial (e.g., differentiated) cells. The cells may be primary cells or cell lines (e.g., mammalian, such as human, or the cell lines described herein). In embodiments, cultured cells may be primordial cells, such as bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPCs), endothelial progenitor cells (EPCs), blasts, intermediate progenitor cells formed in the subventricular zone, neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, progenitor cells, muscle progenitor cells, myoblasts, cardiomyocytes, neural progenitor cells, glial progenitor cells, neural progenitor cells, or hepatocytes.

[0385] In some embodiments, source cells include endothelial cells, fibroblasts, blood cells (e.g., macrophages, neutrophils, granulocytes, leukocytes), stem cells (e.g., mesenchymal stem cells, umbilical cord stem cells, bone marrow stem cells, hematopoietic stem cells, induced pluripotent stem cells, e.g., induced pluripotent stem cells derived from the subject's cells), embryonic stem cells (e.g., embryonic yolk sac, placenta, stem cells from the umbilical cord, fetal skin, adolescent skin, blood, bone marrow, adipose tissue, erythrocyte-producing tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes), alveolar cells, and neurons (e.g., retinal neurons). These include transcellular cells, progenitor cells (e.g., retinal progenitor cells, myeloblasts, myeloprogenitor cells, thymocytes, meiogenic progenitor cells, megakaryoblasts, premegakaryoblasts, melanin-forming blasts, lymphoblasts, myeloprogenitor cells, normoblasts, or angioblasts), originating cells (e.g., cardiac originating cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic originating cells, endothelial originating cells, blast cells), or immortalized cells (e.g., HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cells).

[0386] Cultured cells may be cells derived from epithelial, connective, muscular, or nerve tissues or cells, or combinations thereof. Fusosomes may be generated from cultured cells derived from any eukaryotic (e.g., mammalian) organ system, such as the cardiovascular system (heart, vascular system); the digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, and anus); the endocrine system (hypothalamus, pituitary gland, pineal gland or pineal gland, thyroid gland, parathyroid gland, adrenal gland); the excretory system (kidney, ureter, bladder); the lymphatic system (lymph, lymph nodes, lymphatic vessels, tonsils, pharyngeal tonsils, thymus, spleen); the cutaneous system (skin, hair, nails); the muscular system (e.g., skeletal muscle); the nervous system (brain, spinal cord, nerves); the reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); the respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); the skeletal system (bone, cartilage), and combinations thereof. In the embodiments, the cells are derived from highly mitotic tissue (e.g., healthy, highly mitotic tissue such as epithelium, embryonic tissue, bone marrow, or intestinal crypts). In the embodiments, the tissue sample is a highly metabolic tissue (e.g., skeletal tissue, nerve tissue, or cardiomyocytes).

[0387] In some embodiments, the cells are derived from young donors, for example, donors aged 25, 20, 18, 16, 12, 10, 8, 5, or 1 year or younger. In some embodiments, the cells are derived from fetal tissue.

[0388] In some embodiments, the cells are derived from a subject and administered to the same subject or a subject having similar genetic characteristics (e.g., MHC compatibility).

[0389] In certain embodiments, cells have telomeres of average size exceeding 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000 nucleotides in length (e.g., 4,000–10,000 nucleotides, 6,000–10,000 nucleotides).

[0390] C. Fusogen and Pseudotyping In some embodiments, the fusosomes described herein (e.g., including vesicles or parts of cells) contain one or more fusogens to facilitate the fusion of the fusosome to a membrane, for example, a cell membrane. These compositions may also include surface modifications performed during or after synthesis to include one or more fusogens. Surface modifications may include membrane modifications, for example, the insertion of lipids or proteins into the membrane.

[0391] In some embodiments, fusosomes contain one or more fusogens on their outer surface (e.g., incorporated into the cell membrane) to target a specific cell or tissue type (e.g., T cells). In some embodiments, the specific cell type targeted by one or more fusogens is a T cell, which may optionally be a CD4+ T cell, CD8+ T cell, alpha-beta T cell, gamma-delta T cell, naive T cell, effector T cell, cytotoxic T cell (e.g., CD8+ cytotoxic T cell), regulatory T cell (e.g., thymic regulatory T cell, peripheral regulatory T cell, CD4+Foxp3+ regulatory T cell, or CD4+FoxP3-1 type regulatory T(Tr1) cell), helper T cell (e.g., CD4+ helper T cell, Th1 cell, Th2 cell, Th3 cell, Th9 cell, Th17 cell, Th22 cell, or T follicular helper (Tfh) cell), memory T cell (e.g., stem cell memory T cell, central memory T cell, or effector memory T cell), NKT cell, or mucosa-associated invariant T (MAIT) cell. Fusosomes may contain a targeting domain. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. Fusogens may bind to a partner, such as a feature on the surface of a target cell. In some embodiments, the partner on the surface of the target cell is a portion of the target cell. In certain embodiments, the fusogen is a fusogen or retargeting fusogen that binds to a target cell from among T cells, and optionally, the T cells are CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, naive T cells, effector T cells, cytotoxic T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymic regulatory T cells, peripheral regulatory T cells, CD4+Foxp3+ regulatory T cells, or CD4+FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T(MAIT) cells.In some embodiments, fusosomes containing fusogens will integrate their membranes into the lipid bilayer of target cells.

[0392] In some embodiments, one or more fusogens described herein may be contained within a fusosome.

[0393] The fusosomes (e.g., retroviral vectors) described herein may include fusogens, such as endogenous fusogens or pseudotyped fusogens. i) Protein Fusogens

[0394] In some embodiments, the fusogen comprises proteins (e.g., glycoproteins), lipids, or small molecules. The fusogen may be, for example, a mammalian fusogen or a viral fusogen. In some embodiments, the fusogen comprises protein fusogens, such as mammalian proteins or homologs of mammalian proteins (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher identity), non-mammalian proteins such as viral proteins or homologs of viral proteins (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher identity), native proteins or derivatives of native proteins, synthetic proteins, their fragments, their variants, protein fusions, one or more fusogens or fragments, and any combination thereof. In some embodiments, the viral fusogen is a protein fusion comprising a class I viral membrane fusion protein, a class II viral membrane fusion protein, a class III viral membrane fusion protein, a viral membrane glycoprotein, or other viral fusion proteins, or their homologs, fragments thereof, variants thereof, or one or more proteins or fragments thereof.

[0395] In the embodiment, the fusogen is a viral fusogen, e.g., HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In the embodiment, the fusogen is a mammalian fusogen, e.g., SNARE, Syncytin, myomaker, myomixer, myomerger, or FGFRL1. In the embodiment, the fusogen is active at pH 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In the embodiment, the fusogen is inactive at pH 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In the embodiment, the fusogen fuses to the target cell on its surface. In the embodiment, the fusogen facilitates fusion in a lysosome-independent manner. In the embodiment, the fusogen is a protein fusogen. In the embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glycerides, diacylglycerol, or modified unsaturated fatty acids. In the embodiments, the fusogen is a chemical fusogen, such as PEG. In the embodiments, the fusogen is a small molecule fusogen, such as an NSAID such as halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine. In the embodiments, the fusogen is a recombinant. In the embodiments, the fusogen is biochemically incorporated, for example, the fusogen is provided as a purified protein and contacted with a lipid bilayer under conditions that allow the fusogen to associate with the lipid bilayer. In the embodiments, the fusogen is biosynthetically incorporated, for example, expressed in a source cell under conditions that allow the fusogen to bind to the lipid bilayer.

[0396] In some embodiments, the fusogen (e.g., retargeted fusogen) includes mammalian fusogens. In some embodiments, the fusogen (e.g., retargeted fusogen) includes viral fusogens. In some embodiments, the fusogen (e.g., retargeted fusogen) is a protein fusogen. In some embodiments, the fusogen (e.g., retargeted fusogen) is Nipah virus protein F, measles virus F protein, Tupia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipa virus F protein, Morbillivirus F protein, Respirovirus F protein, and includes a sequence selected from Sendai virus F protein, Rubra virus F protein, or Abura virus F protein, or derivatives thereof.

[0397] In some embodiments, Fusogen (e.g., retargeted Fusogen) is active at pH 4–5, 5–6, 6–7, 7–8, 8–9, or 9–10. In some embodiments, Fusogen (e.g., retargeted Fusogen) is not active at pH 4–5, 5–6, 6–7, 7–8, 8–9, or 9–10.

[0398] In some embodiments, fusogens are present in copy numbers of at least 1, 2, 5, or 10 copies per fusosome.

[0399] In some embodiments, the fusogen (e.g., retargeted fusogen) is Nipah virus protein G, measles protein H, Tupia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, and includes Sendai virus HN protein, Rubra virus HN protein, Abraham virus HN protein, or derivatives thereof. In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupia 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, Henipa virus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubra virus F and HN proteins, or Abura virus F and HN proteins, or derivatives thereof, or any combination thereof.

[0400] Non-mammalian fusogens include viral fusogens, their homologs, their fragments, and fusion proteins containing one or more proteins or their fragments. 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 characteristic trimer of an α-helix hairpin having a central coiled-coil structure. Class I viral fusion proteins include proteins having a central post-fusion 6-helix bundle. Examples of Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, orthomyxovirus-derived hemagglutinin, paramyxovirus-derived F protein (e.g., measles (Katoh et al. BMC Biotechnology 2010, 10:37)), retrovirus-derived ENV protein, and filovirus and coronavirus fusogens. In embodiments, class II viral fusogens, such as dengue fever E glycoprotein, have a structural feature of a β-sheet that refolds to form an elongated outer domain resulting in a hairpin trimer. In embodiments, class II viral fusogens lack a central coiled coil. Class II viral fusogens are found in alphaviruses (e.g., E1 protein) and flaviviruses (e.g., E glycoprotein). Class II viral fusogens include those derived from Semryki forest virus, cymbis, rubella virus, and dengue fever 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 contain an α-helix (e.g., a 6-helix bundle that folds the protein, similar to class I viral fusogens) and a β-sheet with an amphiphilic fusion peptide at its terminal, reminiscent of class II viral fusogens. Class III viral fusogens are found in rhabdoviruses and herpesviruses.In this embodiment, the class IV viral fusogen is a fusion-associated 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), encoded by a non-enveloped reovirus. In this embodiment, the class IV viral fusogen is small enough not to form a hairpin (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0401] Fusogens containing viral envelope proteins (env) generally determine the range of host cells that can infect and transform fusosomes. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, the native env proteins include gp41 and gp120. In some embodiments, the viral env proteins expressed by the source cells described herein are encoded in a vector separate from the viral gag and pol genes, as previously described.

[0402] Examples of usable retrovirus-derived env genes include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (Poultry Epidemic Virus), and influenza virus envelope. Similarly, RNA viruses (e.g., Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae) Alongside RNA virus families (Arenaviridae and Reoviridae), DNA viruses (virus families Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) can utilize envelope-coding genes. 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.

[0403] In some embodiments, the envelope protein for display on the fusosome may be, but is not limited to, any of the following sources: influenza A viruses such as H1N1, H1N2, H3N2 and H5N1 (avian influenza), influenza B and influenza C viruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, monkeypox, lyssavirus such as mononegavirus and rabies virus, Lagos bat virus, mocola virus, dubenhaji virus, European bat virus 1 and 2, Australian bat virus, ephemerovirus, vesiculovirus, vesiculostomatitis virus (VSV), herpesviruses such as herpes simplex virus types 1 and 2, and varicella herpes zoster. , cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, rat gamma herpesvirus, arena viruses 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, hantavirus, viruses that cause hemorrhagic renal syndrome, Rift Valley fever virus, filoviridae (filoviruses) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, flaviviridae including Kasanur forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis viruses, and paramyxoviridae such as Hendra virus and Nipah virus, variola Alphaviruses such as major and variola minor (smallpox), Venezuelan horse encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-related coronavirus (SARS-CoV), West Nile virus, and any virus that causes encephalitis.

[0404] In some embodiments, the source cells described herein produce pseudotyped fusosomes with VSV-G glycoprotein, such as recombinant retroviruses, such as lentiviruses.

[0405] Fusosomes or pseudotyped viruses generally have modifications to one or more of their envelope proteins, for example, the envelope protein being replaced with an envelope protein from another virus. For example, HIV can be pseudotyped with a fusion protein from a rhabdovirus, such as the vesicular stomatitis virus G protein (VSV-G) envelope protein, and because the HIV envelope protein (encoded by the env gene) typically targets the virus to CD4+ presenting cells, HIV can infect a wider range of cells. In some embodiments, the lentiviral envelope protein is pseudotyped with VSV-G. In one embodiment, a source cell produces a recombinant retrovirus, such as a lentivirus, pseudotyped with the VSV-G envelope glycoprotein.

[0406] Furthermore, fusogens or viral envelope proteins can be modified or manipulated to include polypeptide sequences that enable the transduction vector to infect host cells outside its normal range, or more specifically, to restrict transduction to cell or tissue types. For example, fusogens or envelope proteins can be bound in-frame to target sequences such as receptor ligands, antibodies (using the antigen-binding portion of an antibody or recombinant antibody molecules such as single-chain antibodies), and polypeptide portions or their modifications (e.g., if a glycosylation site is present in the target sequence) that, when displayed on the transduction vector coat, facilitate the direct delivery of virion particles to the target cells of interest. In addition, envelope proteins may further include sequences that modulate cellular function. Modulating cellular function with a transduction vector may increase or decrease the transduction efficiency of specific cell types in a mixed cell population. For example, stem cells can be transduced more specifically with envelope sequences containing ligands or binding partners that specifically bind to stem cells, rather than other cell types found in blood or bone marrow. Non-limiting examples include stem cell factor (SCF) and Flt-3 ligand. Other examples include antibodies (e.g., cell-type specific single-chain antibodies) and essentially any antigen (including receptors) that bind to tissues such as lung cancer, liver, pancreas, heart, endothelial, smooth, breast, prostate, epithelial, and vascular cancers.

[0407] The fusogen protein or viral envelope protein can be retargeted by mutating amino acid residues of the fusogen protein or targeting protein (e.g., hemagglutinin protein). In some embodiments, the fusogen is randomly mutated. In some embodiments, the fusogen is rationally mutated. In some embodiments, the fusogen is subjected to directional evolution. In some embodiments, the fusogen is truncated, and only a subset of the peptide is used in the retroviral vector or VLP. For example, by mutating amino acid residues in the measles hemagglutinin protein, the protein's binding properties can be altered, and fusion can be redirected (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).

[0408] In some embodiments, the protein fusogen or viral envelope protein is retargeted by i) mutating amino acids in the native fusogen protein sequence or viral envelope protein sequence, and / or ii) manipulating the fusogen protein or viral envelope protein to include a polypeptide sequence that enables the fusogen protein or viral envelope protein to target, fuse with, or infect host cells outside its normal range.

[0409] In some embodiments, fusosomes contain one or more fusogens on their outer surface (e.g., integrated into the cell membrane) to target specific cell or tissue types. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. Fusogens may bind to partners on the surface of target cells. In some embodiments, fusosomes containing fusogens will integrate their membranes into the lipid bilayer of target cells.

[0410] In some embodiments, the fusogen is a paramyxovirus fusogen. In some embodiments, the fusogen is Nipah virus protein F, measles virus F protein, Tupia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipa virus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubra virus F protein, or Abura virus F protein.

[0411] In some embodiments, the fusogen is a fusogen of the Poxviridae family.

[0412] Additional exemplary fusogens are disclosed in U.S. Patent No. 9,695,446, U.S. Patent Publication No. 2004 / 0028687, U.S. Patent No. 6,416,997, U.S. Patent No. 7,329,807, U.S. Patent Publication No. 2017 / 0112773, U.S. Patent Publication No. 2009 / 0202622, International Publication No. 2006 / 027202, and U.S. Patent Publication No. 2004 / 0009604, all of which are incorporated herein by reference.

[0413] In some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences in Table 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a portion of said sequence, for example, 100, 200, 300, 400, 500, or 600 amino acid lengths. For example, in some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80% identity with any amino acid sequence in Table 1. In some embodiments, the nucleic acid sequences described herein encode amino acid sequences of Table 1, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a portion of such sequences, for example, 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acid lengths.

[0414] In some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOs: 1 to 57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a portion of said sequence, for example, 100, 200, 300, 400, 500, or 600 amino acid lengths. For example, in some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80% identity with any one of SEQ ID NOs: 1 to 57. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence shown in any one of SEQ ID NOs: 1 to 57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a portion of such sequence, for example, 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acid lengths. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0415] In some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences in Table 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a portion of said sequence, for example, 100, 200, 300, 400, 500, or 600 amino acid lengths. For example, in some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80% identity with any amino acid sequence in Table 2. In some embodiments, the nucleic acid sequences described herein encode amino acid sequences of Table 2, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a portion of such sequences, for example, 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acid lengths.

[0416] In some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs. 58 to 133, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to that sequence, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a portion of said sequence, for example, 100, 200, 300, 400, 500, or 600 amino acid lengths. For example, in some embodiments, the Fusogen described herein includes an amino acid sequence having at least 80% identity to the amino acid sequence shown in any one of SEQ ID NOs. 58 to 133. In some embodiments, the nucleic acid sequences described herein encode an amino acid sequence shown in any one of SEQ ID NOs. 58 to 133, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a portion of said sequence, for example, 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acid lengths. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16] [Table 2-17] [Table 2-18] [Table 2-19] [Table 2-20]

[0417] ii) Lipid Fusogens In some embodiments, fusosomes may be treated with a fusionable lipid such as a saturated fatty acid. In some embodiments, the saturated fatty acid has 10 to 14 carbon atoms. In some embodiments, the saturated fatty acid has a longer-chain carboxylic acid. In some embodiments, the saturated fatty acid is a monoester.

[0418] In some embodiments, fusosomes may be treated with unsaturated fatty acids. In some embodiments, the unsaturated fatty acids include 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.

[0419] While we do not wish to be bound by theory, in some embodiments, negative curved lipids promote membrane fusion. In some embodiments, fusosomes contain one or more negative curved lipids in their membrane, for example, exogenous negative curved lipids to the source cell. In embodiments, the negative curved lipid or its precursor is added to a culture medium containing the source cell or fusosome. In embodiments, the source cell is engineered to express or overexpress one or more lipid synthesis genes. The negative curved lipid may be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acid (FA).

[0420] While we do not wish to be bound by theory, in some embodiments, positively curved lipids inhibit membrane fusion. In some embodiments, fusosomes include a reduction in the level of one or more positively curved lipids in the membrane, such as exogenous positively curved lipids. In embodiments, the level is reduced by inhibiting lipid synthesis, for example, by knockout or knockdown of lipid synthesis genes in the source cell. Positively curved lipids may be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

[0421] iii) Chemical Fusogens In some embodiments, fusosomes may be treated with a fusion chemical. In some embodiments, the fusion chemical is polyethylene glycol (PEG) or a derivative thereof.

[0422] In some embodiments, chemical fusogens induce localized dehydration between the two membranes, which leads to undesirable molecular packing of the bilayer. In some embodiments, chemical fusogens induce dehydration in areas near the lipid bilayer, causing the substitution of aqueous molecules between cells and enabling interaction between the two membranes.

[0423] In some embodiments, the chemical fusogen is a cation. Some non-limiting examples of cations include Ca2+, Mg2+, Mn2+, Zn2+, La3+, Sr3+, and H+.

[0424] In some embodiments, chemical fusogens bind to a target membrane by altering its surface polarity, which changes the hydration-dependent intermembrane repulsion.

[0425] In some embodiments, the chemical fusogens are soluble and lipid-soluble. Some non-limiting examples include oleoylglycerol, dioleoylglycerol, trioleoylglycerol, and their variants and derivatives.

[0426] In some embodiments, the chemical fusogen is a water-soluble chemical substance. Some non-limiting examples include polyethylene glycol, dimethyl sulfoxide, and their variants and derivatives.

[0427] In some embodiments, the chemical fusogen is a small organic molecule. Non-limiting examples include n-hexyl bromide.

[0428] In some embodiments, the chemical fusogen does not alter the composition of the fusogen or target membrane, cell viability, or ion transport properties.

[0429] In some embodiments, the chemical fusogen is a hormone or a vitamin. Some non-limiting examples include abscisic acid, retinol (vitamin A1), tocopherol (vitamin E), and their variants and derivatives.

[0430] In some embodiments, the fusosomes include an agent that stabilizes actin and polymerized actin. While we do not wish to be bound by theory, stabilized actin within fusosomes may promote fusion with target cells. In embodiments, the agent that stabilizes polymerized actin is selected from actin, myosin, biotin-streptavidin, ATP, neuronal Wiscott-Aldrich syndrome protein (N-WASP), or formin. See, for example, Langmuir. 2011 Aug 16;27(16):10061-71 and Wen et al., Nat Commun. 2016 Aug 31;7. In embodiments, the fusosomes include actin that is exogenous or overexpressed in the source cell, e.g., wild-type actin or actin containing a polymerization-promoting mutation. In embodiments, the fusosomes include ATP or phosphocreatine, e.g., exogenous ATP or phosphocreatine.

[0431] iv) Small molecule fusogens In some embodiments, fusosomes may be treated with fusionable small molecules. Some non-limiting examples include nonsteroidal anti-inflammatory drugs (NSAIDs) such as halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine.

[0432] In some embodiments, the small molecule fusogen may be present in micelle-like aggregates or may not be present in aggregates.

[0433] v) Fusogen modification In some embodiments, the fusogen is linked to a cleavable protein. In some cases, the cleavable protein can be cleaved by exposure to a protease. The engineered fusion protein can be linked to any domain of a transmembrane protein. The engineered fusion protein can be linked to a protein domain located in the intermembrane space by a cleavage peptide. The cleavage peptide can be cleaved by one or a combination of intermembrane proteases (e.g., HTRA2 / OMI, which requires a nonpolar aliphatic amino acid - valine, isoleucine, or methionine, preferably at the -P1 position, and a hydrophilic residue - arginine, preferably at the -P2 and P3 positions).

[0434] In some embodiments, the fusogen is linked to an affinity tag. In some embodiments, the affinity tag assists in the separation and isolation of fusosomes. In some embodiments, the affinity tag is cleavable. In some embodiments, the affinity tag is non-covalently linked to the fusogen. In some embodiments, the affinity tag is present on the fusosome and separate from the fusogen.

[0435] In some embodiments, the fusogen protein is manipulated by any method known in the Art or any method described herein to include a proteolytic sequence, such as a mitochondrial or cytosolic proteolytic sequence. The fusogen protein may be manipulated to include, but is not limited to, a proteolytic sequence, such as a caspase-2 protein sequence (e.g., Val-Asp-Val-Ala-Asp-|-(SEQ ID NO: 134)) or other proteolytic sequences (e.g., Gasteiger et al., The Proteomics Protocols Handbook; 2005: 571-607), a modified proteolytic sequence having at least 75%, 80%, 85%, 90%, 95% or more identity with the wild-type proteolytic sequence, a cytoplasmic proteolytic sequence, such as ubiquitin, or a modified cytoplasmic proteolytic sequence having at least 75%, 80%, 85%, 90%, 95% or more identity with the wild-type proteolytic sequence. In some embodiments, the composition includes a protein degradation sequence, for example, mitochondria in a source cell or chondriosome, which contains a protein modified to have at least 75%, 80%, 85%, 90%, or 95% identity with wild-type protein degradation; a cytosolic protein degradation sequence, for example, ubiquitin; or a modified cytosolic protein degradation sequence having at least 75%, 80%, 85%, 90%, or 95% identity with wild-type protein degradation.

[0436] In some embodiments, Fusogen may be modified with the overexpression of a specific protein, such as a protease, or a protease domain that recognizes an engineered fusion protein having protease activity. For example, a protease or a protease-derived protease domain, such as MMP mitochondrial processing peptidase, mitochondrial peptidase, intermediate peptidase, or endometrial peptidase.

[0437] Alfonzo,JD&Soll,D.Mitochondrial tRNA import-the challenge to understand has just begun.Biological Chemistry 390:717-722.2009;Langer,T.et al.Characterization of Peptides Released from Mitochondria.THE JOURNAL OF BIOLOGICAL CHEMISTRY.Vol.280,No.4.2691-2699,2005;Vliegh,P.et al.Synthetic therapeutic peptides:science and market.Drug Discovery Today.15(1 / 2).2010;Quiros PMm et al.,New roles for mitochondrial proteases in health,aging and See disease.Nature Reviews Molecular Cell Biology.V16,2015;Weber-Lotfi,F.et al.DNA import competence and mitochondrial genetics.Biopolymers and Cell.Vol.30.N 1.71-73,2014.

[0438] III. Positive Target Cell-Specific Regulatory Elements In some embodiments, the fusosome described herein, for example, a virus, for example, a retrovirus, comprises a nucleic acid (e.g., a gene encoding an exogenous active agent), for example, a retroviral nucleic acid, which includes positive target cell-specific regulatory elements 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 RNA or protein, a tissue-specific mRNA export-promoting site, a tissue-specific translation-enhancing site, or a tissue-specific post-translational modification site.

[0439] In some embodiments, the fusosomes described herein, such as retroviruses, include nucleic acids, such as retroviral nucleic acids, which may include untranslated regions such as origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Schein-Dalgarno sequences or Kozak sequences), introns, polyadenylated sequences, and 5' and 3' untranslated regions (which can interact with host cell proteins to perform transcription and translation and direct, increase, regulate, or control the transcription or expression of operably linked polynucleotides). Such elements may differ in their intensity and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters, may be used.

[0440] In certain embodiments, the regulatory element can direct, increase, regulate, or control the transcription or expression of operably linked polynucleotides in a cell-specific manner. In certain embodiments, the retroviral nucleic acid comprises one or more expression regulatory sequences specific to a particular cell, cell type, or cell lineage, e.g., target cells, i.e., the expression of polynucleotides operably linked to the expression regulatory sequences specific to a particular cell, cell type, or cell lineage is expressed in target cells but not (or expressed at a lower level) in non-target cells.

[0441] In certain embodiments, the retroviral nucleic acid may include exogenous, endogenous, or heterogeneous regulatory sequences such as promoters and / or enhancers.

[0442] In some embodiments, the promoter includes a recognition site to which RNA polymerase binds. RNA polymerase initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, the promoter operating in mammalian cells includes an AT-rich region located about 25–30 bases upstream from the transcription initiation site and / or another sequence, the CNCAAT region, found 70–80 bases upstream from the transcription initiation, where N can be any nucleotide.

[0443] In some embodiments, the enhancer includes a segment of DNA containing a sequence that can provide enhanced transcription and, in some cases, function independently of orientation to another regulatory sequence. The enhancer can function cooperatively or additively with the promoter and / or other enhancer elements. In some embodiments, the promoter / enhancer segment of DNA includes a sequence that can provide both promoter and enhancer functions.

[0444] Exemplary ubiquitous expression regulatory sequences include the cytomegalovirus (CMV) immediate early promoter, Simianvirus 40 (SV40) (e.g., early or late), Moloney's mouse leukemia virus (MoMLV) LTR promoter, Roussarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, vaccinia virus-derived H5, P7.5, and P11 promoters, and elongation factor 1-alpha (E F1a) Promoter, Early Growth Response 1 (EGR1), Ferritin H (FerH), Ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Eukaryotic Translation Initiation Factor 4A1 (EIF4A1), Heat Shock Protein 70kDa 5 (HSPA5), Heat Shock Protein 90kDa Beta, Member 1 (HSP90B1), Heat Shock Protein 70kDa (HSP70), β-Kinesin (β-KIN), Human ROSA Examples include 26 gene loci (Orions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deletion, and d1587rev primer binding site substitution (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)).

[0445] In some embodiments, a promoter can be paired with a heterologous gene to confer the promoter's regulatory function to the heterologous gene. In some embodiments, a cis-regulatory element from the promoter of a first gene can be ligated to a segment of the promoter of a different gene to create a chimeric promoter possessing the characteristics of both promoters.

[0446] In some embodiments, the promoter is a tissue-specific promoter, e.g., a promoter that drives expression in T cells, e.g., CD4+ T cells, CD8+ T cells, alpha-beta T cells, gamma-delta T cells, naive T cells, effector T cells, cytotoxic T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymic regulatory T cells, peripheral regulatory T cells, CD4+Foxp3+ regulatory T cells, or CD4+FoxP3-1 type regulatory T(Tr1) cells), helper T cells (e.g., CD4+ helper T cells, Th1 cells, Th2 cells, Th3 cells, Th9 cells, Th17 cells, Th22 cells, or T follicular helper (Tfh) cells), memory T cells (e.g., stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T (MAIT) cells. Various suitable T cell-specific promoters are listed in Table 3 below. In some embodiments, the fusosomes (e.g., viral vectors) described herein contain in their nucleic acids a promoter having the sequence in Table 3, or a transcriptionally active fragment thereof, or a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the fusosomes (e.g., viral vectors) described herein contain in their nucleic acids a promoter having a transcription factor binding site derived from a region of 3kb or less of the transcription start site of a gene listed in Table 3. In some embodiments, the fusosomes (e.g., viral vectors) described herein contain in their nucleic acids a region of 2.5 kb, 2 kb, 1.5 kb, 1 kb, or 0.5 kb immediately upstream of the transcription start site of a gene listed in Table 3, or a transcriptionally active fragment thereof, or a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. [Table 3]

[0447] In some embodiments, the T cell-specific promoter is a promoter described in the Immgen Consortium, which is incorporated herein by reference in whole, for example, the T cell-specific promoter is the IL2RA(CD25), LRRC32, FOXP3, or IKZF2 promoter. In some embodiments, the T cell-specific promoter or enhancer is a promoter or enhancer described in Schmidl et al., Blood. 2014 Apr 24;123(17):e68-78, which is incorporated herein by reference in whole. In some embodiments, the T cell-specific promoter is any of the transcriptional activity fragments described above. In some embodiments, the T cell-specific promoter is a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any of the aforementioned.

[0448] Internal ribosome entry sites (IRESs) typically facilitate direct internal ribosome entry into the start codon (ATG, etc.) of the cistron (protein-coding region), thereby resulting in gene cap-independent translation. See, for example, Jackson et al. (1990) Trends Biochem Sci 15(12):477-83, and Jackson and Kaminski (1995) RNA 1(10):985-1000. In certain embodiments, the vector contains one or more exogenous genes encoding one or more exogenous activators. In certain embodiments, to achieve efficient translation of each of multiple exogenous proteoactive substances, polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

[0449] The retroviral nucleic acids described herein may also include one or more Kozak sequences, for example, short nucleotide sequences that facilitate the initial binding of mRNA to the small subunit of the ribosome and increase translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is purine (A or G) (Kozak, (1986) Cell. 44(2):283-92, and Kozak, (1987) Nucleic Acids Res. 15(20):8125-48).

[0450] Promoters that respond to heterologous transcription factors and inducers In some embodiments, the retroviral nucleic acid includes elements that enable conditional expression of exogenous activators, including but not limited to any type of conditional expression, such as inducible expression, repressible expression, cell type-specific expression, or tissue-specific expression. In some embodiments, to achieve conditional expression of an exogenous activator, the expression of a cell, tissue, or organism is controlled by subjecting it to a treatment or condition that causes it to express the exogenous activator or to increase or decrease the expression of the exogenous activator.

[0451] Examples of inductive promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoids or estrogen receptors (which can be induced by treatment with the corresponding hormone), metallothione promoters (which can be induced by treatment with various heavy metals), MX-1 promoters (which can be induced by interferon), the "GeneSwitch" mifepristone-controlled system (Sirin et al., 2003, Gene, 323:67), the kmet-inducible gene switch (International Publication No. 2002 / 088346), and tetracycline-dependent regulatory systems.

[0452] The expression of a transgene can be activated or suppressed by the presence or absence of an inducer molecule. In some cases, the inducer molecule activates or suppresses gene expression stepwise, while in other cases, it activates or suppresses gene expression in an all-or-nothing manner.

[0453] The most commonly used inductive promoter / system is the tetracycline (Tet) regulatory system. The Tet system is based on the co-expression of two elements in each target cell: (i) a tetracycline response element (e.g., a gene encoding an exogenous active agent) containing repeats of a Tet operator sequence (TetO) fused to a minimal promoter and connected to the gene of interest; and (ii) a transactivation domain of the herpes simplex virus-derived VP16 protein, a fusion protein of a transcriptional transactivator (tTA), a Tet repressor (TetR). In the first described version, transgene expression was active in the absence of tetracycline or its potent analog, doxycycline (Do) (called the Tet-OFF system), but a modification of four amino acids in the transactivator protein resulted in an inverse tTA (rtTA) that binds to TetO only in the presence of Dox (Tet-ON system). In some embodiments, in the transactivator, the VP16 domain was replaced with a minimal activating domain, potential splice donor and splice acceptor sites were removed, and the protein was codon-optimized, resulting in an improved transactivator variant, rtTA2S-M2, which is highly sensitive to Dox and has reduced baseline activity. Furthermore, various Tet-responsive promoter elements have been generated, including modifying TetO at a 36-nucleotide interval from adjacent operators to enhance regulation. Additional modifications may help to further reduce basal activity and increase the expression dynamic range. For example, the pTet-T11 (abbreviated as TII) variant exhibits a high dynamic range and low background activity.

[0454] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the retroviral nucleic acid includes at least one (typically two) (may be more) sites for recombination mediated by excision or integration proteins, enzymes, cofactors or related proteins involved in the recombination reaction, including site-specific recombinases, e.g., one or more recombination sites that may be wild-type proteins (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.) (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments, and variants thereof. Examples of recombinases, though not limited to them, include Cre, Int, IHF, Xis, Flop, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0455] Riboswitches regulate the expression of exogenous active substances. Some of the compositions and methods provided herein include one or more riboswitches, or polynucleotides comprising one or more riboswitches. Riboswitches are a common function of bacteria that regulate gene expression and are a means of achieving RNA control of biological functions. Riboswitches may reside in the 5' untranslated region of mRNA and can enable regulatory control of gene expression through the binding of small molecule ligands that induce or repress riboswitch activity. In some embodiments, riboswitches control gene products involved in the production of small molecule ligands. Riboswitches typically operate in a cis-mode, but trans-mode riboswitches have been identified. Natural riboswitches consist of two domains: an aptamer domain that binds to a ligand via a three-dimensional folded RNA structure, and a functional switching domain that induces or represses riboswitch activity based on the presence or absence of the ligand. Thus, there are two ligand-sensitive conformations achieved by riboswitches, representing the on-state and the off-state (Garst et al., 2011). Functional switching domains can influence polynucleotide expression by regulating internal ribosome entry sites, accessibility of premRNA splice donors in retroviral gene constructs, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression (Dambach and Winkler 2009). Aptamers and functional switching domains can be used as modular components, allowing synthetic RNA devices to control gene expression as native aptamers, mutated / evolved native aptamers, or fully synthetic aptamers identified from screening random RNA libraries (McKeague et al 2016).

[0456] The purine riboswitch family is one of the largest families with over 500 sequences found (Mandal et al. 2003; U.S. Patent Application Publication No. 20080269258; and International Publication No. 2006055351). Purine riboswitches share a similar structure consisting of three conserved helical / stem structures (PI, P2, P3) and intervening loop / junction elements (J1-2, L2, J2-3, L3, J3-1). The aptamer domains of the riboswitch purine family spontaneously change their affinity / regulation with various purine compounds such as adenine, guanine, adenosine, guanosine, deoxyadenosine, and deoxyguanosine upon sequence changes (Kim et al. 2007).

[0457] In some embodiments, the retroviral nucleic acids described herein include a polynucleotide encoding an exogenous activator operably linked to a promoter and a riboswitch. The riboswitch includes one or more domains, for example, all of the following: a.) an aptamer domain, e.g., an aptamer domain capable of binding to a nucleoside analog antiviral drug, with reduced binding to guanine or 2'-deoxyguanosine compared to a nucleoside analog antiviral drug; b.) a functional switching domain, e.g., a functional switching domain capable of regulating the expression of an exogenous activator, wherein binding of the nucleoside analog by the aptamer domain induces or represses the expression-regulating activity of the functional switching domain, thereby regulating the expression of the exogenous activator. In some embodiments, the exogenous activator may be a polypeptide, miRNA, or shRNA. For example, in one embodiment, the riboswitch is operably linked to a nucleic acid encoding a chimeric antigen receptor (CAR). In the non-limiting examples provided herein, the exogenous gene encodes one or more engineered signaling polypeptides. For example, target polynucleotides encoding riboswitches and one or more manipulated signaling polynucleotides can be found in the genome of source cells, incapacitated recombinant retroviral particles, T cells, and / or NK cells.

[0458] Aptamer domains can be used, for example, as modular components and can be combined with any of the functional switching domains to affect RNA transcripts. In any of the embodiments disclosed herein, a riboswitch may affect RNA transcripts by modulating any of the following activities: internal ribosome entry site (IRES), premRNA splice donor accessibility, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression. In some embodiments, the functional switching domain can control the binding of anti-IRES to IRES (see, e.g., Ogawa, RNA (2011), 17:478-488, the disclosure of which is incorporated herein by reference in its entirety). In any of the embodiments disclosed herein, the presence or absence of a small molecule ligand may result in the riboswitch affecting RNA transcripts. In some embodiments, the riboswitch may include a ribozyme. A riboswitch having a ribozyme can inhibit or enhance transcript degradation of a target polynucleotide in the presence of a small molecule ligand. In some embodiments, the ribozyme may be a pistol-class ribozyme, a hammerhead-class ribozyme, a twist-class ribozyme, a hatchet-class ribozyme, or HDV (hepatitis delta virus).

[0459] IV. Non-target cell-specific regulatory elements In some embodiments, the non-target cell-specific regulatory element or negative TCSRE includes 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.

[0460] In some embodiments, non-target cells contain endogenous miRNAs. Retroviral nucleic acids (e.g., genes encoding exogenous activators) may contain recognition sequences for their miRNAs. Therefore, when retroviral nucleic acids enter non-target cells, the miRNAs may downregulate the expression of exogenous activators. This helps to enhance the specificity between target and non-target cells.

[0461] In some embodiments, miRNAs are small non-coding RNAs of 20–22 nucleotides, typically excised from a foldback RNA precursor structure of about 70 nucleotides known as pre-miRNA. Generally, miRNAs negatively modulate a target in one of two ways, depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with complete or near-complete complementarity to a protein-coding mRNA sequence usually induce RNA-mediated interference (RNAi) pathways. MiRNAs that exert their regulatory effect by binding to an incompletely complementary site within the 3' untranslated region (UTR) of the mRNA target usually repress the expression of the target gene post-transcriptionally, clearly at the translational level, via a RISC complex similar to, or possibly the same as, that used in the RNAi pathway. Consistent with translational regulation, miRNAs using this mechanism reduce the protein levels of the target gene, but the mRNA levels of these genes are minimally affected. miRNAs (e.g., naturally occurring miRNAs or artificially designed miRNAs) can specifically target any mRNA sequence. For example, in one embodiment, those skilled in the art can design a short hairpin RNA construct to be expressed as a primary transcript of human miRNA (e.g., miR-30 or miR-21). This design has been shown to significantly improve knockdown efficiency by adding a Drosha processing site to the hairpin structure (Pusch et al., 2004). The hairpin stem consists of a 22nt dsRNA (e.g., the antisense has complete complementarity to the target of interest) and a 15-19nt loop derived from human miR. Adding the miR loop and miR30 flanking sequence to one or both sides of the hairpin increases Drosha and Dicer processing of the expressed hairpin by more than 10-fold compared to conventional shRNA designs that do not use microRNA. Increased Drosha and Dicer processing leads to increased siRNA / miRNA generation and enhanced potency of the expressed hairpin.

[0462] Hundreds of different miRNA genes are expressed differentially during development and across tissue types. Several studies suggest that miRNAs play a crucial regulatory role in a wide range of biological processes, including developmental timing, cell differentiation, proliferation, apoptosis, carcinogenesis, insulin secretion, and cholesterol biosynthesis. (Bartel 2004 Cell 116:281-97; Ambros 2004 Nature 431:350-55; Du et al.) (See al. 2005 Development 132:4645-52; Chen 2005 N.Engl.J.Med.353:1768-71; Krutzfeldt et al. 2005 Nature 438:685-89). Molecular analysis shows that miRNAs have different expression profiles in different tissues. Using computational methods, we analyzed the expression of approximately 7,000 predicted human miRNA targets. The data suggest that miRNA expression contributes broadly to the tissue specificity of mRNA expression in many human tissues. (See Sood et al. 2006 PNAS USA 103(8):2746-51)

[0463] Therefore, miRNA-based approaches can be used to restrict the expression of exogenous activators to target cell populations by silencing their expression in non-target cell types using endogenous microRNA species. MicroRNAs induce sequence-specific post-transcriptional gene silencing in many organisms by inhibiting messenger RNA (mRNA) translation or causing mRNA degradation. See, for example, Brown et al. 2006 Nature Med. 12(5):585-91, and International Publication No. 2007 / 000668, each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral nucleic acid contains 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 active ingredient does not include GFP, for example, a fluorescent protein, for example, a reporter protein. In some embodiments, the off-target cells are not hematopoietic cells, and / or miRNAs are not present in hematopoietic cells.

[0464] In some embodiments, the method herein comprises tissue-specific expression of an exogenous active agent in target cells, and includes contacting a plurality of retroviral vectors, each containing a nucleotide encoding the exogenous active agent and at least one tissue-specific microRNA (miRNA), with a plurality of cells, including target cells and non-target cells, wherein the exogenous active agent is preferentially, for example, limitedly expressed in the target cells.

[0465] For example, a retroviral nucleic acid may contain at least one miRNA recognition sequence operably linked to a nucleotide sequence having a corresponding miRNA in non-target cells, such as hematopoietic primordial cells (HSPCs) and hematopoietic stem cells (HSCs), which prevents or reduces the expression of the nucleotide sequence in non-target cells but does not prevent or reduce its expression in target cells, such as differentiated cells. In some embodiments, the retroviral nucleic acid contains at least one miRNA sequence target for a miRNA that is present in an effective amount in non-target cells (e.g., the concentration of endogenous miRNA is sufficient to reduce or prevent the expression of the transgene), and also contains a transgene. In embodiments, the miRNA used in this system is strongly expressed in non-target cells such as HSPCs and HSCs, but not in differentiated offspring of bone marrow and lymphoid lineages, for example, thereby preventing or reducing the expression of the transgene in a susceptible stem cell population while maintaining expression and therapeutic effect in target cells.

[0466] In some embodiments, the negative TSCRE or NTSCRE includes a miRNA recognition site. Exemplary miRNAs are provided in Table 4. In some embodiments, the nucleic acid (e.g., fusosomal nucleic acid or retroviral nucleic acid) includes a sequence that is complementary to the miRNA in Table 4, or has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity thereto. In some embodiments, the nucleic acid (e.g., fusosomal nucleic acid or retroviral nucleic acid) includes an endogenous miRNA, for example, a sequence that is perfectly complementary to the seed sequence in the miRNA in Table 4. In embodiments, the seed sequence is at least 6, 7, 8, 9, or 10 nucleotides long. [Table 4]

[0467] In some embodiments, the negative TSCRE or NTSCRE includes a miRNA recognition site for the miRNAs described herein. Exemplary miRNAs include those found in Smigielska-Czepiel et al., Genes Immun. 2014 Mar;15(2):115-25, which are incorporated herein in their entirety by reference, such as miR31, miR363, or miR29c.

[0468] In some embodiments, the fusosome described herein comprises a nucleic acid containing a payload gene and a positive target cell-specific regulatory element, for example, the target cell being a Treg cell, such as a thymus-derived regulatory T cell, a peripheral-derived regulatory T cell, a CD4+Foxp3+ regulatory T cell, or a CD4+FoxP3-1 type regulatory T(Tr1) cell. In some embodiments, the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE), for example, the NTSCRE containing a miRNA recognition site for miRNA expressed in non-Treg cells, such as conventional CD4+ T cells.

[0469] In some embodiments, the negative TSCRE or NTSCRE includes a miRNA recognition site for the miRNA described herein. Exemplary miRNAs include Griffiths-Jones et al.Nucleic Acids Res.2006 Jan 1,34;Chen and Lodish,Semin Immunol.2005 Apr;17(2):155-65;Chen et al.Science.2004 Jan 2;303(5654):83-6;Barad et al.Genome Res.2004 Dec;14(12):2486-2494;Krichevsky et al.,RNA.2003 Oct;9(10):1274-81;Kasashima et al.Biochem Biophys Res Commun.2004 Sep 17;322(2):403-10;Houbaviy et al.,Dev Cell.2003 Aug;5(2):351-8;Lagos-Quintana et al.,Curr Biol.2002 Apr References include 30;12(9):735-9;Calin et al., Proc Natl Acad Sci US A.2004 Mar 2;101(9):2999-3004;Sempere et al. Genome Biol.2004;5(3):R13;Metzler et al., Genes Chromosomes Cancer.2004 Feb;39(2):167-9;Calin et al., Proc Natl Acad Sci US A.2002 Nov 26;99(24):15524-9;Mansfield et al. Nat Genet.2004 Oct;36(10):1079-83;Michael et al. Mol Cancer Res.2003 Oct;1(12):882-91;and those found on www.miRNA.org.

[0470] In some embodiments, the negative TSCRE or NTSCRE comprises miRNA recognition sites for miRNAs selected from miR-1b, miR-189b, miR-93, miR-125b, miR-130, miR-32, miR-128, miR-22, miR124a, miR-296, miR-143, miR-15, miR-141, miR-143, miR-16, miR-127, miR99a, miR-183, miR-19b, miR-92, miR-9, miR-130b, miR-21, miR-30b, miR-16, miR-142-s, miR-99a, miR-212, miR-30c, miR-213, miR-20, miR-155, miR-152, miR-139, miR-30b, miR-7, miR-30c, miR-18, miR-137, miR-219, miR-1d, miR-178, miR-24, miR-122a, miR-215, miR-142-a, miR-223, miR-142, miR-124a, miR-190, miR-149, miR-193, miR-181, let-7a, miR-132, miR-27a, miR-9*, miR-200b, miR-266, miR-153, miR-135, miR-206, miR-24, miR-19a, miR-199, miR-26a, miR-194, miR-125a, miR-15a, miR-145, miR-133, miR-96, miR-131, miR-124b, miR-151, miR-7b, miR-103, and miR-208.

[0471] In some embodiments, the nucleic acid (e.g., retroviral nucleic acid) includes two or more miRNA recognition sites. In some embodiments, the first and second miRNA recognition sites are recognized by the same miRNA, and in some embodiments, the first and second miRNA recognition sites are recognized by different miRNAs. In some embodiments, the first and second miRNA recognition sites are recognized by miRNAs present in the same non-target cell, and in some embodiments, the first and second miRNA recognition sites are recognized by miRNAs present in different non-target cells. In some embodiments, one or both of the first and second miRNA recognition sites are recognized by the miRNAs listed in Table 4. In some embodiments, one or more miRNA recognition sites on fusosomal nucleic acid (e.g., retroviral nucleic acid) are transcribed in cis along with an exogenous activator. In some embodiments, one or more miRNA recognition sites on fusosomal nucleic acid (e.g., retroviral nucleic acid) are located downstream of the poly(A) tail sequence, for example, between the poly(A) tail sequence and the WPRE. In some embodiments, one or more miRNA recognition sites on fusosomal nucleic acid (e.g., retroviral nucleic acid) are located downstream of the WPRE.

[0472] V. Immune regulation In some embodiments, fusosomes, such as the retroviral vectors or VLPs described herein, include elevated CD47. See, for example, U.S. Patent No. 9,050,269, which is incorporated herein in its entirety by reference. In some embodiments, fusosomes, such as the retroviral vectors or VLPs described herein, include elevated complement regulatory proteins. See, for example, Spanish Patent No. 2627445 T3 and U.S. Patent No. 6,790641, each of which is incorporated herein in its entirety by reference. In some embodiments, fusosomes, such as the retroviral vectors or VLPs described herein, include the absence or reduced levels of MHC proteins, such as MHC-1 class 1 or class II. See, for example, U.S. Patent Application Publication No. 20170165348, which is incorporated herein in its entirety by reference.

[0473] Fusosomes, such as retroviral vectors or VLPs, can be recognized by the subject's immune system. In the case of enveloped viral vector particles (e.g., retroviral vector particles), membrane-bound proteins presented on the surface of the viral envelope may be recognized, and the viral particle itself may be neutralized. Furthermore, upon infection of target cells, the viral envelope may integrate with the cell membrane, resulting in the viral envelope proteins being presented on the cell surface or remaining closely associated with the cell surface. Therefore, the immune system may also target cells infected with viral vector particles. Both effects can lead to a decrease in the effectiveness of exogenous agent delivery by viral vectors.

[0474] The viral particle envelope typically originates from the source cell membrane. Therefore, membrane proteins expressed on the cell membrane from which the viral particle budding can potentially be incorporated into the viral envelope. Immunomodulatory protein CD47

[0475] 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 is broadly classified into two categories: phagocytosis, which involves the uptake of particles, and phagocytosis, which involves the uptake of body fluids and solutes.

[0476] Studies using knockout mice lacking the membrane receptor CD47 have shown that professional phagocytic cells can distinguish between non-self and self cells (Oldenborg et al.). 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) found in macrophages (Fujioka et al., 1996, Mol.Cell.Biol.16(12):6887-99, Veillette et al., 1998, J.Biol.Chem.273(35):22719-28, Jiang et al., 1999, J.Biol.Chem.274(2):559-62). CD47-SIRPα interaction appears to inactivate mouse autologous macrophages, but a severe reduction in CD47 (perhaps 90%) has been observed in human blood cells from several Rh genotypes showing little or no evidence of anemia (Mouro-Chanteloup). (Arndt et al., 2003, Blood 101(1):338-344), and there is little or no evidence of enhanced cell interactions with phagocytic monocytes (Arndt et al., 2004, Br.J.Haematol. 125(3):412-4).

[0477] In some embodiments, the retroviral vector or VLP (e.g., viral particles with a radius of less than about 1 μm, less than about 400 nm, or less than about 150 nm) includes, for example, at least a biologically active portion of CD47 on the exposed surface of the retroviral vector or VLP. In some embodiments, the retroviral vector (e.g., lentivirus) or VLP includes a lipid coating. In embodiments, the amount of biologically active CD47 in the retroviral vector or VLP is about 20-250, 20-50, 50-100, 100-150, 150-200, or 200-250 molecules / μm 2 In some embodiments, CD47 is human CD47.

[0478] The methods described herein may...

Claims

[Claim 1] The invention described in the specification.

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