Fusosome compositions for T cell delivery

Abstract

The present disclosure provides, at least in part, methods and compositions for in vivo fusosome delivery. In some embodiments, the fusosomes contain a combination of elements that promote specificity for target cells, such as one or more fusogens, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome compositions contain one or more modifications that reduce an immune response to the fusosomes.

Description

Technical field

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

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application is being submitted with a Sequence Listing in electronic form. The sequence listing is provided as a file titled 186152003240SeqList.TXT, created 14 November 2019, and is 627 kilobytes in size. The information in electronic form of the Sequence Listing is incorporated by reference in its entirety. [Background technology]

[0003] Complex biologics are promising therapeutic candidates for a variety of diseases. However, it is difficult to deliver large biological agents to cells because the plasma membrane functions as a barrier between cells and the extracellular space. There is a need in the art for new methods of delivering complex biologics to the cells of a subject. [Outline of the invention] [Means for solving the problem]

[0004] The present disclosure provides, at least in part, fusosomal methods and compositions for in vivo delivery. In some embodiments, the fusosomes comprise a combination of elements that promote target cell specificity, eg, one or more fusogens, positive target cell-specific regulatory elements, and non-target cell-specific regulatory elements. In some embodiments, the fusosomal composition comprises one or more modifications that reduce the immune response to fusosomes.

[0005] Enumeration of Embodiments 1. Below: a) lipid bilayers containing fusogens; b) the following: (i) a payload gene encoding an exogenous agent, e.g., a payload gene encoding an exogenous agent of Table 5; (ii) 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 The regulatory element increases the expression of the payload gene in the target cell compared to a fusosome that is similar but lacks a positive target cell-specific regulatory element, and the target cell is a T cell, a fusosome.

[0006] 2. 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, wherein the NTCSRE is similar except lacking the NTCSRE reduce expression of a payload gene in non-target cells compared to fusosomes, optionally wherein the target cells are T cells of a first type and the non-target cells are a second, different type of T cells or non-T cells and optionally the target cells are Treg cells and the non-target cells are conventional CD4+ T cells.

[0007] 3 or less: a) lipid bilayers containing fusogens; b) the following: (i) a payload gene encoding an exogenous agent, such as the exogenous agent of Table 5, and (ii) a promoter operably linked to the payload gene, wherein, for example, the promoter sequence of Table 3, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96 A fusosome comprising a nucleic acid comprising a promoter selected from IL2RA, LRRC32, FOXP3, or IKZF2 promoters according to sequences having %, 97%, 98% or 99% identity.

[0008] less than 4: a) lipid bilayers containing fusogens; b) the following: (i) a payload gene encoding an exogenous agent, e.g., a payload gene encoding an exogenous agent of Table 5; (ii) 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 a nucleic acid comprising a non-target cell-specific regulatory element operably linked to a payload gene, wherein the NTCRE reduces expression of the payload gene in non-target cells or tissues compared to otherwise similar fusosomes lacking the NTCRES; Fusosomes, including

[0009] 5. Below: a) lipid bilayers containing fusogens; b) the following: (i) a payload gene encoding an exogenous agent, e.g., a payload gene encoding an exogenous agent of Table 5; (ii) a negative target cell-specific regulatory element (negative TCSRE) that is a non-T cell-specific regulatory element operably linked to the payload gene (e.g., a tissue-specific miRNA recognition sequence), a negative target cell-specific negative TCSRE operably linked to a payload gene that reduces expression of an exogenous agent in non-target cells or tissues compared to an otherwise similar nucleic acid lacking the negative TCSRE A fusosome comprising a nucleic acid comprising regulatory elements.

[0010] 6. 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, wherein the positive target cell-specific regulatory element is a positive target cell-specific increasing expression of payload genes in target cells compared to fusosomes that are similar but lacking regulatory elements, wherein target cells are T cells of a first type and optionally non-target cells are a second, different The fusosome of any of embodiments 4 or 5, wherein the fusosomes are T cells or non-T cells of any type, optionally the target cells are Treg cells and the non-target cells are conventional CD4+ T cells.

[0011] 7. Below: a) a lipid bilayer comprising fusogens, and b) a nucleic acid comprising a payload gene encoding an exogenous agent, such as a payload gene encoding an exogenous agent of Table 5; c) one or both of the following: (i) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer; or (ii) reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%) compared to fusosomes produced from absent or otherwise similar unmodified source cells; %, 70%, 80%, or down to 90%) of a first immunostimulatory protein.

[0012] 8. The fusosome of any of the preceding embodiments, which is one or more of: i) a higher percentage of fusosomes in target cells than in non-target cells, 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 100x fusion; ii) the fusosome is in a higher percentage of target cells than another fusosome, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, twice , 3x, 4x, 5x, 10x, 20x, 50x, or 100x fusion; iii) the fusosomes are at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the target cells after 24, 48, or 72 hours; or fusing with target cells at a rate such that up to 90% delivery; iv) a higher percentage of fusosomes in target cells than in non-target cells, e.g. at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold delivery of nucleic acids, such as retroviral nucleic acids; v) the fusosome has a higher percentage in the target cell than another fusosome, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, twice , 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold deliver nucleic acids, such as retroviral nucleic acids; or vi) the agent 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 Fusosomes deliver nucleic acids, eg, retroviral nucleic acids, to target cells at such a rate.

[0013] 9. One or more (e.g., two or all three) of: 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, The fusosome of any of the preceding embodiments, wherein is applied.

[0014] 10. The nucleic acid is operable to 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), payload gene The central polypurine tube (cPPT) promoter linked to the , payload gene (optionally including an intron before the open reading frame), polyA tail sequence, WPRE, and 3' LTR (e.g., U5 and functional U3). lacking), the fusosome of any of the preceding embodiments.

[0015] 11. Polymerase (e.g., reverse transcriptase, e.g., pol or portion thereof), integrase (e.g., pol or portion thereof, e.g., functional or non-functional variant), matrix protein (e.g., gag or portion thereof) moieties), capsid proteins (e.g. gag or portions thereof), nucleocapsid proteins (e.g. gag or portions thereof), and proteases (e.g. pro). Any form of fusosome.

[0016] 12. The fusosome of embodiment 7, comprising (i) and (ii).

[0017] 13. The fusosome of any of embodiments 7-12, further comprising a second exogenous or overexpressed immunosuppressive protein on the lipid bilayer.

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

[0019] 15. The nucleic acid, e.g., retroviral vector, further comprises a positive target cell-specific regulatory element (e.g., target cell-specific promoter) operably linked to the payload gene, wherein the positive target cell-specific regulatory element is 15. The fusosome of any of embodiments 7-14, which increases expression of a payload gene in a target cell compared to an otherwise similar fusosome lacking a positive target cell-specific regulatory element, and wherein the target cell is a T cell. .

[0020] 16. The nucleic acid, e.g., retroviral nucleic acid, comprises a non-target cell-specific regulatory element (NTCRE) (e.g., non-target cell-specific miRNA recognition sequence) operably linked to a payload gene, wherein NTCSRE comprises reducing expression of a payload gene in non-target cells or tissues compared to but lacking similar fusosomes, optionally wherein the target cells are T cells of a first type and the non-target cells are a second, different type optionally, the target cells are Treg cells and the non-target cells are conventional CD4+ T cells.

[0021] 17. The nucleic acid, e.g., 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, wherein the negative TCSRE is 16. The fusosome of any of embodiments 7-15, which reduces expression of an exogenous agent in non-target cells or tissues compared to an otherwise similar nucleic acid lacking a negative TCSRE, eg, a retroviral nucleic acid.

[0022] 18. The fusosome of any of embodiments 7-17, which when administered to a subject (eg, a human subject or a mouse) is one or more of: i) the fusosomes do not produce a detectable antibody response (e.g. after a single dose or multiple doses) or, e.g., according to a FACS antibody detection assay, e.g. the assays of Example 13 or Example 14, Antibodies to fusosomes are present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of background levels; ii) the fusosomes do not produce a detectable cellular immune response (e.g. T cell response, NK cell response, or macrophage response), or the cellular immune response to fusosomes does not, e.g. 5), NK cell lysis assay (e.g., the assay of Example 6), CD8 killer T cell lysis assay (e.g., the assay of Example 7), or macrophage phagocytosis assay (e.g., the assay of Example 8). a cellular response to fusosomes is present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of background levels; iii) the fusosomes do not produce a detectable innate immune response, e.g. complement activation (e.g. after a single dose or multiple doses) or e.g. ), an innate immune response to fusosomes is present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of background levels; iv) less than 10%, 5%, 4%, 3%, 2% or 1% of fusosomes are inactivated by serum, e.g. according to a serum inactivation assay, e.g. to; v) target cells that have received the exogenous agent from the fusosomes do not produce a detectable antibody response (e.g. after single or multiple doses) or e.g. FACS antibody detection assay, e.g. Example 15 antibodies to target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of background levels, according to the assay of vi) target cells that have received the exogenous agent from the fusosome do not produce a detectable cellular immune response (e.g., a T cell response, NK cell response, or macrophage response), or the cellular response to the target cell is For example, a macrophage phagocytosis 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. Cellular responses to target cells are present at levels less than 10%, 5%, 4%, 3%, 2%, or greater than 1% of background levels (eg, the assay of Example 19).

[0023] 19. The fusosome of embodiment 18, wherein the background level is a comparable level in the same subject prior to administration of the fusosome.

[0024] 20. The fusosome of any of embodiments 7-19, wherein the immunosuppressive protein (eg, the first immunosuppressive protein or the second immunosuppressive protein) is a complement regulatory protein or CD47.

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

[0026] 22. The exogenous agent is 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, carbonic anhydrase, food-based antigens, thyroid stimulating hormone receptor (TSHR), thyroid peroxidase, thyroglobulin, TSH receptor, alpha enolase, alpha b-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, emboplakin, periplakin, alpha-2 -macroglobulin-like-1, citrullinated peptides, non-self antigens, dsDNA, nucleosomal histones, telomeres, Sm core protein, hsp60, 437-460(p277), PPIns / Pins, anti-insulin antigens, glutamate decarboxylase (GAD), Any of the preceding embodiments, which is a binding moiety (e.g., a CAR molecule) that binds an antigen selected from carboxypeptidase H, insulinoma antigen-2, IA-2β, or an antigen in the cytoplasm of neutrophil granulocytes. fusosomes.

[0027] 23. The fusosome of any of the preceding embodiments, wherein the fusogen comprises VSV-G.

[0028] 24. The positive target cell-specific regulatory element is a T-cell-specific promoter, a T-cell-specific enhancer, a T-cell-specific splice site, a T-cell-specific site that extends RNA or protein half-life, a T-cell-specific The fusosome of embodiment 1, 2, 6, 15, 22, or 23, comprising an mRNA nuclear export-enhancing site, a T cell-specific translational enhancement site, or a T cell-specific post-translational modification site.

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

[0030] 26. The fusosome of embodiment 25, wherein the T cell-specific promoter comprises the motifs of Table 3.

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

[0032] 28. Negative TCSRE or NTCSRE is a non-target cell-specific miRNA recognition sequence, non-target cell-specific protease recognition site, non-target cell-specific ubiquitin ligase site, non-target cell-specific transcription repression site, or non-target cell-specific The fusosome of any of embodiments 4-6, or 16-21, comprising a target epigenetic suppression site.

[0033] 29. Embodiments wherein the negative TCSRE or NTCSRE comprises a tissue-specific miRNA recognition sequence, tissue-specific protease recognition site, tissue-specific ubiquitin ligase site, tissue-specific transcriptional repression site, or tissue-specific epigenetic repression site Fusosomes of either 4-6, or 16-21, or 28.

[0034] 30. Negative TCSRE or NTCSRE is a non-target cell-specific miRNA recognition sequence, non-target cell-specific protease recognition site, non-target cell-specific ubiquitin ligase site, non-target cell-specific transcription repression site, or non-target cell-specific 30. The fusosome of any of embodiments 4-6, 16-21, 28, or 29, comprising a selective epigenetic suppression site.

[0035] 31. A practice wherein the negative TCSRE or NTCSRE comprises a non-target cell-specific miRNA recognition sequence bound by the miRNAs of Table 4, e.g., by one or more (e.g., two or more) miR31, miR363, or miR29c Fusosomes of either form 4-6, 16-21, or 28-30.

[0036] 32. Negative TCSRE, or NTCSRE, within a transcribed region (e.g., a transcribed region encoding an exogenous agent), such that the RNA produced by the transcribed region contains miRNA recognition sequences within UTRs or coding regions. 32. The fusosome of any of embodiments 28-31, wherein the fusosome is located in or encoded at.

[0037] 33. The fusosome of any of the preceding embodiments, wherein the nucleic acid, eg retroviral nucleic acid, comprises one or more insulator sequences.

[0038] 34. The nucleic acid, e.g., retroviral nucleic acid, comprises two insulator sequences, e.g., a first insulator sequence upstream of the payload gene and a second insulator sequence downstream of the payload gene, e.g. 34. The fusosome of embodiment 33, wherein the insulator sequence and the second insulator sequence comprise the same or different sequences.

[0039] 35. The fusosome of any of the preceding embodiments, which is not genotoxic or does not increase the rate of tumorigenesis in target cells.

[0040] 36. The fusosome of any of the preceding embodiments, wherein the nucleic acid, eg retroviral nucleic acid, is capable of integrating into the genome of the target cell.

[0041] 37. The fusosome of embodiment 36, wherein the nucleic acid, eg, retroviral nucleic acid, is an integration-competent or non-integration-competent lentivirus.

[0042] 38. Target cells are 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) cells), regulatory T cells (e.g., thymus-derived regulatory T cells, peripheral-derived 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 The fusosome of any of the preceding embodiments, which is selected from memory T cells (or effector memory T cells), NKT cells, or mucosa-associated invariant T (MAIT) cells.

[0043] 39. The fusosome of any of embodiments 4-6 and 9-38, which is one or more of: i) less than 10%, 5%, 4%, 3%, 2%, or 1% of the exogenous agent detectably present in the subject is in non-target cells; ii) at least 90%, 95%, 96%, 97%, 98%, or 99% of the subject's cells detectably contain the exogenous agent are target cells (e.g., cells of a single cell type); is; iii) less than 1,000,000, 500,000, 200,000, 100,000, 50,000, 20,000, or 10,000 of the subject's cells that detectably contain the exogenous agent are non-target cells; iv) the mean level of the exogenous agent in all target cells of the subject is at least 100-fold, 200-fold, 500-fold, or 1,000-fold higher than the mean level of the exogenous agent in all non-target cells of the subject; is; or v) the exogenous agent is not detected in non-target cells of the subject;

[0044] 40. The fusosome of any of the preceding embodiments, wherein the nucleic acid, eg retroviral nucleic acid, encodes a positive TCSRE and / or a NTCSRE or a negative TCSRE.

[0045] 41. The fusosome of any of the preceding embodiments, wherein the nucleic acid, eg, retroviral nucleic acid, comprises a positive TCSRE and / or a complement of a NTCSRE or a negative TCSRE.

[0046] 42. positive TCSRE is at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500% in target cells over non-target cells; 42. The fusosome of any of embodiments 40 or 41, comprising a target cell-specific promoter with an activity of 750%, 1000% or more.

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

[0048] 44. 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 The fusosome of any of the preceding embodiments, which does not deliver nucleic acid, eg, retroviral nucleic acid, to dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0049] 45. For example, using quantitative PCR, e.g., using the assay of Example 1, non-target cell types (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, splenocytes, B cells, hepatocytes, endothelial cells, or one of non-cancerous cells less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% of the one or more) contain nucleic acids, e.g., retroviral nucleic acids, The fusosome of any of the preceding embodiments.

[0050] 46. ​​Target cells have 0.00001-10, 0.0001-10, 0.001-10, 0.01-10, 0.1-10, 0.5-5, 1-4, 1-3, or 1-10 per host genome The fusosome of any of the preceding embodiments, comprising two copies of a nucleic acid, such as a retroviral nucleic acid or a portion thereof, wherein, for example, the copy number of the nucleic acid, such as the retroviral nucleic acid, is assessed after administration in vivo. .

[0051] 47. The fusosome of any of the preceding embodiments, wherein: 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) cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or noncancerous cells) less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01% contains an exogenous agent; or An exogenous agent (e.g., a protein) is isolated from 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, Any of the preceding embodiments that are not detectably present in myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells. fusosomes.

[0052] 48. Fusosomes target nucleic acids, e.g., retroviral nucleic acids, to 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 sexual T cells (e.g., CD8+ cytotoxic T cells), regulatory T cells (e.g., thymus-derived regulatory T cells, peripheral-derived 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 The fusosome of any of the preceding embodiments, wherein the fusosome is delivered to T cells (eg, 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. At least 0.00001%, 0.0001%, 0.001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 5%, e.g., using quantitative PCR, e.g., using the assay of Example 3 %, 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, peripherally-derived 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 ) the fusosome of any of the preceding embodiments, wherein one or more of the cells) comprises a nucleic acid, eg, a 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+ Foxp3+ regulatory T cells, or CD4+ FoxP3-type 1 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., The fusosome of any of the preceding embodiments, wherein the stem cell memory T cell, central memory T cell, or effector memory T cell), NKT cell, or mucosa-associated invariant T (MAIT) cell) comprises an exogenous agent.

[0055] 51. At the time of administration, the ratio of target cells containing nucleic acid, e.g. retroviral nucleic acid, to non-target cells containing nucleic acid, e.g. and at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000 using the assay of Example 3.

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

[0057] 53. The ratio of the median copy number of a nucleic acid, e.g., retroviral nucleic acid or portion thereof, in target cells to the median copy number of a nucleic acid, e.g., retroviral nucleic acid, or portion thereof, in non-target cells is, for example, According to a quantitative PCR assay, for example using the assays of Examples 1 and 3, at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000 The fusosome of any of the preceding embodiments, which is

[0058] 54. The ratio of target cells containing the exogenous RNA agent to non-target cells containing the exogenous RNA agent is at least 1.5, 2, 3, 4, 5, 10, e.g., by reverse transcription quantitative PCR assay , 25, 50, 100, 500, 1000, 5000, 10,000.

[0059] 55. The ratio of the mean exogenous RNA agent level in target cells to the mean exogenous RNA agent level in non-target cells is at least 1.5, 2, 3, 4, 5, 10 by reverse transcription quantitative PCR assay , 25, 50, 100, 500, 1000, 5000, 10,000.

[0060] 56. The ratio of the median exogenous RNA agent level in target cells to the median median exogenous RNA agent level in non-target cells is at least 1.5, 2, 3, 3, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5 than 5 any than than any other median exogenous RNA agent level in any non-target cell than any other median exogenous RNA agent level in target cells, by reverse transcription quantitative PCR assay. The fusosome of any of the preceding embodiments, which is 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000.

[0061] 57. The ratio of target cells containing the exogenous protein agent to non-target cells containing the exogenous protein agent is, for example, by a FACS assay, for example using the assays of Examples 2 and 4, The fusosome of any of the preceding embodiments, which is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000.

[0062] 58. The ratio of the mean exogenous protein agent level of target cells to the mean exogenous protein agent level of non-target cells is determined, e.g., by a FACS assay, e.g., using the assays of Examples 2 and 4. is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000.

[0063] 59. The ratio of the median exogenous protein agent level of target cells to the median exogenous protein agent level of non-target cells is, for example, by FACS assay, e.g., of Examples 2 and 4. The fusosomes of any of the preceding embodiments, which are at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000 using the assay.

[0064] 60. The fusosome of any of the preceding embodiments comprising one or both of: i) exogenous or overexpressed immunosuppressive proteins on the lipid bilayer, such as envelopes; and (ii) absent or reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%) compared to fusosomes produced from otherwise similar unmodified source cells; , 60%, 70%, 80%, or down to 90%).

[0065] 61. The fusosome of any of the preceding embodiments, comprising one or more of: i) a first exogenous or overexpressed immunosuppressive protein on a lipid bilayer, e.g. envelope, and a second exogenous or overexpressed immunosuppressive protein on a lipid bilayer, e.g. envelope; ii) a lipid bilayer, e.g., the first exogenous or overexpressed immunosuppressive protein on the envelope and absent compared to fusosomes produced from otherwise similar unmodified source cells; or a second immunostimulatory protein present at a reduced level (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%); or iii) absent or reduced levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or down to 90%) of the first immunostimulatory protein and compared to fusosomes produced from otherwise similar unmodified source cells. a second immunostimulatory protein that is absent or present at a reduced level (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) .

[0066] 62. The fusosome of any of the preceding embodiments, wherein the fusosomes have been circulating for at least 0.5, 1, 2, 3, 4, 6, 12, 18, 24, 36, or 48 hours after administration to the subject. .

[0067] 63. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulated for 30 minutes.

[0068] 64. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 1 hour.

[0069] 65. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 2 hours.

[0070] 66. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 4 hours.

[0071] 67. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 8 hours.

[0072] 68. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 12 hours.

[0073] 69. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 18 hours.

[0074] 70. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which is in circulation for 24 hours.

[0075] 71. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 36 hours.

[0076] 72. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes after administration The fusosome of any of the preceding embodiments, which has been circulating for 48 hours.

[0077] 73. Body fluids after one or more administrations of fusosomes to a suitable animal model, e.g., an animal model described herein, compared to a reference fusosome, e.g., an unmodified fusosome otherwise similar to the fusosome The fusosome of any of the preceding embodiments, having reduced immunogenicity as measured by reduced sexual response.

[0078] 74. The fusosome of embodiment 73, wherein the reduced humoral response is measured in serum samples by anti-cellular antibody titers, eg, anti-retroviral antibody titers, eg, by ELISA.

[0079] 75. Serum samples from animals administered fusosomes were 1%, 5%, 10%, 20%, 30%, 40%, compared to serum samples from subjects administered unmodified cells, The fusosome of any of the preceding embodiments, having a 50%, 60%, 70%, 80%, 90% or more reduction in anti-fusosomal antibody titer.

[0080] 76. A serum sample from a subject administered fusosomes has increased, e.g., 1%, 2%, 5%, 10%, 20%, 30%, or 40% increased anti-cellular antibody titers from baseline A fusosome according to any of the preceding embodiments, wherein baseline refers to the same subject's serum sample prior to administration of the fusosome.

[0081] 77. The fusosome of any of the preceding embodiments, wherein: Subjects to whom fusosomes or pharmaceutical compositions comprising fusosomes are administered have or are known to have or have been tested for pre-existing antibodies (e.g., IgG or IgM) reactive with fusosomes ; the subject receiving the fusosomes does not have detectable levels of pre-existing antibodies reactive with the fusosomes; the subject receiving fusosomes or a pharmaceutical composition comprising fusosomes has or is known to have or has been tested for antibodies reactive with fusosomes (e.g., IgG or IgM); A subject administered fusosomes or a pharmaceutical composition comprising fusosomes (e.g., at least one, two, three, four, five, or more times) has detectable levels of antibodies reactive with fusosomes does not have; or Antibody levels did not rise by more than 1%, 2%, 5%, 10%, 20%, or 50% between the two time points, the first time point being prior to the first administration of fusosomes and a second time point after one or more doses of fusosomes.

[0082] 78. The fusosome of any of the preceding embodiments, wherein the fusosomes are produced by the method of Example 5, 6, or 7, eg, from cells transfected with HLA-G or HLA-E cDNA.

[0083] 79. Fusosomes generated from NMC-HLA-G cells are lysed at specific time points compared to fusosomes generated from NMC or NMC-empty vector, e.g., PBMC-mediated lysis, NK cell-mediated lysis, and / or the fusosome of any of the preceding embodiments, wherein the rate of CD8+ T cell-mediated lysis is decreased.

[0084] 80. The fusosome of any of the preceding embodiments, wherein the modified fusosome avoids phagocytosis by macrophages.

[0085] 81. The fusosome of any of the preceding embodiments, wherein the fusosome is produced by the method of Example 8, eg, from cells transfected with CD47 cDNA.

[0086] 82. The fusosome of any of the preceding embodiments, wherein the phagocytic index is reduced when macrophages are incubated with retroviral vectors derived from NMC-CD47 compared to NMC-derived vectors or NMC-empty vectors. .

[0087] 83. In macrophage phagocytosis, e.g., 1%, 5%, 10%, 20%, 30%, 40%, 50%, compared to reference fusosomes, e.g., unmodified fusosomes otherwise similar to fusosomes Having a reduction in macrophage phagocytosis of 60%, 70%, 80%, 90% or more, wherein the reduction in macrophage phagocytosis is determined by assaying the in vitro phagocytosis index, e.g., as described in Example 8 The fusosome of any of the preceding embodiments, wherein the fusosome is determined by

[0088] 84. 0, 1, 10, 100, or more phagocytosis when the fusosomal composition is incubated with macrophages in an in vitro assay of macrophage phagocytosis, e.g., as measured by the assay of Example 8 The fusosome of any of the preceding embodiments, having an index.

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

[0090] 86. The fusosome of any of the preceding embodiments, eg, produced by the method of Example 9 from cells transfected with a cDNA encoding a complement regulatory protein, eg, DAF.

[0091] A dose of fusosomes presenting 87.200 pg / ml C3a was higher in the corresponding mouse serum (e.g., HEK293 mouse serum) than in the reference fusosomes (e.g., HEK293 retroviral vector) incubated with the corresponding mouse serum (e.g., HEK293 mouse serum) The fusosomes of any of the preceding embodiments, often modified fusosomes (eg, HEK293-DAF) incubated with HEK-293 DAF mouse serum).

[0092] A dose of 88.200 pg / ml of C3a present fusosomes produced more modified fusosomes (e.g., HEK293-DAF) incubated with naive mouse serum than reference fusosomes (e.g., HEK293 retroviral vector) incubated with naive mouse serum. A fusosome according to any of the preceding embodiments, which is often the case.

[0093] 89. The fusosome of any of the preceding embodiments, wherein the fusosome is resistant to complement-mediated inactivation in patient serum 30 minutes after administration, according to the assay of Example 9.

[0094] 90. At least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of fusosomes complement The fusosome of any of the preceding embodiments, which is resistant to mediated inactivation.

[0095] 91. The complement regulatory protein is a protein that binds decay accelerating factors (DAF, CD55) such as factor H (FH)-like protein-1 (FHL-1) such as C4b binding protein (C4BP) such as complement receptor body 1 (CD35), e.g., membrane cofactor proteins (MCP, CD46), e.g., protectin (CD59), e.g., proteins that inhibit classical and alternative complement pathway CD / C5 convertase enzymes, e.g., proteins that regulate MAC assembly The fusosome of any of embodiments 86-90, comprising one or more of:

[0096] 92. Retroviral vectors produced, for example, by the methods of Example 10 from cells transfected with DNA encoding shRNAs targeting MHC class I, e.g. The fusosome of any of the preceding embodiments, wherein MHC class I expression is reduced compared to a vector control.

[0097] 93. A measure of immunogenicity for fusosomes is serum inactivation, e.g., serum inactivation measured as described herein, e.g., as described in Example 11. The fusosome of any of the embodiments.

[0098] 94. The fusosome of any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosomal samples incubated with serum from fusosomal-naive mice and heat-inactivated serum.

[0099] 95. The fusosomes of any of the embodiments, wherein the percentage of cells that receive the exogenous agent does not differ between fusosome samples incubated with serum from fusosome-naive mice and serum-free control incubations.

[0100] 96. Any of the preceding embodiments, wherein the percentage of cells that receive the exogenous agent is less in the fusosome samples incubated with the positive control serum than in the fusosome samples incubated with serum from fusosomal naïve mice. fusosomes.

[0101] 97. Modified fusosomes, e.g., those modified by the methods described herein, are serum-free following administration of the modified fusosomes multiple times (e.g., more than once, e.g., two or more times). The fusosome of any of the preceding embodiments, having reduced activation (eg, reduced compared to administration of unmodified fusosomes).

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

[0103] 99. A measure of immunogenicity of a fusosome, e.g., serum inactivation after multiple doses, e.g., after multiple doses measured as described herein, e.g., as described in Example 12 The fusosome of any of the preceding embodiments, which is serum-inactivated.

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

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

[0106] 102. The percentage of cells receiving an exogenous agent differs between fusosomal samples incubated with serum from vehicle-treated mice and from mice treated with modified (e.g., HEK293-HLA-G) fusosomes. The fusosome of any of the preceding embodiments.

[0107] 103. Any of the preceding embodiments, wherein the percentage of cells receiving the exogenous agent is less for fusosomes derived from the reference cell (eg, HEK293) than for modified (eg, HEK293-HLA-G) fusosomes. The fusosome.

[0108] 104. The fusosome of any of the preceding embodiments, wherein the measure of immunogenicity to the fusosome is an antibody response.

[0109] 105. A pre-existing antibody that binds and recognizes fusosomes that subjects receiving the fusosomes described herein are measured as described herein, e.g., as described in Example 13 The fusosome of any of the preceding embodiments, comprising:

[0110] 106. Sera from fusosomal naive mice show more signal (e.g., fluorescence) than sera from negative controls, e.g., mice depleted of IgM and IgG, e.g., indicating that immunogenicity has occurred. The fusosome of any of the preceding embodiments.

[0111] 107. According to the preceding embodiment, sera from fusosomally naïve mice show similar signals (e.g., fluorescence) compared to negative controls, e.g., indicating that immunogenicity did not detectably occur. Any fusosome.

[0112] 108. The fusosome of any of the preceding embodiments, wherein the fusosome is a modified fusosome, e.g., modified by a method described herein, e.g., as described in Example 14, e.g. Reduced humoral response (e.g., unmodified fusosomes) after multiple (e.g., more than one, e.g., two or more) administrations of modified fusosomes, as measured as described herein ), fusosomes.

[0113] 109. The fusosome of any of the preceding embodiments, wherein the fusosomes are produced by the method of Example 5, 6, 7, or 14, eg, from cells transfected with HLA-G or HLA-E cDNA.

[0114] 110. The fusosome of any of the preceding embodiments, wherein the humoral response is assessed by determining the level of anti-fusosomal antibodies (eg, IgM, IgG1, and / or IgG2 antibodies).

[0115] 111. Modified (e.g., NMC-HLA-G) fusosomes show higher antiviral IgM or IgG1 / 2 antibody titers (e.g., by FACS) after injection compared to controls, e.g., NMC fusosomes or NMC-empty fusosomes The fusosome of any of the preceding embodiments, wherein the fusosome (as measured by fluorescence intensity) is reduced.

[0116] 112. Recipient cells are not targeted by an antibody response or have an antibody response below a reference level, e.g., when measured, e.g., as described herein, e.g., as described in Example 15 The fusosome of any of the preceding embodiments, which is

[0117] 113. The fusosome of any of the preceding embodiments, wherein the signal (eg, mean fluorescence intensity) is similar in recipient cells from fusosome-treated and PBS-treated mice.

[0118] 114. The fusosome of any of the preceding embodiments, wherein the measure of recipient cell immunogenicity is a macrophage response.

[0119] 115. The fusosome of any of the preceding embodiments, wherein the recipient cell is not targeted by macrophages or is targeted below a reference level.

[0120] 116. The phagocytic index, e.g., measured as described herein, e.g., as described in Example 16, was determined in recipients from mice treated with fusosomes and mice treated with PBS. The fusosome of any of the preceding embodiments, analogous in a cell.

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

[0122] 118. The fusosome of any of the preceding embodiments, wherein the recipient cell does not elicit a PBMC response.

[0123] 119. Mice treated with fusosomes and mice treated with PBS when the percentage of CD3+ / CMG+ cells is measured, e.g., as described herein, e.g., as described in Example 17 The fusosome of any of the preceding embodiments, wherein the fusosome is similar in a recipient cell derived from

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

[0125] 121. The fusosome of any of the preceding embodiments, wherein the recipient cell does not elicit a natural killer cell response, or elicits a natural killer cell response that is, for example, lower than the reference value.

[0126] 122. Mice treated with fusosomes and mice treated with PBS when the percentage of CD3+ / CMG+ cells is measured, e.g., as described herein, e.g., as described in Example 18 The fusosome of any of the preceding embodiments, wherein the fusosome is similar in a recipient cell derived from

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

[0128] 124. The fusosome of any of the preceding embodiments, wherein the recipient cell does not elicit a CD8+ T cell response, or elicits a CD8+ T cell response that is, for example, lower than the reference value.

[0129] 125. Mice treated with fusosomes and mice treated with PBS when the percentage of CD3+ / CMG+ cells is measured, e.g., as described herein, e.g., as described in Example 19 The fusosome of any of the preceding embodiments, wherein the fusosome is similar in a recipient cell derived from

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

[0131] 127. The fusosome of any of the preceding embodiments, wherein (i) a positive target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous agent, or (ii) an exogenous agent A fusosome comprising 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 a nucleic acid, eg, a retroviral nucleic acid.

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

[0133] 129. A method of delivering an exogenous agent to a subject (e.g., a human subject), wherein the fusosome of any of embodiments 1-127 or the pharmaceutical composition of embodiment 128 is administered to the subject. , thereby delivering an exogenous agent to a subject.

[0134] 130. A method of modulating a function in a subject (e.g., a human subject), target tissue or target cell (e.g., T cell, e.g., Treg cell), wherein the subject, target tissue, or target cell is A method comprising contacting, eg, administering to, a fusosome of any of Forms 1-127, or a pharmaceutical composition of embodiment 128.

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

[0136] 132. A method of treating a disease or disorder in a subject (eg, a human subject) comprising administering the fusosomes of any of embodiments 1-127 or the pharmaceutical composition of embodiment 128 to the subject.

[0137] 133. The method of embodiment 132, wherein the disease or disorder is a disease or disorder of Table 5.

[0138] 134. The method of embodiment 132 or 133, wherein the disease or disorder is a disease or disorder treatable by a payload gene encoding an exogenous agent.

[0139] 135. Acute GvHD, alopecia areata, autoimmune uveitis, celiac disease, chronic GvHD, Crohn's disease, endometriosis, eosinophilic esophagitis, Graves' disease, Hashimoto's thyroiditis, multiple 135. The method of any of embodiments 132-134, selected from sclerosteosis, myasthenia gravis, pemphigoid, pemphigus, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis.

[0140] 136. A fusosome of any of embodiments 1-127 or a pharmaceutical composition of embodiment 128 for use in treating a subject (eg, a human subject) having a disease or disorder.

[0141] 137. The fusosome of any of embodiments 1-127 or the pharmaceutical composition of embodiment 128 for the manufacture of an agent for use in treating a subject (e.g., a human subject) with a disease or disorder. use of things.

[0142] 138. The fusosome or pharmaceutical composition for use of embodiment 137 or the use of embodiment 138, wherein the fusosome comprises a payload gene encoding an exogenous agent for treating a disease or disorder.

[0143] 139. Acute GvHD, alopecia areata, autoimmune uveitis, celiac disease, chronic GvHD, Crohn's disease, endometriosis, eosinophilic esophagitis, Graves' disease, Hashimoto's thyroiditis, multiple Fusosomes for use according to embodiment 136 or 138 selected from sclerosis, myasthenia gravis, pemphigoid, pemphigus, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis or a pharmaceutical composition, or the use of embodiment 137 or 138.

[0144] 140. A method of making the fusosomes of any of embodiments 1-127, comprising: a) providing cells containing nucleic acids, e.g. retroviral nucleic acids, and fusogens; b) culturing said cells under conditions that allow the production of fusosomes, and c) separating, enriching or purifying the fusosomes from said cells, thereby producing fusosomes.

[0145] Other features, objects, and advantages of the invention will become apparent from the description and drawings, and from the claims.

[0146] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, eg, in any table, are incorporated by reference. Unless otherwise indicated, sequence accession numbers specified herein, including tables herein, refer to database entries as of May 15, 2018. Where one gene or protein refers to more than one sequence accession number, all sequence variants are included. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0147] The following detailed description of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, specific embodiments described herein, exemplified herein, are described herein. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawing]

[0148]

Figure 1

Figure 2

[0149] The present disclosure provides, at least in part, fusosomal methods and compositions for in vivo delivery. In some embodiments, the fusosomes comprise a combination of elements that promote target cell specificity, such as one or more retargeted fusogens, positive target cell-specific regulatory elements, and non-target cell-specific regulatory elements. Contains regulatory elements. In some embodiments, the fusosomal composition comprises one or more modifications that reduce the immune response to fusosomes.

[0150] I. Definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[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 antigens characteristic of cancer, eg, tumor-associated antigens in neoplastic cells. In some embodiments, the antigen-binding domain binds to antigens characteristic of infectious diseases, eg, virus-associated antigens in virus-infected cells. In some embodiments, the antigen binding domain binds to an antigen characteristic of cells targeted by the subject's immune system in an autoimmune disease, eg, autoantigen. In some embodiments, the antigen binding domain is or comprises an antibody or antigen binding portion thereof. In some embodiments, the antigen binding domain is or comprises a scFv or Fab.

[0152] The terms "cancer", "malignant tumor", "neoplasm", "tumor" and "carcinoma" are used herein to refer to relatively abnormal, uncontrolled and / or autonomous growth. , as a result they are used to refer to cells that exhibit an abnormal growth phenotype characterized by a significant loss of control of cell growth. In some embodiments, a tumor may be or comprise cells that are pre-cancerous (eg, benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be of particular relevance. In some embodiments, the associated cancer may be characterized by a solid tumor. In some embodiments, the tumor may be a diffuse tumor or a liquid tumor. In some embodiments, the associated cancer may be characterized by a hematological neoplasia. In general, examples of different types of cancers known in the art include, for example, leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma and myeloproliferative disorders, sarcoma, melanoma, adenoma, solid tissue carcinoma, mouth cancer. Squamous cell carcinoma of the throat, larynx, and lung, liver cancer, urogenital cancer such as prostate, cervical, bladder, endometrial and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular Included are melanoma, endocrine cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancer, benign lesions such as benign lesion papilloma, and the like.

[0153] As used herein, "CDR" refers to the complementarity determining region, which can be located, for example, within an antibody variable region. Each heavy and light chain variable region has three CDRs, called CDR1, CDR2, and CDR3 for each variable region. A "set of CDRs" or "CDR set" refers to either a single variable region capable of binding antigen or cognate heavy and light chain variable regions of three or six that can bind antigen. refers to a group of CDRs. Certain systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.), and those skilled in the art understand and claim the differences between and within these systems. Able to understand CDR boundaries to the extent necessary to understand and practice the invention.

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

[0155] As used herein, "fusosome" refers to an amphipathic lipid bilayer surrounding a lumen or cavity, and a fusogen that interacts with the amphipathic lipid bilayer. In embodiments, the fusosomes comprise nucleic acids. In some embodiments, fusosomes are membrane-enclosed preparations. In some embodiments, the fusosomes are derived from the source cell.

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

[0157] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-enclosed lumens. In embodiments, the fusogen promotes membrane fusion. In other embodiments, the fusogen creates a connection, eg, a pore, between two lumens (eg, the lumen of the retroviral vector and the cytoplasm of the target cell). In some embodiments, a fusogen comprises a complex of two or more proteins, eg, neither protein has only fusion activity. In some embodiments, a fusogen comprises a targeting domain.

[0158] As used herein, an "insulator sequence" refers to a nucleotide sequence that blocks an enhancer or prevents diffusion of heterochromatin. Insulator sequences can be wild-type or mutant.

[0159] As used herein, the term "effective amount" refers to a pharmaceutical composition sufficient to significantly and positively modify the symptoms and / or condition being treated (e.g., provide a positive clinical response). means quantity. The effective amount of an active ingredient for use in pharmaceutical compositions will depend on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of the concomitant therapy, the particular active ingredient(s) used. , will vary according to the particular pharmaceutically acceptable excipient and / or carrier utilized as well as the knowledge and expertise of the attending physician as well.

[0160] An "exogenous agent" as used herein with respect to a virus, VLP or fusosome refers to an agent not contained in or encoded by the corresponding wild-type virus or fusogen made from the corresponding wild-type source cell. In some embodiments, exogenous agents are non-naturally occurring, such as proteins or nucleic acids with sequences that are altered (e.g., by insertions, deletions, or substitutions) as compared to the naturally occurring protein. . In some embodiments, exogenous agents are not naturally present in the source cell. In some embodiments, exogenous agents are naturally present in the source cell but are exogenous to the virus. In some embodiments, exogenous agents are not naturally present in recipient cells. In some embodiments, the exogenous agent is naturally present in the recipient cells, but not at the desired level or at the desired time. In some embodiments, exogenous agents comprise RNA or protein.

[0161] As used herein, the term "pharmaceutically acceptable" means a reasonable, within the scope of sound medical judgment, without undue toxicity, irritation, allergic reaction, or other problems or complications. Refers to excipients, compositions and / or dosage forms suitable for use in contact with human and animal tissue, commensurate with a reasonable benefit / risk ratio.

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

[0163] As used herein, a "positive target cell-specific regulatory element" (or positive TCSRE) refers to a nucleic acid sequence that increases the level of an exogenous agent in target cells compared to non-target cells. , where the nucleic acid encoding the exogenous agent is operably linked to the positive TCSRE. In some embodiments, a positive TCSRE is a functional nucleic acid sequence, eg, a positive TCSRE can include a promoter or enhancer. In some embodiments, the positive TCSRE encodes a functional RNA sequence, eg, the positive TCSRE may encode splice sites that promote correct splicing of RNA in target cells. In some embodiments, a positive TCSRE encodes a functional protein sequence, or a positive TCSRE can encode a protein sequence that facilitates correct post-translational modification of a protein. In some embodiments, a positive TCSRE decreases the level or activity of an exogenous agent downregulator or inhibitor.

[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 agent in non-target cells compared to target cells. , where the nucleic acid encoding the exogenous agent is operably linked to the negative TCSRE. In some embodiments, the negative TCSRE is a functional nucleic acid sequence, eg, a miRNA recognition site that causes retroviral nucleic acid degradation or inhibition 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 the mRNA encoding the exogenous protein agent, such that the mRNA is non-target Degraded or inhibited in cells. In some embodiments, a negative TCSRE increases the level or activity of an exogenous agent downregulator or inhibitor.

[0165] As used herein, "non-target cell-specific regulatory element" (or NTCSRE) refers to a nucleic acid sequence that reduces levels of an exogenous agent in non-target cells compared to target cells, wherein , a nucleic acid encoding an exogenous agent is operably linked to NTCRES. In some embodiments, NTCSRE is a functional nucleic acid sequence, eg, a miRNA recognition site that causes retroviral nucleic acid degradation or inhibition 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 the mRNA encoding the exogenous protein agent, such that the mRNA is non-target Degraded or inhibited in cells. In some embodiments, NTCSRE increases the level or activity of a downregulator or inhibitor of an exogenous agent. The terms "negative TCSRE" and "NTCRES" are used interchangeably herein.

[0166] As used herein, "non-T cell specific regulatory element" refers to a non-target cell specific regulatory element (NTCSRE), wherein the target cell is a T cell. Thus, a non-T cell-specific regulatory element refers to a nucleic acid sequence that reduces levels of an exogenous agent in non-T cells as compared to T cells, wherein the nucleic acid encoding the exogenous agent operably linked to a T-cell specific regulatory element.

[0167] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting moiety having a sequence that is not part of the naturally occurring form of fusogen. In embodiments, the fusogen comprises a different targeting moiety as 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 comprises a targeting moiety that is not present in the naturally occurring form of fusogen. In embodiments, the fusogen is modified to include a targeting moiety. In embodiments, the fusogen has one or more regions outside the targeting moiety, e.g., in the transmembrane domain, the fusogenically active domain, or the cytoplasmic domain, relative to the naturally occurring form of the fusogen. Contains sequence variations.

[0168] As used herein, a "retroviral nucleic acid", alone or in combination with a helper cell, helper virus, or helper plasmid, contains at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector. It refers to a nucleic acid containing In some embodiments, the retroviral nucleic acid further comprises or encodes an exogenous agent, 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 the 5′LTR (eg, to facilitate integration), U3 (eg, to activate viral genomic RNA transcription), R (eg, the Tat binding region), U5 , 3′LTR (eg, to facilitate integration), packaging sites (eg, psi(Ψ)), RRE (eg, to bind Rev and facilitate nuclear export). Retroviral nucleic acid can include RNA (eg, when part of a virion) or DNA (eg, when introduced into a source cell or after reverse transcription in a recipient cell). In some embodiments, the retroviral nucleic acid is packaged using a helper cell, helper virus, or helper plasmid comprising one or more (eg, all) of gag, pol, and env.

[0169] As used herein, "target cell" refers to the type of cell to which the fusosome (eg, lentiviral vector) is desired to deliver an exogenous agent. In embodiments, the target cells are cells of a particular tissue type or class, eg, T cells, eg, Treg cells. In some embodiments, target cells are diseased cells, eg, cancer cells. In some embodiments, a fusogen, e.g., a retargeted fusogen (alone or in combination with a positive TCSRE, NTCRE, negative TCSRE, or any combination thereof) is compared to non-target cells. lead to preferential delivery of exogenous agents to target cells.

[0170] As used herein, "non-target cell" refers to a type of cell to which the lentiviral vector is not desired to deliver exogenous agents. In some embodiments, non-target cells are cells of a particular tissue type or class. In some embodiments, non-target cells are non-disease cells, eg, non-cancerous cells. In some embodiments, a fusogen, e.g., a retargeted fusogen (alone or in combination with a positive TCSRE, NTCSRE, negative TCSRE, or any combination thereof) is compared to target cells , leading to lower delivery of exogenous agents to non-target cells.

[0171] As used herein, the terms "treat / treat", "treating / treating", or "treatment / treatment" refer to a disease or disorder, e.g. Refers to ameliorating one, eg, delaying, arresting, or reducing the onset of a disease or disorder.

[0172] As used herein, "cell biology" refers to a portion of a cell, including the lumen and cell membrane, or a cell that has partial or complete nuclear inactivation. In some embodiments, cell biology comprises one or more of cytoskeletal components, organelles, and ribosomes. In embodiments, the cell biological material is enucleated cells, microvesicles, or cell ghosts.

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

[0174] In some embodiments, the fusosome is or comprises a virus, eg, a retrovirus, eg, a lentivirus. For example, in some embodiments, the amphipathic lipid bilayer of the fusosomal is or comprises a viral envelope. The viral envelope may comprise a fusogen, eg, a fusogen that is endogenous to the virus or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosomal comprises viral nucleic acid, eg, retroviral nucleic acid, eg, lentiviral nucleic acid. A viral nucleic acid can be a viral genome. In some embodiments, the fusosome further comprises one or more viral nonstructural proteins, eg, in its cavity or lumen.

[0175] Fusosomes can have various properties that facilitate delivery of payloads to target cells. For example, in some embodiments, both the fusosome and the source cell contain sufficient nucleic acid(s) to create a particle that can fuse with the target cell. In embodiments, these nucleic acid(s) have one or more (eg, all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity. encodes a protein;

[0176] Fusosomes can also contain a variety of structures that facilitate delivery of payloads to target cells. For example, in some embodiments, both the fusosome and the source cell contain sufficient nucleic acid(s) to create a particle that can fuse with the target cell. In embodiments, these nucleic acid(s) have one or more (eg, all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity. encodes a protein;

[0177] Fusosomes can also contain a variety of structures that facilitate delivery of payloads to target cells. For example, in some embodiments, a fusosome (e.g., virus, e.g., retrovirus, e.g., lentivirus) contains the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or non- functional variants), proteases, and fusogens. 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 encoded in, for example, helper cells, helper viruses, or helper plasmids. provided in trans by In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) comprises the following nucleic acid sequences: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), payload A central polypurine tube (cPPT) promoter operably linked to the gene, a payload gene (optionally including an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3'LTR (e.g., U5 and functional lacking the typical U3), including one or more (eg, all) of In some embodiments, the fusosomal nucleic acid (eg, retroviral nucleic acid) further comprises one or more insulator sequences. In some embodiments, the fusosomal nucleic acid (eg, 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 polyA tail sequence, eg, between the polyA tail sequence and WPRE.

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

[0179] A. Fusosomes generated from viruses. For example, in some embodiments, a fusosome (e.g., virus, e.g., retrovirus, e.g., lentivirus) contains the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or non- functional variants), proteases, and fusogens. 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 encoded in, for example, helper cells, helper viruses, or helper plasmids. provided in trans by In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) comprises the following nucleic acid sequences: 5'LTR (e.g., containing U5 and lacking a functional U3 domain), Psi packaging element (Psi), payload A central polypurine tube (cPPT) promoter operably linked to the gene, a payload gene (optionally including an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3'LTR (e.g., U5 and functional lacking the typical U3), including one or more (eg, all) of In some embodiments, the fusosomal nucleic acid (eg, retroviral nucleic acid) further comprises one or more insulator sequences. In some embodiments, the fusosomal nucleic acid (eg, 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 polyA tail sequence, eg, between the polyA tail sequence and WPRE.

[0180] i) Lentiviral components and helper cells In some embodiments, the retroviral nucleic acid comprises one or more (eg, all) of the following: a 5′ promoter (eg, to control expression of the entire packaged RNA), a 5′ LTR (eg, R (containing polyadenylation tail signal) and / or U5) containing primer activation signal, primer binding site, psi packaging signal, RRE element for nuclear export, immediate transgene to control expression of the transgene. Upstream promoters, transgenes (or other exogenous agent elements), polypurine tracts, and 3'LTRs (including, for example, mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of cPPT, WPRE, and / or insulator sequences.

[0181] Retroviruses normally replicate by converting their genomic RNA into a linear double-stranded DNA copy by reverse transcription, followed by covalent integration of their genomic DNA into the host genome. Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV ), mouse mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend murine leukemia virus, mouse stem cell virus (MSCV) and Rous sarcoma virus (RSV)), and lentiviruses. be done.

[0182] In some embodiments, the retrovirus is a gammaretrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpharetrovirus. In some embodiments, the retrovirus is a betaretrovirus. In some embodiments, the retrovirus is a deltaretrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0183] Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV types 1 and 2); visna-maedi virus (VMV). Viruses; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV) immune deficiency virus); and simian immunodeficiency virus (SIV). In some embodiments, an HIV-based vector backbone (ie, HIV cis-acting sequence elements) is used.

[0184] In some embodiments, a vector herein is a nucleic acid molecule capable of importing or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked, eg, inserted, into a vector nucleic acid molecule. A vector may contain sequences directing autonomous replication within a cell, or may contain sequences sufficient to render it competent to integrate into host cell DNA. Useful vectors include plasmids (eg, 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 can include nucleic acid molecules (e.g., transfer plasmids) that, for example, typically contain virus-derived nucleic acid elements that facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or into a viral particle that mediates nucleic acid transfer. can. Viral particles usually contain various viral and sometimes host cell components in addition to the nucleic acid(s). A viral vector can, for example, comprise a virus or virus particle capable of transferring nucleic acid into a cell or capable of transferring the transferred nucleic acid (eg, as naked DNA). Viral vectors and transfer plasmids can contain structural and / or functional genetic elements primarily derived from viruses. Retroviral vectors can include viral vectors or plasmids that contain structural and functional genetic elements, or portions thereof, primarily derived from retroviruses. Lentiviral vectors can include viral vectors or plasmids that contain structural and functional genetic elements primarily derived from a lentivirus, or portions thereof, including LTRs.

[0186] In embodiments, a lentiviral vector (eg, a lentiviral expression vector) may comprise a lentiviral transfer plasmid (eg, as naked DNA) or an infectious lentiviral particle. Regarding elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., the sequences of these elements can be present in the form of RNA in the lentiviral particle and in the form of DNA in the DNA plasmid. It should be understood that

[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. be. This results in defective replication of the viral vector. In some embodiments, the vector is capable of transducing a target non-dividing host cell and / or integrating its genome into the host genome.

[0188] The structure of the wild-type retroviral genome often comprises a 5′ terminal repeat (LTR) and a 3′ LTR, between or within which packaging signals that allow packaging of the genome, primer binding sites, host There are gag, pol and env genes that encode integration sites to allow integration into the cellular genome, as well as packaging components that facilitate viral particle assembly. More complex retroviruses have additional functions, such as the rev and RRE sequences of HIV, that can efficiently export integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells. In the provirus, the viral gene is flanked at both ends by regions called long terminal repeats (LTRs). LTRs are involved in proviral integration and transcription. LTRs can also function as enhancer-promoter sequences and control the expression of viral genes. Encapsidation of retroviral RNA occurs by a psi sequence located at the 5' end of the viral genome.

[0189] The LTRs themselves are usually similar (eg, identical) sequences and can be divided into three elements called U3, R, and U5. U3 is derived from a unique sequence at the 3' end of RNA. R is derived from sequences repeated at both ends of the RNA, and U5 is derived from sequences unique to the 5' end of the RNA. The sizes of the three elements vary greatly among retroviruses.

[0190] For viral genomes, the transcription initiation site is usually at the U3-R boundary of one LTR, and the poly(a) addition (termination) site is at the R-U5 boundary of the other LTR. U3 contains most of the proviral transcriptional control elements, including a promoter and multiple enhancer sequences that respond to cellular and possibly viral transcriptional activation proteins. Some retroviruses contain any one or more of the following genes that encode proteins involved in the regulation of gene expression: tot, rev, tax and rex.

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

[0192] In replication-defective retroviral vector genomes gag, pol and env may be absent or non-functional. The R regions at both ends of RNA are usually repetitive sequences. U5 and U3 represent unique sequences 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 (in HIV) one or more of vif, vpr, vpx, vpu, tat, rev and nef. EIAV has (among other things) an additional gene, S2. Proteins encoded by additional genes serve a variety of functions, some of which may overlap those 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 stable stem-loop RNA secondary structures called TARs. Rev regulates and regulates viral gene expression through the Rev response element (RRE) (Martarano et al.1994 J.Virol.68:3102-11). The mechanism of action of these two proteins is believed to be largely the same as similar mechanisms in primate viruses. In addition, the EIAV protein Ttm, encoded by the first exon of tat spliced ​​into the env coding sequence at the start of the transmembrane protein, has been identified.

[0194] In addition to protease, reverse transcriptase and integrase, non-primate lentiviruses contain a fourth pol gene product encoding 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, the recombinant lentiviral vector (RLV), in the presence of packaging components, contains sufficient retroviral genetic material to allow packaging of the RNA genome into viral particles capable of infecting target cells. It is a vector with information. Infection of target cells may involve reverse transcription and integration into the target cell genome. RLVs usually carry non-viral coding sequences that are delivered to target cells by vectors. In embodiments, the RLV is incapable of independent replication to produce infectious retroviral particles within target cells. RLVs usually lack functional gag-pol and / or env genes and / or other genes involved in replication. Vectors can be constructed as split-intron vectors, for example, as described in PCT patent application WO 99 / 15683, which is hereby incorporated by reference in its entirety.

[0196] In some embodiments, the lentiviral vector comprises a minimal viral genome, e.g. Vectors are engineered to remove nonessential elements and retain essential elements to provide the functions necessary to infect, transduce, and deliver the nucleotide sequences of interest to target host cells. ing.

[0197] A minimal lentiviral genome can include, for example, (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vectors used to produce the lentiviral genome in the source cell also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the source cell. obtain. These regulatory sequences may include transcribed retroviral sequences, such as the native sequence associated with the 5'U3 region, or may comprise another viral promoter, such as a heterologous promoter such as the CMV promoter. Some lentiviral genomes contain additional sequences to facilitate efficient virus production. For example, for HIV, rev and RRE sequences may be included. Alternatively or in combination, codon optimization can be used, e.g. It can be codon optimized as described. Alternate arrangements can be used that perform similar or the same functions as the rev / RRE system. For example, functional analogues of the rev / RRE system are found in Mason Pfizer monkey virus. This is known as the CTE and contains RRE-type sequences within the genome that are thought to interact with factors of the infected cell. Cellular factors can be considered rev analogues. Therefore, CTE can be used as an alternative to the rev / RRE system. In addition, the HTLV-I Rex protein can functionally replace the HIV-I Rev protein. Rev and Rex have similar effects as IRE-BP.

[0198] In some embodiments, the retroviral nucleic acid (e.g., lentiviral nucleic acid, e.g., primate or non-primate lentiviral nucleic acid) comprises: (1) the deletion in gag is 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 5 'Contains leader sequence between end of LTR and ATG of gag; (4) is a combination of (1), (2), (3). In one embodiment, the lentiviral vector includes all of features (1) and (2) and (3). This strategy is described in further detail in WO 99 / 32646, which is incorporated herein by reference in its entirety.

[0199] In some embodiments, the primate lentiviral minimal system includes HIV / SIV additional genes vif, vpr, vpx, vpu, tat, for either vector production or transduction of dividing and non-dividing cells. Neither rev nor nef are required. 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 additional genes may allow the creation of vectors without genes associated with disease in lentiviral (such as HIV) infections. In particular, tat is associated with disease. Second, deletion of additional genes allows the vector to package more heterologous DNA. Third, genes of unknown function, such as S2, can be omitted to reduce the risk of unwanted effects. Examples of minimal lentiviral vectors are disclosed in WO99 / 32646 and WO98 / 17815.

[0201] In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (if it is an EIAV vector system), and possibly also vif, vpr, vpx, vpu and nef. In some embodiments, the retroviral nucleic acid also lacks rev, RRE, or both.

[0202] In some embodiments, the retroviral nucleic acid comprises 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 increases reverse transcription activity, the concentration of free dNTPs in the cytoplasm increases, facilitating reverse transcription and integration of the retroviral genome into the target cell genome.

[0203] Different cells have different usage of particular codons. This codon bias corresponds to the relative abundance bias of a particular tRNA in a cell type. Expression can be increased by altering codons within the sequence to align with the relative abundance of the corresponding tRNA. Similarly, it is possible to reduce expression by deliberately choosing codons for which the corresponding tRNA is known to be rare in a particular cell type. Additional translational control is therefore available. Additional discussion of codon optimization can be found, for example, in WO 99 / 41397, which is incorporated herein by reference in its entirety.

[0204] Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by altering these to correspond to commonly used mammalian codons, they can be used in mammalian producer cells. Expression of packaging components can be increased.

[0205] Codon optimization has many other advantages. Due to their sequence variations, the nucleotide sequences encoding the packaging components may have RNA instability sequences (INS) reduced or eliminated from them. At the same time, the amino acid sequences coding for the packaging components are maintained such that the viral components encoded by the sequences remain the same, or at least are sufficiently similar that the function of the packaging components is not compromised. . In some embodiments, codon optimization also overcomes the Rev / RRE requirement for export, making the optimized sequence Rev independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within the vector system (eg, 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 associated with INS are codon optimized. In other embodiments, the sequence is codon-optimized in its entirety, except for the sequence containing the gag-pol frameshift site.

[0207] The gag-pol gene contains two overlapping reading frames that encode gag-pol proteins. Expression of both proteins is dependent on frameshifting during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused, at least in part, by RNA secondary structures that stall the ribosome. Such secondary structures exist downstream of the frameshift site of the gag-pol gene. For HIV, the overlapping region extends downstream of the gag start from nucleotide 1222 (nucleotide 1 is the A of gag ATG) to the end of gag (nt 1503). As a result, the 281 bp fragment that spans the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. In some embodiments, retention of this fragment will allow more efficient expression of the gag-pol protein. For EIAV, the start of the duplication is at nt 1262 (nucleotide 1 is the A of gag ATG). The end of the duplication is at nt 1461. The wild-type sequence can be retained from nt 1156-1465 to ensure that the frameshift site and gag-pol duplication are maintained.

[0208] For example, derivations from optimal codon usage can be made and conservative amino acid changes can be introduced into the gag-pol protein to accommodate convenient restriction sites.

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

[0210] It will be appreciated that many gag-pol sequences can be achieved by those skilled in the art due to the deconstructive nature of the genetic code. Also, a number of retroviral variants have been described that can be used as a starting point for generating codon-optimized gag-pol sequences. Lentiviral genomes can vary considerably. For example, there are many pseudospecies of HIV-I that are still functioning. This also applies to 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 are in the NCBI database maintained by the National Institutes of Health.

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

[0212] As noted above, the packaging components of retroviral vectors can include the expression products of the gag, pol and env genes. In addition, packaging can utilize a short sequence of four stem loops followed by partial sequences from gag and env as packaging signals. Thus, a deleted gag sequence can be included in the retroviral vector genome (in addition to the complete gag sequence on the packaging construct). In embodiments, the retroviral vector is a package containing 255-360 nucleotides gag in the vector that still retains the env sequences, or about 40 nucleotides gag in certain combinations of splice donor mutations gag and env deletions. including ringing signals. In some embodiments, the retroviral vector comprises a gag sequence comprising one or more deletions, eg, the gag sequence comprises approximately 360 nucleotides from the N-terminus.

[0213] A retroviral vector, helper cell, helper virus, or helper plasmid may contain retroviral structural and accessory proteins, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef proteins, or other retroviral proteins. It may contain viral proteins. In some embodiments, the retroviral proteins are derived from the same retrovirus. In some embodiments, the retroviral proteins are derived from more than one retrovirus, eg, 2, 3, 4, or more retroviruses.

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

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

[0216] In various examples, the retroviral nucleic acid comprises (i) a 150-250 (e.g., 168) nucleotide portion of the gag protein comprising a mutated INS1 inhibitory sequence that reduces restriction of nuclear export of RNA compared to wild-type INS1; and (ii) contains a two nucleotide insertion that results in a frameshift and premature termination, and / or (iii) does not contain the INS2, INS3, and INS4 inhibitory sequences of gag.

[0217] In some embodiments, the vectors described herein are hybrid vectors that contain both retroviral (eg, lentiviral) and non-lentiviral viral sequences. In some embodiments, the hybrid vector comprises retroviral, eg, lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0218] According to certain embodiments, most or all of the viral vector backbone sequences are derived from a lentivirus, eg, HIV-1. However, many different sources of retroviral and / or lentiviral sequences may be used, or combined and multiple substitutions and alterations in a particular lentiviral sequence may function as described herein. can be adapted without compromising the ability of the transfer vector to carry out 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. have been developed, many of which can be adapted to produce retroviral nucleic acids.

[0219] Long terminal repeats (LTRs) are usually found at both ends of the provirus. LTRs usually include domains located at the ends of retroviral nucleic acids, which in the context of the native sequence are direct repeats and include the U3, R, and U5 regions. LTRs generally promote retroviral gene expression (eg, promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs can contain numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences for replication and integration of the viral genome. The viral LTR is usually divided into three regions called U3, R, and U5. The U3 region usually contains enhancer and promoter elements. The U5 region is usually the sequence between the primer binding site and the R region and may contain polyadenylation sequences. The R (repeat) region can flank the U3 and U5 regions. LTRs are typically composed of the U3, R and U5 regions and can occur at both the 5' and 3' ends of the viral genome. In some embodiments, the 5' LTR is flanked by 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 insertion of viral RNA into the viral capsid or particle, see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; see pp.2101-2109. Some retroviral vectors use a minimal packaging signal (psi[Ψ] sequence) for encapsidation of the viral genome.

[0221] In various embodiments, the retroviral nucleic acid comprises a modified 5'LTR and / or 3'LTR. Either or both LTRs may contain one or more modifications, including but not limited to one or more deletions, insertions, or substitutions. Modification of the 3'LTR can be accomplished by rendering the virus replication defective (e.g., a virus incapable of complete and efficient replication such that infectious virions are not produced (e.g., progeny of a replication-defective lentivirus), a lentivirus or Often done to improve the safety of retroviral systems.

[0222] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-defective vector, e.g., a retroviral or lentiviral vector, wherein the right (3') LTR enhancer, known as the U3 region, - The promoter region has been modified (eg, by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region can be used as a template for the left (5') LTR U3 region during viral replication, and viral replication is inhibited in the absence of the U3 enhancer promoter. In embodiments, the 3'LTR is modified such that the U5 region is removed, altered, or replaced with, for example, an exogenous poly(A) sequence. The 3'LTR, the 5'LTR, or both the 3' and 5' LTRs can 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 viral particle production. 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. viral (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter is capable of driving high levels of transcription in a Tat-independent manner. In certain embodiments, heterologous promoters have the added advantage of 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 in the presence of the inducer. Inducers include, but are not limited to, one or more compounds under which the host cells are cultured, or physiological conditions such as temperature or pH.

[0224] In some embodiments, the viral vector comprises a TAR (transactivation response) element, eg, located in the R region of a lentiviral (eg, HIV) LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required in embodiments where, for example, the U3 region of the 5'LTR is replaced by a heterologous promoter.

[0225] The R region, eg, the region within the retroviral LTR that begins at the start of the capping group (ie, the start of transcription) and ends just before the start of the polyA tract, can be flanked by 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 can also include FLAP elements, eg, nucleic acids whose sequences include the central polypurin tract and central termination sequences (cPPT and CTS) of a retrovirus, eg, HIV-1 or HIV-2. Suitable FLAP elements are described in US Pat. 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, the central initiation of the plus-stranded DNA at the central polypurine tract (cPPT) and the central termination at the central termination sequence (CTS) form a triple-stranded DNA structure, i.e., one HIV-1 central DNA. It can lead to the formation of flaps. In some embodiments, the retroviral or lentiviral vector backbone comprises one or more FLAP elements upstream or downstream of the gene encoding the exogenous agent. For example, in some embodiments, the transfer plasmid comprises a FLAP element, eg, a FLAP element derived from or isolated from HIV-1.

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

[0228] In some embodiments, expression of heterologous sequences in viral vectors is increased, for example, by incorporating one or more of post-transcriptional regulatory elements, polyadenylation sites, and transcription termination signals into the vector. Various post-transcriptional regulatory elements include, for example, the post-transcriptional regulatory element of woodchuck hepatitis virus (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); Proteins such as post-regulatory elements (Huang et al., Mol. Cell. Biol., 5:3864) (Liu et al., 1995, Genes Dev., 9:1766) can increase the expression of heterologous nucleic acids. and each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral nucleic acids described herein comprise 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 termination and polyadenylation of heterologous nucleic acid transcripts can be included, for example, to increase expression of exogenous agents. Transcription termination signals may be found downstream of the polyadenylation signal. In some embodiments, the vector includes a polyadenylation sequence 3' to the polynucleotide encoding the exogenous agent. Poly A sites may contain DNA sequences that direct both the termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. The polyadenylation sequence promotes mRNA stability by adding a polyA tail to the 3' end of the coding sequence and contributes to improved translation efficiency. Examples of poly A signals that can be used in retroviral nucleic acids include AATAAA, ATTAAA, AGTAAA, bovine growth hormone poly A sequence (BGHpA), rabbit beta-globin poly A sequence (rβgpA), or another suitable heterologous or endogenous poly A sequence. Contains the A sequence.

[0231] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator sequences, such as the insulator sequences described herein.

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

[0233] In some embodiments, the lentiviral nucleic acid comprises a promoter (eg, CMV), an R sequence (eg, including TAR), a U5 sequence (eg, for integration), a PBS sequence (eg, for reverse transcription), a DIS sequences (e.g. for genomic dimerization), psi packaging signals, partial gag sequences, RRE sequences (e.g. for nuclear export), cPPT sequences (e.g. for nuclear import), expression of exogenous agents , a gene encoding an exogenous agent, 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). for integration), and one or more of U5 signals (eg, for integration), all of which, eg, 5′ to 3′.

[0234] ii) vectors engineered to remove splice junctions Some lentiviral vectors have strong splicing and polyadenylation signals that integrate inside active genes and can lead to the formation of aberrant and possibly truncated transcripts.

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

[0236] In some embodiments, the lentiviral nucleic acids described herein comprise a lentiviral backbone with at least two of the splice sites removed, eg, to improve the safety profile of the lentiviral vector. Species of such splice sites and methods of identification are described in WO2012156839A2, all of which are incorporated by reference.

[0237] iii) Retrovirus production method Large-scale viral particle production is often useful to achieve a desired viral titer. Viral particles are transferred to a packaging cell line containing viral structural and / or accessory genes such as the gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes, or other retroviral genes. It can be produced by transfecting a vector.

[0238] In embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding 1, 2, 3, 4, or more viral structural and / or accessory genes. The packaging vector is typically contained in a packaging cell and introduced into the cell via transfection, transduction, or infection. A retroviral, eg, lentiviral, transfer vector can be introduced into the packaging cell line via transfection, transduction, or infection to generate the source cell or cell line. Packaging vectors can be introduced into human cells or cell lines by standard methods including, for example, calcium phosphate transfection, lipofection or electroporation. In some embodiments, the packaging vector is introduced into cells with a major selectable marker such as neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, DHFR, Gln synthase or ADA, followed by , selected in the presence of appropriate drugs to isolate clones. The selectable marker gene can be physically linked to genes encoded by, for example, IRES or self-cleaving viral peptides, by packaging vectors.

[0239] Packaging cell lines include cell lines that do not contain packaging signals, but stably or transiently express viral structural proteins and replication enzymes (eg, gag, pol and env) capable of packaging viral particles. . Any suitable cell line can be used, eg mammalian cells, eg human cells. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells. , BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0240] Source cell lines include cell lines capable of producing recombinant retroviral particles, including packaging cell lines and transfer vector constructs containing packaging signals. Methods for preparing virus stocks are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and N.R. Landau et al. (1992) J. Virol. and are incorporated herein by reference. Infectious viral particles can be harvested from packaging cells by, for example, cell lysis or collection of cell culture supernatants. If desired, the collected viral particles can be concentrated or purified.

[0241] iv) packaging of plasmids and cell lines In some embodiments, the source cell contains one or more plasmids encoding viral structural proteins and replication enzymes (eg, 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, the sequences encoding the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding the gag, pol, and env precursors have the same expression signal, eg, promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, eg, different promoters. In some embodiments, expression of gag, pol, and env precursors is inducible. In some embodiments, plasmids encoding viral structural proteins and replication enzymes are transfected at the same time or at different times. In some embodiments, plasmids encoding viral structural proteins and replication enzymes are transfected from the packaging vector at the same time or at different times.

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

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

[0244] In some embodiments, viral structural gene expression is regulated by a tetracycline (Tet)-dependent system, wherein a Tet-regulated transcriptional repressor (Tet-R) binds to a DNA sequence contained in the promoter, It represses transcription by steric hindrance (Yao et al, 1998; Jones et al, 2005). Addition of doxycycline (dox) releases Tet-R and allows transcription. A number of other suitable transcriptional regulatory promoters, transcription factors, and small molecule inducers are suitable for regulating transcription of viral structural genes.

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

[0246] In some embodiments, a nucleic acid encoding an exogenous agent (eg, a retroviral nucleic acid encoding an exogenous agent) is also integrated into the source cell genome. In some embodiments, the nucleic acid encoding the exogenous agent is maintained episomally. In some embodiments, nucleic acids encoding exogenous agents are transfected into source cells that have Rev, Gag / Pol, and Envelope proteins stably integrated into the genome. See, eg, Milani et al. EMBO Molecular Medicine, 2017, which is hereby incorporated by reference in its entirety.

[0247] In some embodiments, the retroviral nucleic acids described herein are incapable of undergoing reverse transcription. Such nucleic acids, in embodiments, can transiently express exogenous agents. A retrovirus or VLP may contain a disabled reverse transcriptase protein or may contain no reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a disabled primer binding site (PBS) and / or an att site. In embodiments, one or more viral accessory genes including rev, tat, vif, nef, vpr, vpu, vpx and S2 or functional equivalents thereof are disabled or absent from the retroviral nucleic acid. . In embodiments, one or more accessory genes selected from S2, rev and tat are disabled or absent from the retroviral nucleic acid.

[0248] v) Strategies for packaging retroviral nucleic acids Modern retroviral vector systems typically consist of a viral genome with cis-acting vector sequences for transcription, reverse transcription, integration, translation, and packaging of viral RNA into viral particles, and (2) the production of viral particles. consists of a producer cell line that expresses the trans-acting retroviral gene sequences (eg, gag, pol, and env) required for Due to the complete separation of cis- and trans-acting vector sequences, the virus is unable to sustain replication for more than one cycle of infection. Generation of live virus can be avoided by several strategies, such as minimizing overlap between cis- and trans-acting sequences to avoid recombination.

[0249] Viral vector particles lacking or containing sequences lacking viral RNA may result from the removal or exclusion of viral RNA from sequences. In one embodiment, this can be achieved by using the endogenous packaging signal binding site on gag. Alternatively, the endogenous packaging signal binding site is on pol. In this embodiment, the delivered RNA will contain the cognate packaging signal. In another embodiment, a heterologous binding domain (heterologous to gag) located on the delivered RNA and a cognate binding site located on gag or pol are used to package the delivered RNA. can be ensured. Heterologous sequences may be non-viral or viral, in which case they may be derived from a different virus. Vector particles can be used to deliver therapeutic RNA, in which case functional integrase and / or reverse transcriptase are not required. These vector particles can also be used to deliver therapeutic genes of interest, in which case they typically include pols.

[0250] In one embodiment, the gag-pol is modified to replace the packaging signal with the corresponding packaging signal. In this embodiment, the particles can package RNA with a new packaging signal. An advantage of this approach is the ability to package RNA sequences lacking viral sequences, eg RNAi.

[0251] Another approach is to rely on overexpression of packaged RNA. In one embodiment, the RNA to be packaged is overexpressed in the absence of RNA containing the packaging signal. This may result in the packaging of significant levels of therapeutic RNA, 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 a retroviral gag and pol protein, wherein the gag protein or pol protein recognizes the corresponding sequence in the RNA sequence to generate the viral vector. It contains a heterologous RNA binding domain that can facilitate packaging of RNA sequences into particles.

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

[0254] In some embodiments, the methods herein comprise detecting or confirming the absence of replication competent retroviruses. Although this method allows gene products to be expressed in specific cells infected with replication-competent retroviruses such as gammaretroviruses or lentiviruses, viral vectors used to transduce cells with heterologous nucleic acids Viral genes, such as structural or packaging genes, that are not present and / or not expressed, or not expected to be present and / or expressed, in cells that do not contain a replication-competent retrovirus. It may involve assessing RNA levels of one or more target genes. A replication competent retrovirus can 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 gene. See WO2018023094 A1 for detailed disclosure.

[0255] vi) suppression of the gene encoding the exogenous agent in the source cell Proteins (over)expressed in the source cell may indirectly or directly affect vector virion population and / or infectivity. Incorporation of exogenous agents into vector virions can also affect downstream processing of vector particles.

[0256] In some embodiments, tissue-specific promoters are used to restrict expression of exogenous agents in source cells. In some embodiments, heterologous translational control systems are used in eukaryotic cell culture to suppress translation of exogenous agents in source cells. More specifically, a retroviral nucleic acid can comprise a binding site operably linked to a gene encoding an exogenous agent, wherein the binding site is a binding site that permits translation of the exogenous agent to occur in the source cell. It can interact with an RNA binding protein in such a way as to be suppressed or prevented.

[0257] In some embodiments, the RNA binding protein is a tryptophan RNA binding attenuating protein (TRAP), eg, a bacterial tryptophan RNA binding attenuating protein. The use of RNA binding proteins (eg, bacterial trp operon regulator protein, tryptophan RNA binding attenuation protein, TRAP) and the RNA targets it binds repress or prevent translation of the transgene within the source cell. This system is called Transgene Repression In vector Production cell system or TRIP system.

[0258] In embodiments, placing the binding site for an RNA binding protein (e.g., TRAP binding sequence, tbs) upstream of the NOI translation initiation codon allows the internal expression cassette to be generated without adversely affecting vector RNA production or stability. specific suppression of translation of mRNAs derived from The number of nucleotides between the tbs and the translation start codon of the gene encoding the exogenous agent can vary from 0-12 nucleotides. tbs can be placed downstream of an internal ribosome entry site (IRES) to repress translation of genes encoding exogenous agents of multicistronic mRNAs.

[0259] vii) kill switch system and amplification In some embodiments, polynucleotides or cells harboring genes encoding exogenous agents utilize suicide genes, e.g., inducible suicide genes, to directly risk toxic and / or uncontrolled proliferation. Reduce. In certain embodiments, the suicide gene is not immunogenic to host cells harboring exogenous agents. Examples of suicide genes include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using specific chemical inducers of dimerization (CIDs).

[0260] In certain embodiments, the vectors comprise gene segments that render target cells, eg, immune effector cells, eg, T cells, susceptible to negative selection in vivo. For example, transduced cells can be eliminated as a result of a change in an individual's in vivo state. A negative selectable phenotype can result from the insertion of a gene that confers sensitivity to an administered agent, eg, 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); cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)). ).

[0261] In some embodiments, the transduced cells, e.g., immune effector cells such as T cells, comprise a polynucleotide that further comprises a positive marker that allows selection of cells with a negative selectable phenotype in vitro. . A positive selectable marker can be a gene that, when introduced into target cells, expresses a dominant phenotype that allows positive selection of cells that carry the gene. Genes of this type include, inter alia, the hygromycin-B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene from Tn5, which encodes resistance to the antibiotic G418, dihydrofolate reductase (DHFR). (neo or aph), the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0262] In some embodiments, a positive selectable marker and a negative selectable element are linked such that loss of the negative selectable element entails loss of the positive selectable marker as well. For example, positive and negative selectable markers can be fused such that loss of one results in loss of the other. An example of a fusion polynucleotide that yields as an expression product a polypeptide that provides both the desired positive and negative selection functions described above is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that confers hygromycin B resistance to positive selection in vitro and ganciclovir sensitivity to negative selection in vivo. See Lupton S.D., et al, Mol. and Cell. Biology 1 1:3374-3378, 1991. Further, in embodiments, the polynucleotide encoding the chimeric receptor comprises a fusion gene, particularly a fusion gene that confers hygromycin B resistance to positive selection in vitro and ganciclovir sensitivity to negative selection in vivo. , in retroviral vectors such as the HyTK retroviral vector described in Lupton S.D., et al, (1991), supra. See also publications PCT U591 / 08442 and PCT / U594 / 05601 describing the use of bifunctional selectable fusion genes derived from the fusion of dominant positive selectable markers and negative selectable markers.

[0263] Suitable positive selectable markers are derived from genes selected from the group consisting of hph, nco and gpt and suitable negative selectable markers are from cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. derived from a gene selected from the group consisting of Other suitable markers are bifunctional selectable fusion genes, where 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 the selectable marker.

[0264] viii) Strategies for modulating lentiviral integration Retroviral and lentiviral nucleic acids are disclosed that lack or are disabled for critical proteins / sequences to prevent integration of the retroviral or lentiviral genome into the target cell genome. For example, the highly conserved DDE motif of retroviral integrase (Engelman and Craigie (1992) J. Virol. 66:6361-6369; Johnson et al. (1986) Proc. Natl. Acad. Sci. USA 83:7648). -7652; Khan et al. (1991) Nucleic Acids Res. 19:851-860) allow the production of integration-defective retroviral nucleic acids.

[0265] For example, in some embodiments, the retroviral nucleic acids herein comprise a lentiviral integrase that contains a mutation that renders the integrase incapable of catalyzing 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 an illustrative type I mutation is a mutation that affects any of the three residues involved in the catalytic core domain of integrase: DX. 39-58 DX 35 E (D64, D116 and E152 residues of HIV-1 integrase). In certain embodiments, the mutation rendering the integrase incapable of catalyzing the integration of the viral genome into the cellular genome consists of replacing one or more amino acid residues of the DDE motif of the catalytic core domain of the integrase. and preferably the replacement of the first aspartic acid residue of the DEE motif with an asparagine residue. In some embodiments, the retroviral vector does not contain an integrase protein.

[0266] In some embodiments, the retrovirus integrates into an active transcription unit. In some embodiments, the retrovirus does not integrate near the transcription start site, the 5' end of the gene, or the DNAsel cleavage site. In some embodiments, retroviral integration does not activate proto-oncogenes or inactivate tumor suppressor genes. In some embodiments, retroviruses are not genotoxic. In some embodiments, the lentivirus integrates into an intron.

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

[0268] In some embodiments, the DNA encoding the exogenous agent is genomically integrated. In some embodiments, the DNA encoding the exogenous agent is maintained episomally. In some embodiments, the ratio of exogenous agent-encoding episomal DNA integration is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, 100.

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

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

[0271] ix) maintenance of episomal virus In poorly integrated retroviruses, circular cDNA byproducts of reverse transcription (e.g., 1-LTR and 2-LTR) can accumulate in the cell nucleus without being integrated into the host genome (Yanez-Munoz R J et al. al., Nat. Med. 2006, 12:348-353). These intermediates are then sent to the same frequency (e.g., 10 3 ~10 5 / cell) can integrate into cellular DNA.

[0272] In some embodiments, episomal retroviral nucleic acids are non-replicating. Episomal viral DNA can be modified to be maintained in replicating cells by including a eukaryotic origin of replication and a matrix-binding region (S / MAR) to associate with the scaffold / nuclear matrix.

[0273] Thus, in some embodiments, the retroviral nucleic acids described herein comprise a eukaryotic origin of replication or a variant thereof. Examples of eukaryotic origins of replication of interest are the β-globin gene origin of replication described by Aladjem et al (Science, 1995, 270:815-819), Price et al Journal of Biological Chemistry, 2003, 278(22):19649-59, a consensus sequence derived from self-replicating sequences associated with alpha-satellite sequences previously isolated from monkey CV-1 cells and human dermal fibroblasts. , the origin of replication of the human c-myc promoter region has been described by McWinney and Leffak (McWinney C. and Leffak M., Nucleic Acid Research 1990, 18(5):1233-42). In embodiments, the variant substantially retains 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, an autonomous replication assay based on bromodeoxyuridine incorporation and density shift (Araujo F.D. et al., supra; Frappier L. .et al.)

[0274] In some embodiments, the retroviral nucleic acid comprises a scaffold / matrix attachment region (S / MAR) or variant thereof, e.g., a non-consensus-like AT-rich DNA element several hundred base pairs in length, which is a protein They organize the nuclear DNA of the eukaryotic genome into chromatin domains by periodic attachment to the scaffolding or matrix of the cell nucleus. They are typically found in non-coding regions such as flanking regions, chromatin boundary regions, introns and the like. An example of the S / MAR region is the human IFN-γ gene (hIFN-γ large ), the S / MAR 0.7 Kbp minimal region of the human IFN-γ gene (hIFN-γ short), the 0.2 Kbp minimal region of the S / MAR of the human human dehydrofolate reductase gene (hDHFR) as described by Mesner L.D. et al., Proc Natl Acad Sci USA, 2003, 100:3281-86). In embodiments, functionally equivalent variants of S / MAR are sequences selected based on a set of six rules that together or singly are suggested to contribute to S / MAR function (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 freely available at genomecluster.secs.oakland.edu / MAR-Wiz. In embodiments, the variant retains substantially the same function of the S / MAR from which it was derived, in particular the ability to specifically bind to the nuclear matrix. One skilled in the art can determine whether a particular variant is capable of specifically binding to the nuclear matrix by, for example, the in vitro or in vivo MAR assays described by Mesner et al. (Mesner L.D. et al, supra). can decide. In some embodiments, a particular sequence is a variant of S / MAR if the particular variant exhibits a propensity for DNA strand separation. This property can be determined using specific programs based on methods of equilibrium statistical mechanics. The stress-induced double destabilization (SIDD) analytical technique states that "[...] an imposed level of superhelical stress increases the free energy required to open the duplex at each position along the DNA sequence. Calculate the extent of reduction Results are expressed as SIDD profiles, where sites of strong destabilization are deep minima [. ..] The SIDD algorithm and mathematical foundations (Bi and Benham (2004) Bioinformatics 20, 1477), as well as analysis of SIDD profiles, are freely available at WebSIDD (www.genomecenter.ucdavis.edu / benham). This can be done using internet resources, so in some embodiments a polynucleotide is considered a variant of an S / MAR sequence if it exhibits a SIDD profile similar to S / MAR.

[0275] B. Cell-derived fusosomes The composition of fusosomals can be produced from cells in culture, such as cultured mammalian cells, such as cultured human cells. Cells can be progenitor cells or non-progenitor (eg, differentiated) cells. Cells can be primary cells or cell lines (eg, mammalian, eg, human, cell lines described herein). In embodiments, the cultured cells are progenitor cells such as bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPC), endothelial progenitor cells (EPC), blast cells, intermediate progenitor cells formed in the subventricular zone, neuronal 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, cardiac myoblasts, nerves progenitor cells, glial progenitor cells, neural progenitor cells, and hepatoblasts.

[0276] In some embodiments, the source cells are 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 cells of a subject), embryonic stem cells (e.g., stem cells from embryonic yolk sac, placenta, umbilical cord, fetal skin, pubertal skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g. hepatocytes), alveolar cells, neurons (e.g. retinal neurons), progenitor cells (e.g. retinal progenitors) cells, myeloblasts, myeloid progenitor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanogenic blasts, lymphoblasts, myeloid progenitor cells, normoerythroblasts, or angioblasts), Progenitor cells (e.g., cardiac progenitor cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor 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 can be cells derived from epithelial, connective, muscle, or neural tissue or cells, and combinations thereof. Fusosomes can be obtained from cultured cells derived from any eukaryotic (e.g. mammalian) organ system, e.g. cardiovascular system (heart, vascular system); , colon, rectum and anus); endocrine system (hypothalamus, pituitary, pineal or pineal, thyroid, parathyroid, adrenal); excretory system (kidneys, ureters, bladder); lymphatic system (lymph, lymph nodes, lymphatic vessels, tonsils, pharyngeal tonsils, thymus, spleen); integumentary system (skin, hair, nails); muscular system (e.g., skeletal muscle); nervous system (brain, spinal cord, nerves); reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); skeletal system (bones, cartilage), and combinations thereof. In embodiments, the cells are derived from highly mitotic tissue (eg, highly mitotic healthy tissue such as epithelium, embryonic tissue, bone marrow, intestinal crypts, etc.). In embodiments, the tissue sample is a highly metabolic tissue (eg, skeletal tissue, neural tissue, cardiomyocytes).

[0278] In some embodiments, the cells are derived from young donors, eg, donors aged 25, 20, 18, 16, 12, 10, 8, 5, 1 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 subjects with similar genetic characteristics (eg, MHC matching).

[0280] In certain embodiments, the cells are more than 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length (eg, 4,000-10,000 nucleotides in length, 6,000-10,000 nucleotides in length). Have average sized telomeres.

[0281] In some embodiments, the fusosomes are generated from cell clones identified, sorted, or selected based on a desired phenotype or genotype for use as a source of the fusosomal compositions described herein. . For example, cell clones are identified, sorted, or selected based on low mitochondrial mutational burden, long telomere length, differentiation status, or certain genetic characteristics (eg, genetic characteristics matching the recipient).

[0282] The fusosomal compositions described herein can be composed of fusosomes from one cell or tissue source or from a combination of sources. For example, fusosomal compositions may be derived from heterologous sources (e.g., animals, tissue cultures of cells of the aforementioned species), allogeneic, autologous, specific tissues (liver, skeletal, neural, adipose) that result in different protein concentrations and distributions. etc.), may include fusosomes from cells of different metabolic states (eg, glycolytic, respiratory). The composition may also comprise different metabolic states, eg, bound or unbound fusosomes, as described elsewhere herein.

[0283] In some embodiments, fusosomes are produced from source cells that express a fusogen, eg, a fusogen described herein. In some embodiments, the fusogen is located in the membrane of the source cell, eg, a lipid bilayer membrane, eg, a cell surface membrane, or an intracellular membrane (eg, a lysosomal membrane). In some embodiments, fusosomes are produced from source cells that have fusogens disposed on the cell surface membrane.

[0284] In some embodiments, the fusosomes are exosomes, microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, giant plasma membrane vesicles, apoptotic bodies, mitoparticles, pyrenocytes, lysosomes, or other membrane-encapsulated It is produced by inducing vesicle budding.

[0285] In some embodiments, fusosomes are produced by inducing cell enucleation. Nucleus enucleation can be performed both genetically and chemically (e.g., using actinomycin D, 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), mechanical methods (e.g. squeezing or aspiration, Lee et 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 the complete removal of the nucleus, but also the displacement of the nucleus from its typical position so that the cell contains the nucleus but is non-functional.

[0286] In embodiments, making fusosomes comprises producing cell ghosts, giant plasma membrane vesicles, or apoptotic bodies. In embodiments, the fusosomal composition comprises one or more of 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 is performed using chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation or density centrifugation), freeze-thaw, or sonication), or combinations thereof. It can be carried out using the following methods, including but not limited to:

[0288] In some embodiments, fusosomes can be generated from source cells that express fusogens by any one, all, or a combination of the following methods, for example, as described herein. i) inducing budding of filamentous particles, exosomes, or other membrane-enclosed vesicles; ii) inducing nuclear inactivation, e.g. enucleation, by any or a combination of the following methods: a) genetic methods; b) chemical methods, for example using actinomycin D; or c) mechanical methods, such as squeezing or sucking; or iii) inducing cell fragmentation, for example by any or a combination of the following methods: a) chemical methods; b) mechanical methods such as centrifugation (eg ultracentrifugation or density centrifugation); freeze-thaw; or sonication.

[0289] i) Modification of cells prior to fusosome generation In some aspects, the modification is performed on a subject, tissue, or cell, such as modification of the cell, prior to fusosome production. Such modifications can be effective, for example, in improving fusion, fusogen expression or activity, cargo structure or function, or target cell structure or function.

[0290] a) physical alteration; In some embodiments, the cells are physically modified prior to producing fusosomes. For example, as described elsewhere herein, fusogens can be attached to the surface of cells.

[0291] In some embodiments, the cells are treated with chemicals prior to producing fusosomes. For example, cells can be treated with chemical or lipid fusogens such that the chemical or lipid fusogens non-covalently or covalently interact with the surface of the cell or become embedded within the surface of the cell. be In some embodiments, cells are treated with agents to enhance the fusogenic properties of lipids in cell membranes.

[0292] In some embodiments, the cell, prior to producing fusosomes, has one or more covalent binding molecules for synthetic or endogenous small molecules or lipids that enhance targeting of fusosomes to organs, tissues, or cell types. or physically modified with a non-covalent attachment site.

[0293] In embodiments, the fusosomes contain increased or decreased levels of endogenous molecules. For example, fusosomes can contain endogenous molecules that are naturally present in the naturally occurring source cell, but are present at higher or lower levels than fusosomes. In some embodiments, the polypeptide is expressed from exogenous nucleic acid in the source cell or fusosome. In some embodiments, the polypeptide is isolated from a source and loaded or bound to the source cell or fusosome.

[0294] In some embodiments, the cell, prior to producing fusosomes, extracts an endogenous fusogen within the cell (e.g., in some embodiments endogenous to the source cell, in some embodiments endogenous to the target cell), treated with chemical agents, eg, small molecules, to increase expression or activity. In some embodiments, the small molecule can increase the expression or activity of an endogenous fusogenic transcriptional activator. In some embodiments, the small molecule may decrease the expression or activity of an endogenous fusogen regulatory transcriptional repressor. In some embodiments, the small molecule is an epigenetic modifier that increases endogenous fusogen expression.

[0295] In some embodiments, fusosomes are produced from cells treated with a fusion-terminating compound, such as lysophosphatidylcholine. In some embodiments, fusosomes are generated from cells treated with a dissociation reagent that does not cleave fusogens, eg, accutase.

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

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

[0298] b) genetic modification In some embodiments, the cell is genetically modified prior to producing fusosomes to increase expression of endogenous fusogens within the cell (e.g., in some embodiments, endogenous to the source cell). Yes, and in some embodiments, endogenous to the target cell). In some embodiments, the genetic modification may increase the expression or activity of an endogenous fusogenic transcriptional activator. In some embodiments, the genetic modification may reduce the expression or activity of an endogenous fusogen regulatory transcriptional repressor. In some embodiments, the activator or repressor is nuclease-inactive cas9 (dCas9) linked to a transcriptional activator or repressor that targets endogenous fusogens by guide RNA. In some embodiments, the genetic modification epigenetically modifies the endogenous fusogen gene to increase its expression. In some embodiments, the epigenetic activator is nuclease-inactive cas9 (dCas9) linked by a guide RNA to an epigenetic modifier that targets endogenous fusogens.

[0299] In some embodiments, the cells are genetically modified prior to producing fusosomes to increase expression of exogenous fusogens within the cells, eg, delivery of transgenes. In some embodiments, nucleic acids, e.g., DNA, mRNA or siRNA, increase or In order to reduce it, it is introduced into the cell before it produces fusosomes. In some embodiments, the nucleic acid targets a fusogen repressor, eg, shRNA, siRNA constructs. In some embodiments, the nucleic acid encodes an inhibitor of fusogen repressor.

[0300] In some embodiments, the method comprises introducing into the source cell a nucleic acid that is exogenous to the source cell encoding the fusogen. Exogenous nucleic acid can be, for example, DNA or RNA. In some embodiments, an exogenous nucleic acid can be, for example, DNA, gDNA, cDNA, RNA, pre-mRNA, mRNA, miRNA, siRNA, and the like. In some embodiments, exogenous DNA can be linear DNA, circular DNA, or artificial chromosomes. In some embodiments, the DNA is maintained episomally. In some embodiments, the DNA is integrated into the genome. Exogenous RNA can be chemically modified RNA, and can contain, for example, one or more backbone modifications, sugar modifications, non-canonical bases, or a cap. Backbone modifications include, for example, phosphorothioates, N3' phosphoramidites, boranophosphates, phosphonoacetates, thio-PACE, morpholino phosphoramidites, or PNAs. 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, pseudo Uridine, 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 by reference in its entirety. incorporated herein.

[0301] In some embodiments, cells are treated with chemical agents, eg, small molecules, to increase the expression or activity of fusogens that are exogenous to the intracellular source cell prior to producing fusosomes. In some embodiments, the small molecule can increase the expression or activity of an exogenous fusogenic transcriptional activator. In some embodiments, the small molecule may reduce the expression or activity of an exogenous fusogen regulatory transcriptional repressor. In some embodiments, the small molecule is an epigenetic modifier that increases expression of exogenous fusogens.

[0302] In some embodiments, the nucleic acid encodes a modified fusogen. For example, tunable fusogens, eg, fusogens with specific cell-type, tissue-type, or local microenvironmental activity. Such modulatable fusion activity includes activation of fusion activity by exposure to low pH, high pH, ​​heat, infrared light, extracellular enzymatic activity (eukaryotic or prokaryotic), or small molecules, proteins, or lipids. and / or initiation may be included. In some embodiments, small molecules, proteins, or lipids are displayed on target cells.

[0303] In some embodiments, the cell is genetically modified prior to producing fusosomes to alter (i.e., upregulate or downregulate) expression of a signaling pathway (e.g., Wnt / beta-catenin pathway). be done. In some embodiments, the cells are genetically modified prior to producing fusosomes to alter (eg, upregulate or downregulate) expression of one or more genes of interest. In some embodiments, the cell is genetically modified prior to producing fusosomes to alter (e.g., upregulate or downregulate) expression of a nucleic acid (e.g., miRNA or mRNA) or nucleic acid of interest. be done. In some embodiments, nucleic acid, eg, DNA, mRNA or siRNA, is transferred to the cell prior to generating fusosomes, eg, to increase or decrease expression of a signaling pathway, gene, or nucleic acid. In some embodiments, the nucleic acid targets a repressor of a signaling pathway, gene, or nucleic acid or suppresses a signaling pathway, gene, or nucleic acid. In some embodiments, the nucleic acid encodes a signal transduction pathway, gene, or transcription factor that upregulates or downregulates the nucleic acid. In some embodiments, the activator or repressor is nuclease-inactive cas9 (dCas9) linked by a guide RNA to a transcriptional activator or repressor that targets a signaling pathway, gene, or nucleic acid. In some embodiments, genetic modification epigenetically alters an endogenous signaling pathway, gene, or nucleic acid to its expression. In some embodiments, the epigenetic activator is nuclease-inactive cas9 (dCas9) linked by a guide RNA to an epigenetic modifier that targets a signaling pathway, gene, or nucleic acid. In some embodiments, the DNA of the cell is modified with fusosomes to alter (e.g., upregulate or downregulate) expression of signaling pathways (e.g., Wnt / beta-catenin pathway), genes, or nucleic acids. Edited before generating. 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 recombinant methods. A nucleic acid sequence encoding a desired gene can be obtained using recombinant methods, e.g., by screening libraries from cells expressing the gene, by deriving the gene from a vector known to contain it, or It can be obtained by isolation directly from the cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0305] Expression of a natural or synthetic nucleic acid is usually accomplished by operably linking the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for the expression of the desired nucleic acid sequence.

[0306] In some embodiments, cells may be genetically modified with one or more expression regions, eg, genes. In some embodiments, the cells may be genetically modified with exogenous genes (eg, capable of expressing 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 and / or endogenous genes that are endogenous to the target cell. . In some embodiments, cells may be genetically modified with exogenous genes and / or regulatory nucleic acids that regulate the expression of exogenous genes. In some embodiments, cells may be genetically modified with regulatory nucleic acids that regulate the expression of exogenous and / or endogenous genes. It is appreciated that the cells described herein can be genetically modified to express a variety of exogenous genes encoding proteins or regulatory molecules that can act, for example, on gene products of the endogenous or exogenous genome of the target cell. understood by traders. In some embodiments, such genes confer characteristics on the fusosome, eg, 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, an endogenous gene or regulatory nucleic acid modulates the expression of another endogenous gene. In some embodiments, cells differ from versions of endogenous genes and / or regulatory nucleic acids on other chromosomes (e.g., inducibly, tissue-specifically, constitutively, or at higher levels or It may be genetically modified to express endogenous genes and / or regulatory nucleic acids that are expressed (at lower levels).

[0307] Promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically these are located in a region 30-110 bp upstream of the start site, although some promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are flipped or moved relative to each other. The thymidine kinase (tk) promoter allows up to 50 bp of spacing between promoter elements before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0308] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, 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 immediate early promoter, Rous Other constitutive promoter sequences may also be used, including but not limited to sarcoma virus promoters and human gene promoters such as, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters.

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

[0310] The expression vector introduced into the source may also contain a selectable marker gene or reporter to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected via the viral vector. Either or both genes can be included. In other embodiments, selectable markers can be carried on separate pieces of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in the 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 assess the function of regulatory sequences. Generally, a reporter gene is a gene encoding a polypeptide that is not present in, or expressed by, the recipient source and whose expression is manifested by some readily detectable property, such as enzymatic activity. . Reporter gene expression is assayed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes include 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). An encoding gene may be included. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, constructs with minimal 5' flanking regions that give the highest levels of expression of the reporter gene are identified as promoters. Such promoter regions can be linked to a reporter gene and used to assess agents for their ability to modulate promoter-driven transcription.

[0312] In some embodiments, cells can be genetically modified to alter expression of one or more proteins. Expression of one or more proteins may be altered for a particular time, eg, the developmental or differentiation state of the source. In some embodiments, the fusosome is a source of cells genetically modified to alter expression of one or more proteins, e.g., fusogenic proteins or non-fusogenic proteins that affect fusion activity, structure or function. generated from Expression of one or more proteins may be restricted to a particular location or spread throughout the source.

[0313] In some embodiments, expression of the fusogenic protein is altered. In some embodiments, the fusosomes have altered expression of fusogenic proteins, e.g., at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, Produced from cells with increased or decreased expression of fusogens of 90% or more.

[0314] In some embodiments, cells can be engineered to express cytosolic enzymes (eg, proteases, phosphatases, kinases, demethylases, methyltransferases, acetylases) that target fusogenic proteins. In some embodiments, the cytosolic enzyme affects one or more fusogens by altering post-translational modifications. Post-translational protein modifications of proteins can affect nutrient availability and responsiveness to redox conditions, and protein-protein interactions. In some embodiments, the fusosome has an altered post-translational modification, e.g. Fusogens with further increased or decreased post-translational modifications are included.

[0315] Methods of introducing modifications into cells include physical, biological and chemical methods. For example, Geng.&Lu,Microfluidic electroporation for cellular analysis and delivery.Lab on 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. See Nature Reviews Genetics.15:541-555.2014. Suitable methods of modifying cells for use in generating fusosomes as described herein include, for example, diffusion, osmosis, osmotic pulses, osmotic shock, hypotonic lysis, hypotonic dialysis, iontophoresis, electrophoresis, poration, sonication, microinjection, calcium precipitation, membrane intercalation, lipid-mediated transfection, detergent treatment, viral infection, receptor-mediated endocytosis, use of protein transduction domains, particle calcination, membrane fusion , freeze-thaw, mechanical disruption and filtration.

[0316] Confirmation of the presence of genetic alterations involves a variety of assays. Such assays include, for example, Southern and Northern blotting, molecular biological assays such as RT-PCR and PCR; biochemical assays such as detecting the presence or absence of peptides of

[0317] The disclosure provides, in some aspects, a fusosome comprising: (a) a lipid bilayer, (b) a lumen surrounded by the lipid bilayer (e.g., comprising the cytosol); (c) an exogenous fusogens that are sexually or overexpressed, e.g., fusogens arranged in a lipid bilayer, where the fusosomes are derived from the source cell; and where the fusosomes are partially or completely nuclear inactivated ( e.g. nuclear removal).

[0318] The disclosure provides, in some aspects, 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 cytoplasm; a lumen containing a sol and surrounded by a lipid bilayer; (c) exogenous or overexpressed fusogens arranged in the lipid bilayer; (d) containing nucleic acids, e.g., payload genes. 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 total protein; When in contact with a cell population comprising non-target cells, the cargo is at least 10-fold more present in the target cells than the non-target or reference cells, or (ii) the plurality of fusosomes is present in the non-target or reference cells At least 50% higher rate of fusion with target cells, where target cells are alphabeta T cells, effector T cells, CD8+ cytotoxic T cells, thymus-derived regulatory T cells, peripherally derived 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 be done.

[0319] The disclosure provides, in some aspects, 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 cytoplasm; (c) an exogenous or overexpressed fusogen located in the lipid bilayer; (d) an exogenous agent of Table 5; A nucleic acid comprising a payload gene encoding, wherein the fusosomes do not contain a nucleus; and wherein the amount of viral capsid proteins in the fusosomal composition is less than 1% of total protein.

[0320] The disclosure provides, in some aspects, 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 cytoplasm; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; (d) a nucleic acid containing a payload gene, wherein , the nucleic acid comprises NTCSRE operably linked to a payload gene, wherein NTCSRE is bound by a non-target cell-specific miRNA recognition sequence, e.g., a miRNA that is present in non-target cells at higher levels than in target cells. non-target cell-specific miRNA sequences, e.g., non-target cell-specific miRNA recognition sequences bound by the miRNAs of Table 4, wherein the target cells are T cells of the first type, optionally non- The target cell is a second different type of T cell or non-T cell; where the fusosomes do not contain a nucleus; and where the amount of viral capsid protein in the fusosomal composition is 1% of total protein. is less than

[0321] In some embodiments, the miRNA is present in non-target cells (e.g., as described herein) at levels at least 10, 100, 1,000, or 10,000-fold higher than the levels of the miRNA present in target cells (e.g., T cells). non-target cells). In some embodiments, the miRNA is not detectably present in target cells (eg, T cells, eg, T cells described herein). In some embodiments, the miRNA is absent from target cells (eg, T cells, eg, T cells described herein).

[0322] The disclosure provides, in some aspects, 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 cytoplasm; a lumen containing a sol and surrounded by a lipid bilayer; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; (d) a nucleic acid containing a payload gene; , comprising a promoter operably linked to a payload gene, wherein the promoter is a T cell-specific promoter, 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+ Foxp3+ regulatory sexual 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 specific for 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 a promoter, a nucleic acid; wherein the fusosome is free of the nucleus; and wherein the amount of viral capsid protein in the fusosomal composition is less than 1% of total protein.

[0323] The disclosure provides, in some aspects, 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 cytoplasm; a lumen containing a sol and surrounded by a lipid bilayer; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; (d) a nucleic acid containing a payload gene; , the 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 fusosomal composition is less than 1% of total protein.

[0324] The disclosure provides, in some aspects, 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 cytoplasm; (c) an exogenous or overexpressed fusogen arranged in the lipid bilayer; (d) a nucleic acid comprising: (i) payload gene; (ii) NTCRE operably linked to the payload gene, e.g., NTCRE is a non-target cell-specific miRNA bound by a non-target cell-specific miRNA recognition sequence, e.g., the miRNA of Table 4 containing a recognition sequence, and (iii) optionally a positive target cell-specific regulatory element, e.g., a positive target cell-specific regulatory element (e.g., a target cell-specific promoter) operably linked to the payload gene, wherein The positive target cell-specific regulatory element increases the expression of the payload gene in the target cell compared to an otherwise similar fusosome lacking the positive target cell-specific regulatory element, where the target cell is the first type of T cell; optionally the non-target cell is a second different type of 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 where the fusosomes do not contain a nucleus; and where the amount of viral capsid protein in the fusosome composition is less than 1% of total protein.

[0325] In some embodiments, there is one or more of the following: i) the fusosomes comprise or are contained in the cell biological material; ii) the fusosomes comprise enucleated cells; iii) the fusosomes comprise inactivated nuclei; iv) the fusosomes comprise a higher proportion of target cells than non-target cells, e.g., at least 1%, 2%, 3%, e.g., in the assay of Example 42 %, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2x, 3x, 4x, 5x, 10x, fusosomes are fused 20-fold, 50-fold, or 100-fold; v) the fusosomes are at a higher proportion, e.g., 10%, 20%, 30%, with target cells than with other fusosomes, e.g., in the assay of Example 42; 40%, 50%, 60%, 70%, 80% or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold fused; vi) fusosomes However, for example, in the assay of Example 42, agents within fusosomes were found to be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 70%, 24, 48, or 72 hours later in target cells. vii) the fusogen is at least 10, 50, 100, 500, 1,000, e.g., as measured by the assay of Example 26; 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、若しくは1,000,000,000コピー、又はそれ以下のコピー数で存在する;viii)フソソームは、例えば、実施Example When measured by an assay of 88, at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 200,000, 200,000, 200,000, 500,000, 5,000,000, 10,000,000 ix) the ratio of the copy number of fusogen to the copy number of the therapeutic agent is 1,000,000:1 to 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, 1:10,000~1:100,000, or 1:100,000~ 1:1,000,000; x) fusosomes contain a lipid composition substantially similar to that of the source cell or CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS , PA, PC, PE, PG, PI, PS, CE, SM and TAG 10%, 15%, 20%, 25%, 30%, 35% of the corresponding lipid levels in the source cells , 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%; xii) the fusosomes are 10%, 20%, 30%, 40%, or 50% of the corresponding proportion in the source cell, e.g., as measured using the assay of Example 40; xiii) fusosomes, e.g., 10%, 20%, 30%, 40% of the corresponding ratio in the source cell when measured using the assay of Example 41 , or a ratio of protein to nucleic acid (e.g., DNA) that is within 50%; xiv) fusosomes, e.g., 10% of the corresponding ratio in the source cell when measured using the assay of Example 91; comprising a lipid to nucleic acid (e.g., DNA) ratio that is within 20%, 30%, 40%, or 50%; 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the half-life of the cell, e.g. %, with a half-life within 100%; xvi) fusosomes are at least 1%, 2%, 3% higher than a negative control, e.g., otherwise similar fusosomes, in the absence of glucose, e.g., when measured using the assay of Example 50 , 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more (e.g., about 11.6% more) glucose (e.g., labeled transport glucose, e.g., 2-NBDG) across membranes; xvii) fusosomes, e.g. Lumen within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of activity containing esterase activity in xviii) fusosomes are 1%, 2%, 3%, 4%, 5%, 10%, 20% of the citrate synthase activity in a reference cell, e.g. a source cell, e.g. as described in Example 53 , 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the metabolic activity level; 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of respiration level in reference cells, e.g. source cells xx) fusosomes up to 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, Either the fusosomes contain an annexin V staining level of 12,000, 11,000, or 10,000 MFI, or the fusosomes are at least 5%, 10%, or greater than the annexin V staining level of otherwise similar fusosomes treated with menadione in the assay of Example 55. %, 20%, 30%, 40%, or 50% lower annexin V staining levels, or the fusosomes are at least 5% lower than the annexin V staining levels of menadione-treated macrophages in the assay of Example 55, xxi) fusosomes containing 10%, 20%, 30%, 40% or 50% lower annexin V staining levels than those of the source cells, e.g., by the assay of Example 33, at least 1%, 2%, have a miRNA content level of 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more; xxii) fusosomes are Soluble:insoluble protein ratios are 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less, or, for example, 1%-2%, 2%-3%, 3%-4%, 4%-5% of that of the source cells or within 80% to 90% or greater; have an LPS level of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001% or less of the LPS content of the source cells, as determined by analytical analysis; 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40% more than otherwise similar fusosomes in the absence of insulin using 49 assays %, 50%, 60%, 70%, 80%, 90%, 100% more signaling, e.g. delivery of extracellular signals in response to insulin, e.g. AKT phosphorylation, or glucose in response to insulin (e.g. , labeled glucose, e.g., 2-NBDG) capable of uptake; When administered to a subject, e.g., a mouse, targeting the nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, eye, or the like, for example, after 24, 48, or 72 hours by the assay of Example 64, e.g., at least 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50% of the population of administered fusosomes, 60%, 70%, 80% or 90% of fusosomes are present in the target tissue; At least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, than induced juxtacrine signaling xxvii) the fusosomes have a juxtacrine signaling level that is 90% or 100% greater; xxvii) the fusosomes are at least 1 %, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% greater paracrine signaling levels ; xxviii) fusosomes are 1%, 2%, 3%, 4%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, 5%, than than actin levels polymerized in reference cells, e.g., source cells or C2C12 cells, e.g., by the assay of Example 58; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% xxix) fusosomes polymerize actin at levels within , 90%, or 100%; , 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or have a membrane potential within , having a membrane potential of about −20 to −150 mV, −20 to −50 mV, −50 to −100 mV, or −100 to −150 mV; at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or a percentage of cells or cells of the same type as the source cell At a rate of 90%, extravasation from blood vessels is possible, where the source cells are neutrophils, lymphocytes, B cells, macrophages or NK cells; xxxii) fusosomes are, e.g., at least 1% higher than reference cells, e.g. mouse embryonic fibroblasts, using the assay of Example 48; , 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater than 100% secreted protein xxxiii) the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards; xxxiv) the fusosomes were manufactured according to Good Manufacturing Practices (GMP); xxxv) the fusosomes are below the prescribed limit xxxvi) the fusosomes have a level of contaminants below a predetermined reference value, e.g. are substantially free of contaminants; xxxvii) the fusosomes are xxxviii) source cells include neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, bone marrow or xxxix) the source cells are 293 cells, HEK cells, human endothelial cells, or human epithelial cells, monocytes, macrophages, dendrites; cells, or other than stem cells.

[0326] The present disclosure also provides, in some embodiments, fusosomes comprising: a) a lipid bilayer and an aqueous solution, e.g., a lumen that is miscible with water and in which the fusosomes are derived from the source cell, b) lipids Exogenous or overexpressed fusogens arranged in bilayers and c) organelles arranged within the lumen, eg a therapeutically effective number of organelles.

[0327] In some embodiments, one or more of the following are present: i) the source cells are endothelial cells, macrophages, neutrophils, granulocytes, leukocytes, stem cells (e.g., mesenchymal stem cells, bone marrow stem cells, induced polycythemia); embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, such as retinal neurons; selected from endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxisomes, hydrogenosomes, melanosomes, mitosomes, nematocysts, peroxisomes, proteasomes, vesicles, and stress granules; iii) fusosomes are 5um , having a size greater than 10um, 20um, 50um, or 100um; fusosomes have a density other than g / ml, e.g., fusosomes have a density of 1.25 g / ml + / - 0.05; v) fusosomes are not captured by the circulating scavenger system or Kupffer cells within the sinuses of the liver; vi ) the source cells are other than 293 cells; vii) the source cells have not been transformed or immortalized; viii) the source cell is transformed or immortalized using a method other than adenovirus-mediated immortalization, e.g. immortalized by spontaneous mutation or telomerase expression; or is other than a secretory granule protein; x) the fusosomes do not contain Cre or GFP, such as EGFP; xi) the fusosomes further contain an exogenous protein other than Cre or GFP, such as EGFP; xii) the fusosomes For example, they further contain exogenous nucleic acids (eg, RNA, eg, mRNA, miRNA, or siRNA) or exogenous proteins (eg, antibodies, eg, antibodies) within their lumen; or xiii) fusosomes do not contain mitochondria.

[0328] The disclosure also provides, in some aspects, a fusosome comprising: (a) a lipid bilayer, (b) a lumen surrounded by the lipid bilayer (e.g., comprising a cytosol), (c) Exogenous or overexpressed fusogens, such as fusogens, are arranged within the lipid bilayer, and (d) a functional nucleus, where fusosomes are derived from the source cell.

[0329] In some embodiments, there is one or more of the following: i) source cells are other than dendritic cells or tumor cells, e.g., source cells are endothelial cells, macrophages, neutrophils, granulocytes, selected from leukocytes, stem cells (e.g. mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells), myeloblasts, myoblasts, hepatocytes, or neurons, such as retinal neurons; ii) Fusogens are other than fusogenic glycoproteins; iii) Fusogens are mammalian proteins other than Fertilin-beta; iv) Fusosomes are less immunogenic, e.g., as described herein v) the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards; vi) the fusosomes were manufactured according to Good Manufacturing Practices (GMP); and, eg, substantially free of pathogens; or viii) the fusosomes have a contaminant level below a predetermined reference value, eg, are substantially free of contaminants.

[0330] The disclosure also provides, in some aspects, 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 the cytosol and surrounded by a lipid bilayer; (c) exogenous or overexpressed fusogens arranged in the lipid bilayer; (d) cargo; and where fusosomes does not contain a nucleus; where the amount of viral capsid protein in the fusosomal composition is less than 1% of total protein; Deliver cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population when contacted with the population and when contacted with a reference target cell population that has not been treated with an inhibitor of endocytosis .

[0331] The disclosure also provides, in some aspects, 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 the cytosol and surrounded by a lipid bilayer; (c) exogenous or overexpressed retargeted fusogens arranged in the lipid bilayer; (d) cargo; 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 total protein; When in contact with a cell population comprising cells, the cargo is at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold more abundant in target cells than in non-target cells, or (ii) multiple fusosomes fuses with target cells at least 50% higher than non-target cells.

[0332] The disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes comprises: (a) a lipid bilayer; (b) (c) an exogenous or overexpressed fusogen, where the fusogen is located within the lipid bilayer; and (d) a cargo; where the fusosome contains a nucleus. and wherein one or more (e.g., at least 2, 3, 4, or 5): i) the fusogen is present in a copy number of at least 1,000 copies; ii) the fusosome is present in a copy number of at least 1,000 copies iii) the fusosome is one of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM and TAG contain lipids within 75% of the corresponding lipid levels in the source cells; iv) fusosomes contain proteomic composition similar to that of the source cells; v) fusosomes in the absence of insulin, negative control , e.g., 10% more than otherwise similar fusosomes, signaling, e.g., delivery of extracellular signals in response to insulin, e.g., AKT phosphorylation, or glucose in response to insulin (e.g., labeled glucose, e.g., 2-NBDG) capable of uptake; vi) fusosomes are available in tissues such as liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovaries, brain, reproductive organs, central nervous system, peripheral nervous system; , skeletal muscle, endothelium, inner ear, or eye, etc., and when administered to a subject, e.g., a mouse, after 24 hours, e.g., at least 0.1% of the population of administered fusosomes, or 10% of the fusosomes target present in tissue; or source cells are neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g. selected from retinal neurons;

[0333] In embodiments, one or more of the following: i) the source cells are other than 293 cells; ii) the source cells have not been transformed or immortalized; iii) the source cells have been adenovirus-mediated immortalized. iv) the fusogen is other than VSVG, SNARE proteins, or secretory granule proteins; v) the therapeutic agent is other than Cre or EGFP; vi) the therapeutic agent is, for example, an intraluminal nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., an antibody, e.g., an antibody); or vii) fusosomes do not contain mitochondria.

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

[0335] In embodiments, one or more of the following: i) the therapeutic agent is a soluble protein expressed by the source cell; ii) the fusogen is TAT, TAT-HA2, HA-2, gp41, Alzheimer's beta other than amyloid peptides, Sendai virus proteins, or amphipathic net-negative peptides (WAE 11); iii) fusogens are mammalian fusogens; iv) fusosomes contain, in their lumen, comprising a polypeptide selected from an enzyme, an antibody, or an antiviral polypeptide; v) the fusosome does not contain an exogenous therapeutic transmembrane protein; or vi) the fusosome does not contain CD63 or GLUT4, or the fusosome is 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% or less (e.g., about 0.048% below) including CD63.

[0336] In embodiments, the fusosomes are: i) virus-free, non-infectious, or non-propagating in host cells; ii) not viral vectors; iii) not VLPs (virus-like particles); iv) viral structural proteins. e.g., does not contain proteins derived from gag, e.g., viral capsid proteins, e.g., viral capsid proteins, e.g., viral nucleocapsid proteins, or the amount of viral capsid proteins is reduced, e.g., using the assay of Example 93. and, e.g., by mass spectrometry, less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of total protein; vi) does not contain viral nonstructural proteins, such as pol or fragments or variants thereof, viral reverse transcriptase protein, viral integrase protein, or viral protease protein; vii) does not contain viral nucleic acids, such as viral RNA or viral DNA; Not included; VIII) Virus structural protein veloses 10, 50, 50, 500, 1,000, 2,000, 5,000, 5,000, 10,000, 200,000, 200,000, 500,000, 500,000, 5,000,000, 10,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000 or contains less than 1,000,000,000 copies; or ix) the fusosome is not a virosome.

[0337] In some embodiments, the fusosomes are about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, Less than 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of viral capsid protein (e.g., about 0.05% viral capsid protein) contains (or is identified as containing); In embodiments, the viral capsid protein is a rabbit endogenous lentivirus (RELIK) capsid and cyclophilin A complex. In embodiments, the viral capsid protein:total protein ratio is (or is identified as) about 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 do not comprise (or are identified as not comprising) gag protein, or fragments or variants thereof, or the amount of gag protein, or fragments or variants thereof, is less than, e.g., Example 93 less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of total protein by assay of

[0339] In embodiments, the ratio of fusogen copy number to viral structural protein copy number 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 100:1~50:1, 50:1~20:1, 20:1~10:1, 10:1~5:1 Between or 1:1. In embodiments, the ratio of fusogen copy number to viral matrix protein copy number 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: i) the fusosome does not comprise water-immiscible droplets; ii) the fusosome comprises an aqueous lumen and a hydrophilic exterior; iii) the fusogen is a protein fusogen. or iv) the organelle is mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxisomes, hydrogenosomes, melanosomes, mitosomes, cnidarian cells, peroxisomes , proteasomes, vesicles, and stress granules.

[0341] In embodiments, one or more of the following: i) the fusogen is a mammalian fusogen or a viral fusogen; ii) the fusosome is made by loading the fusosome with a therapeutic or diagnostic agent. iii) the source cells were not loaded with therapeutic or diagnostic agents; iv) fusosomes were loaded with doxorubicin, dexamethasone, cyclodextrin, polyethylene glycol, microRNAs such as miR125, VEGF receptor, ICAM -1, does not contain E-selectin, iron oxide, fluorescent proteins such as GFP or RFP, nanoparticles, or RNases, or does not contain exogenous forms of any of the above; or v) the fusosomes contain one or more Further included are exogenous therapeutic agents that have post-translational modifications, eg, glycosylation.

[0342] In embodiments, the fusosomes are unilamellar or multilamellar.

[0343] In embodiments, one or more of: i) the fusosomes are not exosomes; ii) the fusosomes are microvesicles; iii) the fusosomes comprise non-mammalian fusogens; v) the fusosomes contain exogenous fusogen; vi) the size of the fusosomes 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 population of fusosomes is at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; including acrosomes, autophagosomes, centrioles, glycosomes, glyoxisomes, hydrogenosomes, melanosomes, mitosomes, cnidarian cells, peroxisomes, proteasomes, vesicles, and stress granules; viii) fusosomes, the cytoskeleton or components thereof , e.g., actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; Isolation and characterization of exosomes from cell culture supernatants and biological fluids.” Curr Protoc Cell Biol.2006 Apr;Chapter 3: Unit 3.22, e.g. or have a density of at least 1.18-1.25 g / ml, or 1.05-1.12 g / ml; x) the lipid bilayer is enriched in ceramide or sphingomyelin, or a combination thereof, compared to the source cell; or the lipid bilayer is not enriched (e.g., depleted) in glycolipids, free fatty acids or phosphatidylserine, or combinations thereof, compared to the source cell; xi) fusosomes are shown in Examples Phosphatidylserine (PS) or CD40 ligand, as measured by 92 assays xii) fusosomes are enriched in PS compared to source cells, e.g., Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via At least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, e.g. %, or 90% are positive for PS; xiii) the fusosomes are substantially free of acetylcholinesterase (AChE), e.g., 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, containing less than 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, or 1000 AChE activity units / ug protein; xiv) Fusosomes contain tetraspanin family proteins such as CD63 , CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, Actinin-4, Mitofilin, Syntenin-1, TSG101, ADAM10, EHD4, Syntenin -1, TSG101, EHD1, flotillin-1, heat shock 70-kDa proteins (HSC70 / HSP73, HSP70 / HSP72), or any combination thereof, for example, by the assay of Example 89. 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5% or 10% compared to non-enriched cells or any one or more of these proteins % of any individual exosomal marker protein and / or 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or containing less than 25% of the total exosomal marker proteins of any of the proteins of interest, or not enriched for any one or more of these proteins; xv) fusosomes, e.g., using the assay of Example 36, , 250, 100, 50, 20, 10 , less than 5 or 1 ng GAPDH / ug total protein, or less than the level of GAPDH in the source cell, e.g., 1%, 2.5%, 5%, 10% less than the level of GAPDH per ng / ug total protein in the source cell , 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% lower levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH); Enriched with one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasomal proteins, or one or more mitochondrial proteins, or any combination thereof, e.g., amounts of calnexin of 500, 250, 100 , 50, 20, 10, 5, or less than 1 ng calnexin / ug total protein or fusosomes are less than 1 ng / ug compared to source cells, e.g., using the assays of Examples 37 or 90. %, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin per total protein or fusosomes The average fractional content of calnexin in -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 4×10 -4 less than or the fusosomes contain about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99% less amount of calnexin per total protein than the parental cell; xvii) the fusosomes contain an exogenous agent (e.g., exogenous protein, mRNA, or siRNA), e.g., as measured using the assay of Example 34; or xviii) the fusosomes contain e.g. M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442 fixed to the surface of mica by atomic force microscopy for at least 30 min. can be

[0344] In embodiments, one or more of the following: i) the fusosomes are exosomes; ii) the fusosomes are not microvesicles; or is less than 1500 nm, or the mean size of the population of fusosomes is less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; iv) the fusosomes do not contain organelles; , the cytoskeleton or its components, e.g., actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; , Thery et al., "Isolation and characterization of exosomes from cell cultures and biological fluids." Curr Protoc Cell Biol.2006 Apr;Chapter 3: Unit 3.22, e.g. vii) the lipid bilayer is not enriched (e.g., depleted) in ceramide or sphingomyelin, or a combination thereof, or lipid bilayer compared to the source cell; The overlay is enriched in glycolipids, free fatty acids or phosphatidylserine, or a combination thereof, compared to the source cell; viii) fusosomes contain phosphatidylserine (PS) or CD40 as measured by ix) fusosomes are not enriched in PS compared to source cells (e.g., depleted) (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles.Proc Natl Acad Sci USA 112:E1433-E1442, e.g. less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% are positive for PS; x) fusosomes, e.g., by the assay of 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 AChE activity units / protein ug acetylcholinesterase (AChE); xi) Fusosomes are 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, mitofilin, syntenin-1, TSG101, ADAM10, EHD4, syntenin-1, TSG101, EHD1, flotillin-1, heat shock 70-kDa proteins (HSC70 / HSP73, HSP70 / HSP72) or any thereof and compared to the source cells, e.g., by the assay of Example 89, for example, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5% or >10% of any individual exosome marker protein and / or 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20% or contain less than 25% of the total exosomal marker proteins of any of the proteins of interest, or are enriched for any one or more of these proteins; xii) the fusosomes are, for example, using the assay of Example 36 Greater than 500, 250, 100, 50, 20, 10, 5 or 1 ng GAPDH / ug total protein, or less than the level of GAPDH in the source cell, e.g., less than the level of GAPDH per ng / ug total protein in the source cell also at least 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% higher glyceraldehyde 3-phosphate dehydrogenase ( GAPDH); xiii) fusosomes containing one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasomal proteins, or one or more mitochondrial proteins, or any combination thereof; If the combination is not enriched (e.g., depleted), e.g., the amount of calnexin is 500, 250, 100, 50, 20, 10, 5, or 1 ng calnexin / ug total protein, fusosomes 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, in ng / ug units compared to source cells, e.g. 60%, 70%, 80%, or 90% less calnexin per total protein, or an average fractional content of calnexin in fusosomes of about 1 x 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 4×10 -4 less than or the fusosomes contain about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99% less amount of calnexin per total protein than the parental cell; or xiv) fusosomes are subjected to atomic force for at least 30 minutes, e.g., by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. It cannot be immobilized on the surface of mica by microscopy.

[0345] In embodiments, one or more of the following: i) the fusosomes do not contain VLPs; ii) the fusosomes do not contain viruses; iii) the fusosomes do not contain replication-competent viruses; iv) the fusosomes contain viral proteins; e.g., free of viral structural proteins, e.g., capsid proteins or viral matrix proteins; v) fusosomes do not contain capsid proteins from enveloped viruses; vi) fusosomes do not contain nucleocapsid proteins; Not a viral fusogen.

[0346] In embodiments, the fusosomes comprise the cytosol.

[0347] In embodiments, one or more of the following: i) the fusosomes or source cells do not form teratomas when implanted into a subject, e.g., by the assay of Example 65; For example, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% more than the reference cells using the assay of Example 45. chemotaxis within %, 80%, 90%, 100% or more; Derived from human cells, e.g., the source cells are neutrophils, using the assay of Example 46; iv) the fusosomes are capable of phagocytosis, e.g., the source cells are macrophages. Forty-seven assays can be used to detect fusosomal phagocytosis within 0.5, 1, 2, 3, 4, 5, or 6 hours.

[0348] In embodiments, the fusosomes or fusosomal compositions are administered to a subject, e.g., a human subject, for no more than 5 days, e.g., no more than 4 days, no more than 3 days, no more than 2 days, no more than 1 day, e.g. 1, 2, 3, 4, 5, 6 or more of any of the above characteristics for 72 hours.

[0349] In embodiments, the fusosomes have one or more of the following characteristics: a) contain one or more endogenous proteins, such as membrane proteins or cytosolic proteins, from the source cell; b) at least 10, 20, 50 , comprising 100, 200, 500, 1000, 2000, or 5000 different proteins; c) comprising at least 1, 2, 5, 10, 20, 50, or 100 different glycoproteins; d) the number of proteins in the fusosome at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight is naturally occurring protein; e) at least 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 different RNAs; or f) at least 2, 3, 4, 5, 10, or 20 different lipids, e.g. CL, Cer, DAG, HexCer, LPA, LPC, LPE , LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG.

[0350] In embodiments, the fusosomes have been engineered to have one, two, three, four, five, or more of the following properties, or the fusosomes are not naturally occurring cells and the nucleus is Does not naturally possess 1, 2, 3, 4, 5 or more of the following properties: a) Partial nuclear inactivation is associated with nuclear functions such as transcription or DNA replication. or at least 50%, 60%, 70%, 80%, 90% or more of both, wherein transcription is measured by the assay of Example 24 and DNA replication is measured by the assay of Example 25 b) whether the fusosomes are transcriptionally incompetent, e.g. have less than 30%, 40%, 50%, 60%, 70%, 80%, or 90% transcriptional activity; c) the fusosomes are incapable of nuclear DNA replication, e.g., using the assay of Example 25 , less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the nuclear DNA replication of a reference cell, e.g., a source cell have nuclear DNA replication; d) fusosomes lack chromatin, e.g. , has a chromatin content of less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%; , has an amount of nuclear membrane less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of a reference cell, e.g., a source cell or a Jurkat cell; f) the fusosome lacks a functional nuclear pore complex or has nuclear import or export activity of at least 50%, 40%, 30%, 20%, 10%, 5% by the assay of Example 31; reduced by 4%, 3%, 2% or 1% or the fusosomes lack nuclear pore proteins such as NUP98 or importin 7; g) fusosomes do not contain histones or histone levels are 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30% of the histone levels (e.g., H1, H2a, H2b, H3, or H4) of the source cells, e.g., by the assay of Example 32. %, 40%, 50%, 60%, 70%, 80%, or 90 h) the fusosomes contain less than 20, 10, 5, 4, 3, 2, or 1 chromosome; i) the nuclear function has been eliminated; j) the fusosomes are enucleated mammals k) the nucleus is extruded, for example by mechanical force, or removed or inactivated by radiation or by chemical ablation; or derived from mammalian cells with partially deleted DNA.

[0351] In embodiments, the fusosomes comprise mtDNA or vector DNA. In embodiments, the fusosomes do not contain DNA.

[0352] In embodiments, the source cells are primary cells, immortalized cells or cell lines (eg myeloblastic cell lines such as C2C12). In embodiments, the fusosomes are derived, for example, from source cells with modified genomes that have been rendered less immunogenic (eg, by genome editing, eg, to remove MHC proteins or MHC complexes). In embodiments, the source cells are derived from cell cultures that have been treated with anti-inflammatory signals. In embodiments, the source cells are derived from cell cultures that have been treated with immunosuppressants. In embodiments, the source cells are substantially non-immunogenic, eg, using the assays described herein. In embodiments, the source cells contain an exogenous agent, eg, a therapeutic agent. In embodiments, the source cell is a recombinant cell.

[0353] In embodiments, the fusosome further comprises an exogenous agent, eg, a therapeutic agent, eg, a protein or nucleic acid (eg, DNA, chromosome (eg, human artificial chromosome), RNA, eg, mRNA or miRNA). According to the embodiment, the external causal effect substance is at least 10, 20, 50, 100, 500, 1,000, 2,000, 5,000, 5,000, 10,000, 20,000, 50,000, 200,000, 500,000, 500,000, 5,000,000, 10,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000,000. , 500,000,000, or 1,000,000,000 or less copies, e.g. Present at an average level of 200,000, 500,000 or 1,000,000 or less copies. In embodiments, the fusosomes have one or more endogenous molecules, e.g., proteins or nucleic acids, whose levels have been altered, e.g., increased or decreased, e.g., by treating mammalian cells with siRNAs or gene-editing enzymes. have. In embodiments, the endogenous agent is, for example, at least , 100,000,000, 500,000,000, or 1,000,000,000 or less copies present (e.g., contained by the fusosome) or at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000 per fusosome, Present at an average level of 10,000, 20,000, 50,000, 100,000, 200,000, 500,000 or 1,000,000 or less copies. In embodiments, the endogenous molecule (eg, RNA or protein) is at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 10 greater than its concentration in the source cell. 3 , 5.0×10 3 ,Ten 4 , 5.0×10 4 ,Ten 5 , 5.0×10 5 ,Ten 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0×10 8 Present in high concentrations.

[0354] In embodiments, the active agent is a protein, protein complex (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 (eg DNA, chromosomes or RNA, eg mRNA, siRNA or miRNA) or small molecules. In embodiments, exogenous agents include site-specific nucleases, such as Cas9 molecules, TALENs, or ZFNs.

[0355] In embodiments, the fusogen is a viral fusogen, such as HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, such as a SNARE, Syncytin, myomaker, myomixer, myomerger, or FGFRL1. In embodiments, the fusogen is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusogen is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusosome fuses to the target cell on the surface of the target cell. In embodiments, the fusogen promotes fusion in a lysosome-independent manner. In embodiments, the fusogen is a protein fusogen. In embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glycerides, diacylglycerols, or modified unsaturated fatty acids. In embodiments, the fusogen is a chemical fusogen, such as PEG. In embodiments, the fusogen is a small molecule fusogen, eg, an NSAID such as halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine. In embodiments, the fusogen is recombinant. In embodiments, the fusogen is biochemically incorporated, e.g., the fusogen is provided as a purified protein and contacted with the lipid bilayer under conditions that allow association of the fusogen with the lipid bilayer. In embodiments, the fusogen is biosynthetically integrated, eg, expressed in the source cell under conditions that allow the fusogen to associate with the lipid bilayer.

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

[0357] In some embodiments involving fusosomal compositions, the plurality of fusosomes are the same. In some embodiments, the multiple fusosomes are different. In some embodiments, the plurality of fusosomes is derived from one or more source cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes are 10%, 20% of the average diameter of the fusosomes in the fusosome composition. , 30%, 40%, or 50% of the diameter. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes is 10%, 20% of the average volume of fusosomes in the fusosome composition. , 30%, 40%, or 50% of the volume. In some embodiments, the fusosomal composition is about 90%, 80%, 70% within 10%, 50%, or 90% variation of the source cell population in size distribution, e.g., based on Example 28. , with variations in size distribution of less than 60%, 50%, 40%, 30%, 20%, 10%, 5%. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes is 10%, 20% of the average fusogen copy number among fusosomes in the fusosomal composition %, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the fusogen copy number. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have an average copy number of the therapeutic agent in the fusosomes of the fusosome composition of 10. %, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% copy number of the therapeutic. In some embodiments, the fusosomal composition contains at least 10 5 ,Ten 6 ,Ten 7 ,Ten 8 ,Ten 9 ,Ten 10 ,Ten 11 ,Ten 12 ,Ten 13 ,Ten 14 , or 10 15 or more fusosomes. In some embodiments, the fusosomal composition has 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 fusosomal composition reduces the cell number of the target cell population by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, Deliver cargo to 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

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

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

[0361] In some embodiments, the inhibitor of endocytosis is a lysosomal acidification inhibitor, eg, bafilomycin A1. In some embodiments, the inhibitor of endocytosis is a dynamin inhibitor, eg, Dynasore.

[0362] In some embodiments, the target cell population is at physiological pH (eg, 7.3-7.5, eg, 7.38-7.42).

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

[0364] In some embodiments, the cargo is a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., an inhibitor of endocytosis, e.g., macromolecules at a concentration of 25 μM). A pinocytosis inhibitor, e.g., 5-(N-ethyl-N-isopropyl) amiloride (EIPA)), or an inhibitor of the actin-independent pathway (e.g., an inhibitor of endocytosis, e.g., actin polymerization at a concentration of 6 μM). enter cells via inhibitors of e.g. latrunculin B).

[0365] In some embodiments, the plurality of fusosomes further comprises a targeting moiety. In embodiments, the targeting moiety is contained by the fusogen or contained by a separate molecule.

[0366] In some embodiments, when multiple fusosomes contact a cell population comprising target and non-target cells, the cargo is at least 10-fold more abundant in target cells than in non-target cells.

[0367] In some embodiments, cargo is at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold more present, and / or the cargo is 2-, 5-, 10-, 20-, or 50-fold more present in target cells than in reference cells.

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

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

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

[0371] In some embodiments, the plurality of fusosomes has 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 plurality of fusosomes has an average size of less than 100 nm, 80 nm, 60 nm, 40 nm, or 30 nm.

[0372] In some embodiments, the fusogen (eg, retargeted fusogen) comprises a mammalian fusogen. In some embodiments, the fusogen (eg, retargeted fusogen) comprises a viral fusogen. In some embodiments, the fusogen (eg, 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, henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, comprising a sequence selected from Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or derivatives thereof.

[0373] In some embodiments, a fusogen (eg, a retargeted fusogen) is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. . In some embodiments, the fusogen (eg, retargeted fusogen) is not active at pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. .

[0374] In some embodiments, the fusogen is present in a copy number 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 proteins, morbillivirus H proteins, respirovirus HN proteins, including Sendai virus HN proteins, rubulavirus HN proteins, oil virus HN proteins, or derivatives thereof. In some embodiments, the fusogen (e.g., retargeted fusogen) is 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, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubula virus including sequences selected from F and HN proteins, or Avravirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0376] In some embodiments, cargo comprises exogenous proteins or exogenous nucleic acids. 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 contains a therapeutic agent. In some embodiments, the cargo is present at a copy number of at least 1, 2, 5, 10, 20, 50, 100, or 200 copies per fusosome (eg, up to about 1,000 copies per fusosome). In some embodiments, the ratio of fusogen (e.g., retargeted fusogen) copy number to cargo copy number is 1000:1 to 1:1, or 500:1 to 1:1, or 250:1 to 1:1. 1~1:1, or 150:1~1:1, or 100:1~1:1, or 75:1~1:1, 50:1~1:1, 25:1~1:1, 20 : 1~1:1, or 15:1~1:1, or 10:1~1:1, or 5:1~1:1, or 2:1~1:1, or 1:1~1: 2.

[0377] In some embodiments, the fusosomal composition comprises viral capsid proteins or DNA-integrating polypeptides. In some embodiments the cargo comprises a viral genome.

[0378] In some embodiments, fusosomes can deliver nucleic acids to target cells, eg, for gene therapy, eg, to stably modify the target cell's genome.

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

[0380] In embodiments, the fusosomal composition comprises at least 10 5 ,Ten 6 ,Ten 7 ,Ten 8 ,Ten 9 ,Ten 10 ,Ten 11 ,Ten 12 ,Ten 13 ,Ten 14 , or 10 15 of fusosomes. In embodiments, the fusosomal composition comprises at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, IL, 2 L, 5 L, 10 L, 20 L, or 50 L.

[0381] In embodiments, the fusosomes are derived, for example, from mammalian cells whose genomes have been modified to reduce immunogenicity (e.g., by genome editing, e.g., to remove MHC proteins or MHC complexes). In embodiments, the source cells are derived from cell cultures that have been treated with anti-inflammatory signals. In embodiments, the method further comprises contacting the source cells of step a) with an immunosuppressive substance or anti-inflammatory signal, eg before or after inactivating the nucleus, eg enucleating the cells.

[0382] In one aspect, provided herein is a fusosome composition comprising a plurality of fusosomes derived from a source cell, wherein the plurality of fusosomes are: (a) a lipid bilayer; a lumen containing the cytosol and surrounded by a lipid bilayer; (c) exogenous or overexpressed fusogens located within the lipid bilayer; (d) containing cargo; nucleus-free; the amount of viral capsid protein in the fusosomal composition is less than 1% of total protein; multiple fusosomes when contacted with a target cell population in the presence of an inhibitor of endocytosis; When contacted with a reference target cell population that has not been treated with an inhibitor of endocytosis, it delivers cargo to at least 30% the number of cells in the target cell population compared to the reference target cell population.

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

[0384] The composition of fusosomals can be produced from cells in culture, such as cultured mammalian cells, such as cultured human cells. Cells can be progenitor cells or non-progenitor (eg, differentiated) cells. Cells can be primary cells or cell lines (eg, mammalian, eg, human, cell lines described herein). In embodiments, the cultured cells are progenitor cells such as bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPC), endothelial progenitor cells (EPC), blast cells, intermediate progenitor cells formed in the subventricular zone, neuronal 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, cardiac myoblasts, nerves progenitor cells, glial progenitor cells, neural progenitor cells, and hepatoblasts.

[0385] In some embodiments, the source cells are 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 cells of a subject), embryonic stem cells (e.g., stem cells from embryonic yolk sac, placenta, umbilical cord, fetal skin, pubertal skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g. hepatocytes), alveolar cells, neurons (e.g. retinal neurons), progenitor cells (e.g. retinal progenitors) cells, myeloblasts, myeloid progenitor cells, thymocytes, meiocytes, megakaryoblasts, promegakaryoblasts, melanogenic blasts, lymphoblasts, myeloid progenitor cells, normoerythroblasts, or angioblasts), Progenitor cells (e.g., cardiac progenitor cells, satellite cells, radial glial cells, bone marrow stromal cells, pancreatic progenitor cells, endothelial progenitor 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 can be cells derived from epithelial, connective, muscle, or neural tissue or cells, and combinations thereof. Fusosomes can be obtained from cultured cells derived from any eukaryotic (e.g. mammalian) organ system, e.g. cardiovascular system (heart, vascular system); , colon, rectum and anus); endocrine system (hypothalamus, pituitary, pineal or pineal, thyroid, parathyroid, adrenal); excretory system (kidneys, ureters, bladder); lymphatic system (lymph, lymph nodes, lymphatic vessels, tonsils, pharyngeal tonsils, thymus, spleen); integumentary system (skin, hair, nails); muscular system (e.g., skeletal muscle); nervous system (brain, spinal cord, nerves); reproductive system (ovaries, uterus, mammary glands, testes, vas deferens, seminal vesicles, prostate); respiratory system (pharynx, larynx, trachea, bronchi, lungs, diaphragm); skeletal system (bones, cartilage), and combinations thereof. In embodiments, the cells are derived from highly mitotic tissue (eg, highly mitotic healthy tissue such as epithelium, embryonic tissue, bone marrow, intestinal crypts, etc.). In embodiments, the tissue sample is a highly metabolic tissue (eg, skeletal tissue, neural tissue, cardiomyocytes).

[0387] In some embodiments, the cells are derived from young donors, eg, donors aged 25, 20, 18, 16, 12, 10, 8, 5, 1 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 subjects with similar genetic characteristics (eg, MHC matching).

[0389] In certain embodiments, the cells are more than 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length (eg, 4,000-10,000 nucleotides in length, 6,000-10,000 nucleotides in length). Have average sized telomeres.

[0390] C. Fusogen and Pseudotyping In some embodiments, the fusosomes (e.g., including portions of vesicles or cells) described herein are conjugated with one or more fusogens, e.g., to facilitate fusosome fusion to a membrane, e.g., a cell membrane. including. These compositions may also include surface modifications during or after synthesis to include one or more fusogens. Surface modifications can include modifications to membranes, such as the insertion of lipids or proteins into membranes.

[0391] In some embodiments, the fusosome comprises one or more fusogens on its outer surface (eg, integrated into the cell membrane) to target specific cells or tissue types (eg, T cells). In some embodiments, the specific cell type targeted by one or more fusogens are T cells, 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. thymus-derived regulatory T cells, peripherally-derived regulatory T cells, CD4+ Foxp3+ regulatory CD4 + FoxP3-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 (eg, stem cell memory T cells, central memory T cells, or effector memory T cells), NKT cells, or mucosa-associated invariant T (MAIT) cells. Fusosomes may contain a targeting domain. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. Fusogens can bind to partners, eg, surface features of target cells. In some embodiments, the target cell surface partner is a target cell moiety. In certain embodiments, the fusogen is a fusogen or a retargeting fusogen that binds to target cells from among T cells, optionally the T cells are CD4+ T cells, CD8+ T cells, alphabeta 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, peripherally-derived regulatory T cells , CD4 + Foxp3 + regulatory T cells, or CD4 + FoxP3-type 1 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) are cells. In some embodiments, fusosomes containing fusogens will integrate membranes into the lipid bilayer of target cells.

[0392] In some embodiments, one or more fusogens described herein can be contained in fusosomes.

[0393] A fusosome (eg, a retroviral vector) described herein can comprise a fusogen, eg, an endogenous fusogen or a pseudotyped fusogen. i) protein fusogen

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

[0395] In embodiments, the fusogen is a viral fusogen, such as HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, such as a SNARE, Syncytin, myomaker, myomixer, myomerger, or FGFRL1. In embodiments, the fusogen is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusogen is not active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. In embodiments, the fusosome fuses to the target cell on the surface of the target cell. In embodiments, the fusogen promotes fusion in a lysosome-independent manner. In embodiments, the fusogen is a protein fusogen. In embodiments, the fusogen is a lipid fusogen, such as oleic acid, glycerol monooleate, glycerides, diacylglycerols, or modified unsaturated fatty acids. In embodiments, the fusogen is a chemical fusogen, such as PEG. In embodiments, the fusogen is a small molecule fusogen, eg, an NSAID such as halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine. In embodiments, the fusogen is recombinant. In embodiments, the fusogen is biochemically incorporated, e.g., the fusogen is provided as a purified protein and contacted with the lipid bilayer under conditions that allow association of the fusogen with the lipid bilayer. In embodiments, the fusogen is biosynthetically integrated, eg, expressed in the source cell under conditions that allow the fusogen to associate with the lipid bilayer.

[0396] In some embodiments, the fusogen (eg, retargeted fusogen) comprises a mammalian fusogen. In some embodiments, the fusogen (eg, retargeted fusogen) comprises a viral fusogen. In some embodiments, the fusogen (eg, 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, henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, comprising a sequence selected from Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or derivatives thereof.

[0397] In some embodiments, a fusogen (eg, a retargeted fusogen) is active at a pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. . In some embodiments, the fusogen (eg, retargeted fusogen) is not active at pH of 4-5, 5-6, 6-7, 7-8, 8-9, or 9-10. .

[0398] In some embodiments, the fusogen is present in a copy number 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 proteins, morbillivirus H proteins, respirovirus HN proteins, including Sendai virus HN proteins, rubulavirus HN proteins, oil virus HN proteins, or derivatives thereof. In some embodiments, the fusogen (e.g., retargeted fusogen) is 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, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubula virus including sequences selected from F and HN proteins, or Avravirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0400] Non-mammalian fusogens include viral fusogens, homologues thereof, fragments thereof, and fusion proteins comprising one or more proteins or fragments thereof. Viral fusogens include class I fusogens, class II fusogens, class III fusogens, and class IV fusogens. In embodiments, class I fusogens, such as human immunodeficiency virus (HIV) gp41, have a characteristic post-fusion conformation with a characteristic trimer of an α-helical hairpin with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, orthomyxovirus-derived hemagglutinin, paramyxovirus-derived F protein (e.g., measles (Katoh et al. BMC Biotechnology 2010, 10:37). ), ENV proteins from retroviruses, and fusogens of filoviruses and coronaviruses. In embodiments, class II viral fusogens, such as the Dengue E glycoprotein, have structural features of β-sheets that form elongated ectodomains that refold to give trimers of hairpins. In embodiments, class II virus fusogens lack a central coiled-coil. Class II viral fusogens are found in alphaviruses (eg, E1 protein) and flaviviruses (eg, E glycoprotein). Class II viral fusogens include those derived from Semliki Forest virus, Simbis, Rubella virus, and Dengue virus. In embodiments, class III viral fusogens, such as vesicular stomatitis virus G glycoprotein, combine structural features found in classes I and II. In embodiments, class III virus fusogens have an α-helix (e.g., protein folds forming six-helix bundles similar to class I virus fusogens) and a β-sheet with amphipathic fusion peptides at its ends. containing and reminiscent of class II virus fusogens. Class III viral fusogens are found in rhabdoviruses and herpesviruses. In embodiments, the Class IV viral fusogen is a fusion-associated small transmembrane (FAST) protein (doi:10.1038 / sj.emboj.7600767, Nesbitt, Rae L., "Targeted Intracellular Therapeutic Delivery Using Liposomes Formulated with Multifunctional FAST proteins"). (2012). Electronic Thesis and Dissertation Repository. Paper 388) and is encoded by a non-enveloped reovirus. In embodiments, Class IV viral fusogens are small enough not to form hairpins (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0401] Fusogens, including viral envelope proteins (env), generally determine the range of host cells that can be infected and transformed by fusosomes. For lentiviruses such as HIV-1, HIV-2, SIV, FIV, EIV, native env proteins include gp41 and gp120. In some embodiments, the viral env proteins expressed by the source cells described herein are encoded on vectors separate from the viral gag and pol genes, as previously described.

[0402] Examples of env genes from retroviruses that can be used include, but are not limited to, MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (fowl plague virus), and influenza. A viral envelope is included. Similarly, RNA viruses (e.g., Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae) , Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae RNA virus family), along with DNA viruses (Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae). Genes encoding envelopes from the viral families (Herpesviridae, Poxyiridae, and Iridoviridae) are available. 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 , EIAV and the like.

[0403] In some embodiments, envelope proteins for display on fusosomes include, but are not limited to, any of the following sources: H1N1, H1N2, H3N2 and H5N1 (bird flu), etc. influenza A, influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, intestinal adenovirus Viruses, parvovirus, dengue virus, monkeypox, mononegavirus, lyssaviruses such as rabies virus, Lagos bat virus, mokola virus, Dubenhage virus, European bat virus 1 and 2, Australian bat virus, ephemerovirus, vesiculovirus Viruses, vesicular stomatitis virus (VSV), herpes viruses such as herpes simplex virus type 1 and 2, Varicella zoster, cytomegalovirus, Epstein-Barr virus (EBV), human herpes virus (HHV), human herpes virus type 6 and type 8, human immunodeficiency virus (HIV), papilloma virus, murine gamma herpes virus, arena viruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Savia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machu Bunyaviridae including povirus, lymphocytic choriomeningitis virus (LCMV), Crimean-Congo hemorrhagic fever virus, hantaviruses, viruses that cause hemorrhagic kidney syndrome, Rift Valley fever virus, Ebola hemorrhagic fever and Marburg hemorrhagic fever Filoviridae (Filoviruses), Flaviviridae including Kyasanur forest disease virus, Omsk hemorrhagic fever virus, viruses that cause tick-borne encephalitis and Paramyxoviridae such as Hendra virus and Nipah virus, variola major and variola minor (natural Pox) alphaviruses such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephalitis-causing virus.

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

[0405] A fusosomal or pseudotyped virus generally has modifications in one or more of its envelope proteins, eg, the envelope protein has been 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, which is normally encoded by the env gene. HIV infects a wider range of cells because it targets the virus to CD4+ presenting cells. In some embodiments, the lentiviral envelope protein is pseudotyped with VSV-G. In one embodiment, the source cell produces a recombinant retrovirus, such as a lentivirus, pseudotyped with the VSV-G envelope glycoprotein.

[0406] In addition, fusogenic or viral envelope proteins allow the transduction vector to target and infect host cells outside its normal range, or more specifically limit transduction to cell or tissue types. can be modified or engineered to contain polypeptide sequences that allow For example, fusogenic or envelope proteins are displayed on receptor ligands, targeting sequences such as antibodies (using recombinant antibody-type molecules such as antigen-binding portions of antibodies or single-chain antibodies), and transducing vector coats. can be linked in-frame with a polypeptide moiety or modification thereof (eg, where a glycosylation site is present in the target sequence) that facilitates direct delivery of the virion particle to the target cell of interest. Furthermore, envelope proteins can further comprise sequences that regulate cellular function. Modulation of cell function with transduction vectors can increase or decrease the transduction efficiency of particular cell types in mixed cell populations. For example, stem cells can be more specifically transduced with an envelope sequence that contains a ligand or binding partner that specifically binds to stem cells rather than other cell types found in blood or bone marrow. Non-limiting examples are stem cell factor (SCF) and Flt-3 ligand. Other examples include, for example, antibodies (e.g., cell-type specific single-chain antibodies), and tissues such as lung, liver, pancreatic, cardiac, endothelial, smooth, breast, prostate, epithelial, and vascular cancers. Essentially any antigen (including receptor) that binds is included.

[0407] A protein fusogen or viral envelope protein can be retargeted by mutating amino acid residues of the fusogen protein or targeting protein (eg, 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 directed evolution. In some embodiments, the fusogen is truncated and only a subset of peptides are used in the retroviral vector or VLP. For example, amino acid residues in the measles hemagglutinin protein can be mutated to alter the binding properties of the protein and redirect fusion (doi:10.1038 / nbt942, Molecular Therapy vol.16 no.8, 1427-1436 Aug.2008 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 obtained by i) mutating amino acids in the native fusogen or viral envelope protein sequence, and / or ii) the fusogen or viral envelope protein is in its normal range. It is retargeted by engineering the fusogenic protein or viral envelope protein to contain polypeptide sequences that allow it to target, fuse, or infect foreign host cells.

[0409] In some embodiments, fusosomes comprise one or more fusogens on their outer surface (eg, incorporated 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 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, Henipavirus F protein, Morbillivirus F protein, Les It is a pirovirus F protein, a Sendai virus F protein, a rubula virus F protein, or an oil virus F protein.

[0411] In some embodiments, the fusogen is a poxviridae fusogen.

[0412] Additional exemplary fusogens are described in U.S. Pat. No. 2009 / 0202622, WO 2006 / 027202, and US Patent Application Publication No. 2004 / 0009604, all of which are incorporated herein by reference.

[0413] In some embodiments, the fusogens described herein are amino acid sequences of Table 1, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% Amino acid sequences with % sequence identity, or portions of said sequences, such as at least 80%, 85%, 90%, 95%, 96% for a length of 100, 200, 300, 400, 500, or 600 amino acids , includes amino acid sequences with 97%, 98%, or 99% sequence identity. For example, in some embodiments, a fusogen described herein comprises an amino acid sequence having at least 80% identity to any of the amino acid sequences in Table 1. In some embodiments, the nucleic acid sequences described herein are the amino acid sequences of Table 1, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or Amino acid sequences having 99% sequence identity, or portions of said sequences, such as at least 80%, 85% for a length of 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids , 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0414] In some embodiments, the fusogens described herein have an amino acid sequence set forth in any one of SEQ ID NOs: 1-57, or at least 80%, 85%, 90%, 95%, 96% amino acid sequences having %, 97%, 98% or 99% sequence identity, or portions of said sequences, such as at least 80%, 85 for a length of 100, 200, 300, 400, 500 or 600 amino acids It includes amino acid sequences with %, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. For example, in some embodiments, the fusogens described herein comprise an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-57. In some embodiments, the nucleic acid sequences described herein are amino acid sequences set forth in any one of SEQ ID NOs: 1-57, or at least 80%, 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98% or 99% sequence identity, or parts of said sequences, such as 40, 50, 60, 80, 100, 200, 300, 400, 500 or 600 amino acids Encode amino acid sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the length. [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 fusogens described herein are amino acid sequences of Table 2, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% Amino acid sequences with % sequence identity, or portions of said sequences, such as at least 80%, 85%, 90%, 95%, 96% for a length of 100, 200, 300, 400, 500, or 600 amino acids , includes amino acid sequences with 97%, 98%, or 99% sequence identity. For example, in some embodiments, a fusogen described herein comprises an amino acid sequence having at least 80% identity to any of the amino acid sequences in Table 2. In some embodiments, the nucleic acid sequences described herein are the amino acid sequences of Table 2, or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or Amino acid sequences having 99% sequence identity, or portions of said sequences, such as at least 80%, 85% for a length of 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids , 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0416] In some embodiments, the fusogens described herein have an amino acid sequence set forth in any one of SEQ ID NOS: 58-133, or at least 80%, 85%, 90%, 95%, 96% amino acid sequences having %, 97%, 98% or 99% sequence identity, or portions of said sequences, such as at least 80%, 85 for a length of 100, 200, 300, 400, 500 or 600 amino acids It includes amino acid sequences with %, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. For example, in some embodiments, the fusogens described herein comprise an amino acid sequence having at least 80% identity to the amino acid sequence set forth in any one of SEQ ID NOs:58-133. In some embodiments, the nucleic acid sequences described herein are amino acid sequences set forth in any one of SEQ ID NOS: 58-133, or at least 80%, 85%, 90%, 95%, Amino acid sequences with 96%, 97%, 98% or 99% sequence identity, or parts of said sequences, such as 40, 50, 60, 80, 100, 200, 300, 400, 500 or 600 amino acids Encode amino acid sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over the length. [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 can be treated with fusogenic lipids such as saturated fatty acids. In some embodiments, saturated fatty acids have 10-14 carbons. In some embodiments, saturated fatty acids have longer chain carboxylic acids. In some embodiments, saturated fatty acids are monoesters.

[0418] In some embodiments, fusosomes can be treated with unsaturated fatty acids. In some embodiments, the unsaturated fatty acids comprise C16-C18 unsaturated fatty acids. In some embodiments, unsaturated fatty acids include oleic acid, glycerol monooleate, glycerides, diacylglycerols, modified unsaturated fatty acids, and any combination thereof.

[0419] While not wishing to be bound by theory, in some embodiments negative curvature lipids promote membrane fusion. In some embodiments, the fusosome comprises one or more negatively curved lipids in the membrane, eg, negatively curved lipids exogenous to the source cell. In embodiments, negatively curved lipids or their precursors are added to the medium containing the source cells or fusosomes. In embodiments, the source cell is engineered to express or overexpress one or more lipogenic genes. Negatively curved lipids can be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acids (FA).

[0420] While not wishing to be bound by theory, in some embodiments positive curvature lipids inhibit membrane fusion. In some embodiments, the fusosomes comprise reduced levels of one or more positively curved lipids, eg, exogenous positively curved lipids, in the membrane. In embodiments, levels are reduced by inhibiting lipid synthesis, eg, by knocking out or knocking down lipid synthesis genes in the source cell. Positively curved lipids can be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

[0421] iii) chemical fusogen In some embodiments, fusosomes can be treated with fusogenic chemicals. In some embodiments, the fusogenic chemical is polyethylene glycol (PEG) or a derivative thereof.

[0422] In some embodiments, chemical fusogens induce local dehydration between two membranes, which leads to unfavorable molecular packing of the bilayer. In some embodiments, chemical fusogens induce dehydration of regions near the lipid bilayer, causing displacement of aqueous molecules between cells and allowing interactions between the two membranes.

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

[0424] In some embodiments, chemical fusogens bind to target membranes by altering surface polarity, which alters hydration-dependent transmembrane repulsion.

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

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

[0427] In some embodiments, chemical fusogens are small organic molecules. A non-limiting example includes n-hexyl bromide.

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

[0429] In some embodiments, chemical fusogens are hormones or vitamins. Some non-limiting examples include abscisic acid, retinol (vitamin A1), tocopherol (vitamin E), and variants and derivatives thereof.

[0430] In some embodiments, the fusosomes comprise agents that stabilize actin and polymerized actin. While not wishing to be bound by theory, stabilized actin within fusosomes can facilitate fusion with target cells. In embodiments, the agent that stabilizes polymerized actin is selected from actin, myosin, biotin-streptavidin, ATP, neuronal Wiskott-Aldrich syndrome protein (N-WASP), or formin. 2011 Aug 16;27(16):10061-71 and Wen et al., Nat Commun.2016 Aug 31;7. In embodiments, the fusosomes comprise actin that is exogenous or overexpressed to the source cell, such as wild-type actin or actin containing mutations that promote polymerization. In embodiments, the fusosomes contain ATP or phosphocreatine, eg, exogenous ATP or phosphocreatine.

[0431] iv) small molecule fusogens In some embodiments, fusosomes can be treated with fusogenic 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 micellar-like aggregates or may be aggregate-free.

[0433] v) fusogenic modification In some embodiments, the fusogen is linked to a cleavable protein. In some cases, a cleavable protein can be cleaved by exposure to a protease. Engineered fusion proteins can bind to any domain of a transmembrane protein. The engineered fusion protein can be linked by a truncated peptide to a protein domain located within the intermembrane space. Cleavage peptides are preferred for transmembrane proteases (e.g., HTRA2 / OMI requiring non-polar aliphatic amino acids-valine, isoleucine or methionine at the -P1 position, hydrophilic residues-arginine at the -P2 and P3 positions). (preferred in ), or a combination thereof.

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

[0435] In some embodiments, the fusogenic protein is engineered by any method known in the art or described herein to include proteolytic sequences, such as mitochondrial or cytosolic degradation sequences. be. The fusogenic protein has a proteolytic sequence, such as the 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), modified proteolytic sequences with at least 75%, 80%, 85%, 90%, 95% or more identity to the wild-type proteolytic sequence, cytoplasmic proteolysis Although it can be engineered to contain a sequence, such as ubiquitin, or a modified cytoplasmic proteolytic sequence that has at least 75%, 80%, 85%, 90%, 95% or more identity to the wild-type proteolytic sequence. , but not limited to. In some embodiments, the composition comprises a protein that has been modified with a proteolytic sequence, e.g., at least 75%, 80%, 85%, 90%, 95% or more identity to a wild-type proteolytic sequence. , mitochondria in the source cell or chondrisome, cytosolic proteolytic sequences such as ubiquitin, or modified having at least 75%, 80%, 85%, 90%, 95% or more identity to the wild-type proteolytic sequence contains cytosolic proteolytic sequences.

[0436] In some embodiments, fusogens can be modified with a protease domain that recognizes overexpression of a particular protein, eg, a protease, eg, an engineered fusion protein with protease activity. For example, a protease or a protease domain derived from a protease, eg, MMP mitochondrial processing peptidase, mitochondria, intermediate peptidase, inner membrane peptidase.

[0437] Alfonzo,J.D.&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 P.M.m et al.,New roles for mitochondrial proteases in health , aging and disease. Nature Reviews Molecular Cell Biology. V16, 2015; Weber-Lotfi, F. et al. DNA import competence and mitochondrial genetics.

[0438] III. Positive Target Cell-Specific Regulatory Elements In some embodiments, the fusosomes, e.g., viruses, e.g., retroviruses, described herein extend the half-life of tissue-specific promoters, tissue-specific enhancers, tissue-specific splice sites, RNA or proteins. Nucleic acids containing positive target cell-specific regulatory elements such as tissue-specific sites, tissue-specific mRNA export-enhancing sites, tissue-specific translational enhancement sites, or tissue-specific post-translational modification sites (e.g., exogenous agents genes encoding), including, for example, retroviral nucleic acids.

[0439] In some embodiments, the fusosomes, e.g., retroviruses, described herein comprise regions such as origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, Polyadenylation sequences, 5' and 3' untranslated regions (which interact with host cellular proteins to carry out transcription and translation and direct, increase, regulate or control the transcription or expression of an operably linked polynucleotide) can contain untranslated regions such as retroviral nucleic acids. Such elements may differ in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, can be used.

[0440] In certain embodiments, control elements are capable of directing, increasing, regulating, or controlling transcription or expression of operably linked polynucleotides in a cell-specific manner. In certain embodiments, the retroviral nucleic acid comprises one or more expression control sequences specific for a particular cell, cell type, or cell lineage, e.g., target cell, i.e., a particular cell, cell type, or cell lineage. Expression of a polynucleotide operably linked to a strain-specific expression control sequence is expressed in target cells, but not (or expressed at a lower level) in non-target cells.

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

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

[0443] In embodiments, an enhancer comprises a segment of DNA containing sequences that can provide enhanced transcription and, optionally, can function independently of its orientation relative to other regulatory sequences. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. In some embodiments, the promoter / enhancer segment of DNA includes sequences that can provide both promoter and enhancer functions.

[0444] Exemplary ubiquitous expression control sequences include the cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV). ) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-alpha (EF1a) 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 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 ( HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (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, d1587rev primer binding site replacement (MND) promoters (Challita et al., J Virol. 69(2):748-55 (1995)).

[0445] In some embodiments, a promoter may be paired with a heterologous gene to confer a regulatory function of that promoter on the heterologous gene. In some embodiments, cis-regulatory elements from the promoter of a first gene may be joined to segments of the promoter of a different gene to create a chimeric promoter with properties of both promoters.

[0446] In some embodiments, the promoter is a tissue-specific promoter, e.g., 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., thymus-derived regulatory T cells, peripheral-derived regulatory T cells, CD4+Foxp3+ regulatory T cells, or CD4+FoxP3- type 1 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) , the promoter driving expression in memory T cells (eg, 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 have in their nucleic acids a promoter having the sequence of Table 3, or a transcriptionally active fragment thereof, or at least 70%, 75 %, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the fusosomes (e.g., viral vectors) described herein have in their nucleic acids transcription factor binding sites derived from regions within 3 kb of the transcription start sites of the genes listed in Table 3. including promoters with In some embodiments, the fusosomes (e.g., viral vectors) described herein have in their nucleic acids 2.5 kb, 2 kb, 1.5 kb immediately upstream of the transcription start site of the genes listed in Table 3, A region within 1 kb, or 0.5 kb, or a transcriptionally active fragment thereof, or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% thereof includes variants that have the identity of [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 its entirety, e.g., the T cell-specific promoter is IL2RA (CD25), LRRC32, FOXP3, or IKZF2 promoters. 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 its entirety. incorporated into the specification. In some embodiments, the T-cell specific promoter is a transcriptionally active fragment of any of the above. In some embodiments, the T cell-specific promoter is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or A variant with 99% identity.

[0448] An internal ribosome entry site (IRES) normally facilitates direct internal ribosome entry into the initiation codon (such as ATG) of a cistron (protein-coding region), thereby resulting in cap-independent translation of the gene. See, eg, 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 agents. In certain embodiments, to achieve efficient translation of each of a plurality of exogenous protein agents, the polynucleotide sequences are separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides. can be

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

[0450] Promoters Responsive to Heterologous Transcription Factors and Inducers In some embodiments, the retroviral nucleic acid comprises conditional expression of exogenous agents, including but not limited to inducible expression, repressible expression, cell type specific expression, or tissue specific expression. Contains elements that allow for any type of conditional expression. In some embodiments, to achieve conditional expression of the exogenous agent, the cell, tissue, or organism is caused to express the exogenous agent or to increase or decrease expression of the exogenous agent. Expression is controlled by subjecting it to a treatment or condition.

[0451] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as the promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible by interferon), 'GeneSwitch' mifepristone regulatable system (Sirin et al., 2003, Gene, 323:67), cumate induction sex gene switch (WO 2002 / 088346), tetracycline-dependent regulatory system, and the like.

[0452] Transgene expression can be activated or repressed by the presence or absence of an inducer molecule. In some cases, the inducer molecule activates or represses gene expression in a stepwise fashion, and in some cases, the inducer molecule activates or represses gene expression in an all-or-nothing fashion. .

[0453] A commonly used inducible 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 containing repeats of the Tet operator sequence (TetO) fused to a minimal promoter and connected to the gene of interest ( For example, genes encoding exogenous agents) and (ii) the transcriptional transactivator (tTA), a fusion protein of the Tet repressor (TetR), and the transactivation domain of the VP16 protein from herpes simplex virus. In the first described version, expression of the transgene was active in the absence of tetracycline or its potent analogue doxycycline (Do) (termed the Tet-OFF system), whereas the expression of the transgene within the transactivator protein was active. Four amino acid modifications resulted in reverse tTA (rtTA) that binds TetO only in the presence of Dox (Tet-ON system). In some embodiments, in the transactivator, the VP16 domain is replaced by a minimal activation domain, potential splice donor and splice acceptor sites are removed, the protein is codon optimized, and Dox resulted in an improved transactivator variant, rtTA2S-M2, with increased sensitivity to and reduced baseline activity. In addition, a variety of Tet-responsive promoter elements have been generated that contain alterations of TetO at 36-nucleotide intervals from the flanking operator to enhance regulation. Additional modifications may serve to further reduce basal activity and increase expression dynamic range. As an example, the pTet-T11 (abbreviation: 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 has one or more recombination sites (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 variants, derivatives (e.g., fusion proteins comprising recombinant protein sequences or fragments thereof). ), fragments, and variants thereof, for recombination mediated by excision or integration proteins, enzymes, cofactors or associated proteins involved in the recombination reaction. Contains part(s). Examples of recombinases include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1 and ParA.

[0455] Riboswitches that regulate the expression of exogenous agents Some of the compositions and methods provided herein involve one or more riboswitches or polynucleotides that include one or more riboswitches. Riboswitches are a common function of bacteria to regulate gene expression and are a means of achieving RNA control of biological functions. Riboswitches can reside in the 5' untranslated region of mRNA and can allow regulatory control of gene expression through the binding of small molecule ligands that induce or repress riboswitch activity. In some embodiments, the riboswitch controls gene products involved in the production of small molecule ligands. Although riboswitches typically operate in the cis mode, riboswitches have been identified that operate in the trans mode. Natural riboswitches are composed of two domains, an aptamer domain that binds ligands through a three-dimensional folded RNA structure, and a function-switching domain that induces or represses the activity of the riboswitch based on the presence or absence of ligand. It is configured. Therefore, there are two ligand-sensitive conformations achieved by riboswitches, representing an on state and an off state (Garst et al., 2011). Functional switching domains influence polynucleotide expression by regulating internal ribosome entry sites, pre-mRNA splice donor accessibility in retroviral gene constructs, translation, termination of transcription, 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 regulate 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; US20080269258; and WO2006055351). Purine riboswitches consist of three conserved helical / stem structures (PI, P2, P3) and an intervening loop / junctional element (Jl-2, L2, J2-3, L3, J3-1). share a structure. Aptamer domains of the purine family of riboswitches are naturally altered in affinity / regulation by various purine compounds such as adenine, guanine, adenosine, guanosine, deoxyadenosine, and deoxyguanosine upon sequence variation (Kim et al. 2007). ).

[0457] In some embodiments, a retroviral nucleic acid described herein comprises a polynucleotide encoding an exogenous agent operably linked to a promoter and a riboswitch. Riboswitches include one or more, e.g., all of the following: a.) aptamer domains, e.g. '-Aptamer domain with reduced binding to deoxyguanosine; b.) a function-switching domain, e.g., a function-switching domain capable of modulating the expression of an exogenous agent, wherein the aptamer domain binds to nucleoside analogues; Binding induces or represses the expression modulating activity of the functional switching domain, thereby modulating expression of the exogenous agent. In some embodiments, an exogenous agent can be a polypeptide, miRNA, or shRNA. For example, in one embodiment, a riboswitch is operably linked to a nucleic acid encoding a chimeric antigen receptor (CAR). In the non-limiting examples provided herein, exogenous genes encode one or more engineered signaling polypeptides. For example, a target polynucleotide encoding a riboswitch and one or more engineered signaling polynucleotides can be found in the genome of the source cell, a recombinant replication-incompetent retroviral particle, T cells and / or NK cells. can.

[0458] Aptamer domains, for example, can be used as modular components and combined with any of the functional switching domains to affect RNA transcripts. In any of the embodiments disclosed herein, the riboswitch can affect RNA transcripts by modulating any of the following activities: internal ribosome entry site (IRES), pre-mRNA splice donor accessibility, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression. In some embodiments, the functional switching domain can regulate binding of an anti-IRES to an IRES (see, e.g., Ogawa, RNA (2011), 17:478-488, the disclosure of which is incorporated herein by reference). incorporated herein in its entirety). In any of the embodiments disclosed herein, the presence or absence of a small molecule ligand provides the potential for riboswitch influence on RNA transcripts. In some embodiments, a riboswitch can comprise a ribozyme. Riboswitches with ribozymes can inhibit or enhance transcript degradation of target polynucleotides in the presence of small molecule ligands. In some embodiments, the ribozyme can be the pistol class of ribozymes, the hammerhead class of ribozymes, the twist class of ribozymes, the hatchet class of ribozymes, 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 is a tissue-specific miRNA recognition sequence, tissue-specific protease recognition site, tissue-specific ubiquitin ligase site, tissue-specific transcription repression site, or tissue Contains specific epigenetic suppression sites.

[0460] In some embodiments, non-target cells contain endogenous miRNAs. A retroviral nucleic acid (eg, a gene encoding an exogenous agent) can contain a recognition sequence for that miRNA. Therefore, miRNAs may downregulate the expression of exogenous agents when retroviral nucleic acids enter non-target cells. This helps increase specificity between target and non-target cells.

[0461] In some embodiments, miRNAs are small noncoding RNAs of 20-22 nucleotides, typically excised from a foldback RNA precursor structure of about 70 nucleotides known as pre-miRNA. In general, miRNAs negatively regulate their targets in one of two ways, depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind with perfect or near-perfect complementarity to protein-encoding mRNA sequences typically induce the RNA-mediated interference (RNAi) pathway. miRNAs that exert regulatory effects by binding to imperfect complementary sites within the 3′ untranslated region (UTR) of mRNA targets are commonly found in RISC complexes similar to, or possibly the same as, those used in the RNAi pathway. , it represses target gene expression at the post-transcriptional and translational level. Consistent with translational regulation, miRNAs using this mechanism reduce protein levels of target genes, whereas mRNA levels of these genes are minimally affected. miRNAs (eg, naturally occurring miRNAs or artificially designed miRNAs) can specifically target any mRNA sequence. For example, in one embodiment, one can design a short hairpin RNA construct that is expressed as a human miRNA (eg, miR-30 or miR-21) primary transcript. This design adds a Drosha processing site to the hairpin structure and has been shown to greatly improve knockdown efficiency (Pusch et al., 2004). The hairpin stem consists of a 22 nt dsRNA (eg, the antisense has perfect complementarity to the target of interest) and a 15-19 nt loop derived from a human miR. Addition of miR loops and miR30 flanking sequences 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 without microRNAs. Increased Drosha and Dicer processing lead to increased siRNA / miRNA production and increased potency of the expressed hairpins.

[0462] Hundreds of different miRNA genes are differentially expressed during development and between tissue types. Several studies have suggested important regulatory roles for miRNAs in a wide range of biological processes, such as developmental timing, cell differentiation, proliferation, apoptosis, oncogenesis, insulin secretion, and cholesterol biosynthesis. (Bartel 2004 Cell 116:281-97; Ambros 2004 Nature 431:350-55; Du et 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 indicates 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 broadly contributes to the tissue specificity of mRNA expression in many human tissues. (See Sood et al. 2006 PNAS USA 103(8):2746-51)

[0463] Thus, miRNA-based approaches limit the expression of exogenous agents to target cell populations by silencing their expression in non-target cell types using endogenous microRNA species. can be used for MicroRNAs induce sequence-specific post-transcriptional gene silencing in many organisms by either inhibiting translation of messenger RNA (mRNA) or causing mRNA degradation. See, eg, Brown et al.2006 Nature Med.12(5):585-91., and WO2007 / 000668, each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral nucleic acid comprises one or more (eg, multiple) tissue-specific miRNA recognition sequences. In some embodiments, tissue-specific miRNA recognition sequences are about 20-25, 21-24, or 23 nucleotides in length. In embodiments, the tissue-specific miRNA recognition sequences have perfect complementarity to miRNAs present in non-target cells. In some embodiments, the exogenous agent does not include GFP, eg, does not include a fluorescent protein, eg, does not include a reporter protein. In some embodiments, off-target cells are not hematopoietic cells and / or the miRNA is absent in hematopoietic cells.

[0464] In some embodiments, the methods herein comprise tissue-specific expression of an exogenous agent in a target cell, comprising nucleotides encoding the exogenous agent and at least one tissue-specific microRNA (miRNA) with a plurality of cells, including target cells and non-target cells, wherein the exogenous agent is expressed preferentially, eg, exclusively, to the target cells.

[0465] For example, a retroviral nucleic acid can include at least one miRNA recognition sequence operably linked to a nucleotide sequence having the corresponding miRNA in non-target cells, e.g., hematopoietic progenitor cells (HSPCs), hematopoietic stem cells (HSCs). As such, it prevents or reduces expression of the nucleotide sequence in non-target cells, but not in target cells, eg, differentiated cells. In some embodiments, the retroviral nucleic acid is present in non-target cells in an effective amount (e.g., the concentration of the endogenous miRNA is sufficient to reduce or prevent transgene expression). It contains the miRNA sequence target and also contains the transgene. In embodiments, the miRNAs used in this system are strongly expressed in non-target cells such as HSPCs and HSCs, but not in differentiated progeny of e.g. prevent or reduce transgene expression in susceptible stem cell populations while maintaining

[0466] In some embodiments, a negative TSCRE or NTSCRE comprises 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) is complementary to or at least 50%, 60%, 70%, 75%, 80% , 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity. In some embodiments, the nucleic acid (eg, fusosomal nucleic acid or retroviral nucleic acid) comprises a sequence that is fully complementary to a seed sequence within an endogenous miRNA, eg, the miRNAs of Table 4. In embodiments, the seed sequence is at least 6, 7, 8, 9, or 10 nucleotides long. [Table 4]

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

[0468] In some embodiments, the fusosomes described herein comprise a nucleic acid comprising a payload gene and a positive target cell-specific regulatory element, e.g., the target cell is a Treg cell, e.g., a thymus-derived regulatory T cell. , peripherally-derived regulatory T cells, CD4 + Foxp3 + regulatory T cells, or CD4 + FoxP3-1 type regulatory T (Tr1) cells. In some embodiments, the nucleic acid further comprises a non-target cell-specific regulatory element (NTSCRE), e.g., the NTSCRE provides miRNA recognition sites for miRNAs expressed in non-Treg cells, e.g., conventional CD4+ T cells. include.

[0469] In some embodiments, a negative TSCRE or NTSCRE comprises a miRNA recognition site for a 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; 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 30;12(9):735-9;Calin et al.,Proc Natl Acad Sci U S 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 USA.2002 Nov 26;99(24):15524-9 2004 Oct;36(10):1079-83; Michael et al. Mol Cancer Res.2003 Oct;1(12):882-91; and those found at www.miRNA.org is mentioned.

[0470] In some embodiments, the negative TSCRE or NTSCRE is 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, a nucleic acid (eg, retroviral nucleic acid) comprises two or more miRNA recognition sites. In some embodiments, the first miRNA recognition site and the second miRNA recognition site are recognized by the same miRNA, and in some embodiments, the first miRNA recognition site and the second miRNA recognition site are Recognized by different miRNAs. In some embodiments, the first miRNA recognition site and the second miRNA recognition site are recognized by miRNAs present in the same non-target cell; miRNA recognition sites are recognized by miRNAs present in different non-target cells. In some embodiments, one or both of the first miRNA recognition site and the second miRNA recognition site are recognized by the miRNAs of Table 4. In some embodiments, one or more miRNA recognition sites on a fusosomal nucleic acid (eg, retroviral nucleic acid) are transcribed in cis with an exogenous agent. In some embodiments, one or more miRNA recognition sites on a fusosomal nucleic acid (eg, retroviral nucleic acid) are located downstream of the polyA tail sequence, eg, between the polyA tail sequence and WPRE. In some embodiments, one or more miRNA recognition sites on a fusosomal nucleic acid (eg, retroviral nucleic acid) are located downstream of WPRE.

[0472] V. Immunomodulation In some embodiments, the fusosomes, eg, retroviral vectors or VLPs described herein, comprise elevated CD47. See, eg, US Pat. No. 9,050,269, which is incorporated herein by reference in its entirety. In some embodiments, the fusosomes, eg, retroviral vectors or VLPs described herein, comprise elevated complement regulatory proteins. See, for example, Spanish Patent No. 2,627,445 T3 and US Patent No. 6,790,641, each of which is incorporated herein by reference in its entirety. In some embodiments, the fusosomes, eg, retroviral vectors or VLPs described herein, lack or contain reduced levels of MHC proteins, eg, MHC-1 class 1 or class II. See, eg, US Patent Application Publication No. 20170165348, which is incorporated herein by reference in its entirety.

[0473] Fusosomes, such as retroviral vectors or VLPs, may be recognized by the subject's immune system. In the case of enveloped viral vector particles (eg, retroviral vector particles), membrane-bound proteins displayed on the surface of the viral envelope can be recognized and the viral particles themselves can be neutralized. Furthermore, upon infection of a target cell, the viral envelope may integrate with the cell membrane such that viral envelope proteins are displayed on the cell's surface or remain closely associated with the cell's surface. Therefore, the immune system may also target cells infected with viral vector particles. Both effects can lead to decreased efficacy of exogenous agent delivery by viral vectors.

[0474] The viral particle envelope is usually derived from the membrane of the source cell. Therefore, membrane proteins expressed in the cell membrane from which the viral particle buds may be incorporated into the viral envelope. immunomodulatory protein CD47

[0475] Internalization of extracellular substances into cells is carried out by a process commonly 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 falls into two general categories: phagocytosis, which involves the uptake of particles, and pinocytosis, which involves the uptake of fluids and solutes.

[0476] Based on studies with knockout mice lacking the membrane receptor CD47, professional phagocytes have been shown to distinguish between nonself and self (Oldenborg et al., 2000, Science 288(5473):2051-4). ). CD47 is a ubiquitous member of the Ig superfamily that interacts with the immunoinhibitory receptor SIRPα (signal-regulating protein) found on 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). Although the CD47-SIRPα interaction appears to inactivate autologous macrophages in mice, severe reductions in CD47 (perhaps 90%) are associated with several Rh genotypes that show little or no evidence of anemia in humans. It is found in blood cells (Mouro-Chanteloup et al., 2003, Blood 101(1):338-344), with little or no evidence of enhanced cellular interactions with phagocytic monocytes (Arndt et al. , 2004, Br. J. Haematol. 125(3):412-4).

[0477] In some embodiments, a retroviral vector or VLP (e.g., a viral particle having a radius of less than about 1 μm, less than about 400 nm, or less than about 150 nm) is coated with CD47, e.g., on the exposed surface of the retroviral vector or VLP. It contains at least a biologically active portion. In some embodiments, the retroviral vector (eg, lentivirus) or VLP comprises a lipid coat. 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 is. In some embodiments, the CD47 is human CD47.

[0478] The methods described herein can include avoiding phagocytosis of particles by phagocytic cells. The method comprises exposing a retroviral vector or VLP containing CD47 to phagocytic cells and determining whether the viral particles escape phagocytosis by the phagocytic cells or are compared to an otherwise similar unmodified retroviral vector or VLP. and expressing at least one peptide comprising at least a biologically active portion of CD47 in a retroviral vector or VLP so as to exhibit reduced phagocytosis. In some embodiments, the half-life of the retroviral vector or VLP in the subject is increased compared to the unmodified retroviral vector or VLP.

[0479] Deletion of MHC Major histocompatibility complex class I (MHC-I) is a host cell membrane protein that can integrate into the viral envelope and is highly polymorphic in nature, making it a major target of the body's immune response (McDevitt H.O. (2000) Annu.Rev.Immunol.18:1-17 MHC-I molecules exposed to the plasma membrane of the source cell may be incorporated into the viral particle envelope during vector budding. These MHC-I molecules, derived and incorporated into the viral particle, can then be transferred to the plasma membrane of the target cell, or MHC-I molecules can be absorbed and bound by the viral particle to the target cell membrane. As a result of their tendency to remain, they may remain closely associated with the target cell membrane.

[0480] The presence of exogenous MHC-I molecules on or near the plasma membrane of transduced cells can induce an alloreactive immune response in a subject. This can result in immune-mediated killing or phagocytosis of transduced cells, either by ex vivo gene transfer followed by administration of transduced cells to a subject, or by direct in vivo administration of viral particles. be. Furthermore, when MHC-I with viral particles are administered to the blood stream in vivo, the viral particles can be neutralized by pre-existing MHC-I specific antibodies before they reach their target cells.

[0481] Thus, in some embodiments, the source cell is modified (eg, genetically engineered) to reduce expression of MHC-I on the surface of the cell. In embodiments, the source comprises a genetically engineered disruption of the gene encoding β2-microglobulin (β2M). In embodiments, the source cell contains a genetically engineered disruption of one or more genes encoding the MHC-I α chain. Cells may contain engineered disruptions in all copies of the gene encoding β2-microglobulin. Cells may contain engineered disruptions in all copies of the gene encoding the MHC-I α chain. Cells can contain both a genetically engineered disruption of the gene encoding β2-microglobulin and a genetically engineered disruption of the gene encoding the MHC-I α chain. In some embodiments, the retroviral vector or VLP comprises a reduced number of surface-exposed MHC-I molecules. The number of surface-exposed MHC-I molecules can be reduced such that the immune response to MHC-I is reduced to a therapeutically relevant extent. In some embodiments, the enveloped viral vector particle substantially lacks surface-exposed MHC-I molecules.

[0482] Overexpression of HLA-G / E In some embodiments, the retroviral vector or VLP has on its envelope a tolerogenic protein, such as an ILT-2 or ILT-4 agonist, such as HLA-E or HLA-G, or any other of ILT-2 or ILT-4 agonists. In some embodiments, the retroviral vector or VLP is HLA-E, HLA-G, ILT-2 compared to a reference retrovirus, e.g. Or the expression of ILT-4 is increased.

[0483] In some embodiments, the retroviral composition has decreased MHC class I compared to unmodified retrovirus and increased HLA-G compared to unmodified retrovirus.

[0484] In some embodiments, the retroviral vector or VLP has increased expression of HLA-G or HLA-E, e.g., a reference cell retrovirus, e.g. In comparison, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of HLA-G or HLA-E expression Resulting in an increase, HLA-G or HLA-E expression is assayed in vitro using flow cytometry, eg FACS.

[0485] In some embodiments, retroviruses with increased HLA-G expression exhibit reduced immunogenicity, eg, as measured by reduced immune cell infiltration in teratoma formation assays.

[0486] complement regulatory protein Complement activity is normally controlled by several complement regulatory proteins (CRPs). These proteins prevent pseudo-inflammation and host tissue damage. One group of proteins, including CD55 / decay accelerating factor (DAF) and CD46 / membrane cofactor protein (MCP), inhibits C3 / C5 convertases of the canonical and alternative pathways. Another set of proteins, including CD59, regulate MAC assembly. CRP has been used to prevent rejection of xenograft tissue and has also been shown to protect viruses and viral vectors from complement inactivation.

[0487] Examples of complement regulatory factors present in the membrane include decay accelerating factor (DAF) or CD55, factor H (FH)-like protein-1 (FHL-1), C4b binding protein (C4BP), complement receptor 1 ( CD35), membrane cofactor protein (MCP) or CD46, and CD59 (protectin) (eg, to prevent membrane attack complex (MAC) formation and protect cells from lysis).

[0488] albumin binding protein In some embodiments, the lentivirus binds albumin. In some embodiments, the lentivirus comprises a protein on its surface that binds albumin. In some embodiments, the lentivirus comprises an albumin binding protein on its surface. In some embodiments, the albumin binding protein is a streptococcal albumin binding protein. In some embodiments, the albumin binding protein is a streptococcal albumin binding domain.

[0489] Expression of nonfusogenic proteins in the lentiviral envelope In some embodiments, the lentivirus is engineered to contain one or more proteins on its surface. In some embodiments, the protein affects immune interaction with a subject. In some embodiments, the protein affects lentiviral pharmacology in a subject. In some embodiments the protein is a receptor. In some embodiments the protein is an agonist. In some embodiments the protein is a signaling molecule. In some embodiments, the protein on the lentiviral surface comprises OKT3 or IL7.

[0490] In some embodiments, the mitogenic transmembrane protein and / or cytokine-based transmembrane protein is present in the source cell, which can be incorporated into the retrovirus as it buds from the source cell membrane. Mitogenic transmembrane proteins and / or cytokine-based transmembrane proteins can be expressed as separate cell surface molecules on the source cell rather than being part of the viral envelope glycoproteins.

[0491] Exemplary features In some embodiments of any of the aspects delineated herein, the retroviral vector, VLP, or pharmaceutical composition is substantially non-immunogenic. Immunogenicity can be quantified, for example, as described herein.

[0492] In some embodiments, retroviral vectors or VLPs are fused with target cells to produce recipient cells. In some embodiments, recipient cells fused to one or more retroviral vectors or VLPs are evaluated for immunogenicity. In embodiments, recipient cells are analyzed for the presence of antibodies on the cell surface, eg, by staining with anti-IgM antibodies. In other embodiments, immunogenicity is assessed by a PBMC cytolysis assay. In embodiments, recipient cells are incubated with peripheral blood mononuclear cells (PBMC) and then assessed for cell lysis by PBMC. In other embodiments, immunogenicity is assessed by a natural killer (NK) cell lysis assay. In embodiments, recipient cells are incubated with NK cells and then evaluated for cell lysis by NK cells. In other embodiments, immunogenicity is assessed by a CD8+ T cell lysis assay. In embodiments, recipient cells are incubated with CD8+ T cells and then assessed for lysis of cells by CD8+ T cells.

[0493] In some embodiments, the retroviral vector or VLP is compared to a reference retroviral vector or VLP, e.g., produced from an otherwise unmodified source cell similar to the source cell, or HEK293 cells. , contain high levels of immunosuppressive substances (eg, immunosuppressive proteins). In some embodiments, the level increases by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 3-fold, 5-fold , 10x, 20x, 50x, or 100x. In some embodiments, the retroviral vector or VLP contains an immunosuppressive substance not present in the reference cell. In some embodiments, the retroviral vector or VLP is compared to a reference retroviral vector or VLP, e.g., produced from an otherwise unmodified source cell similar to the source cell, or HEK293 cells. , containing reduced levels of immunostimulatory substances (eg, immunostimulatory proteins). In some embodiments, the reduced level is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, compared to a reference retroviral vector or VLP. 90%, 95%, 98%, or 99%. In some embodiments, the immunostimulatory substance is substantially absent from the retroviral vector or VLP.

[0494] In some embodiments, the retroviral vector or VLP or the source cell from which the retroviral vector or VLP is derived is 1, 2, 3, 4, 5, 6, 7, 8, Having 9, 10, 11, 12, or more of the following characteristics: a. MHC class I or MHC class II compared to a reference retroviral vector or VLP, e.g., an unmodified retroviral vector or VLPs derived from otherwise similar source cells, HeLa cells or HEK293 cells. less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% expression; b. compared to VLPs derived from a reference retroviral vector or VLP, e.g., an unmodified retroviral vector, or cells otherwise similar to the source cell, or HEK cells, or reference cells described herein , but not limited to LAG3, ICOS-L, ICOS, Ox40L, OX40, CD28, B7, CD30, CD30L 4-1BB, 4-1BBL, SLAM, CD27, CD70, HVEM, LIGHT, B7-H3 or Less than 50%, 40%, 30%, 20%, 15%, 10%, or 5% expression of one or more co-stimulatory proteins, including B7-H4; c. Expression of a surface protein that inhibits macrophage phagocytosi...

Claims

1. below: a) a lipid bilayer comprising a retargeted paramyxoviral fusogen, said fusogen linked to a targeting domain, said targeting domain being an antibody, an antigen-binding portion of an antibody, or a receptor ligand, said retargeted paramyxoviral fusogen directing preferential delivery of an exogenous agent to T cells over non-T cells; and b) a nucleic acid, (i) a payload gene encoding the exogenous agent; and (ii) a positive target cell-specific regulatory element operably linked to the payload gene, wherein the positive target cell-specific regulatory element increases expression of the payload gene in the T cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element. A nucleic acid comprising containing, fusosomes.

2. below: (i) the fusosome further comprises a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer; and / or The fusosome of claim 1, wherein (ii) a first immunostimulatory protein is not present in the fusosome or the fusosome further comprises a first immunostimulatory protein that is present at a reduced level, the reduced level being at least a 10% reduction compared to fusosomes produced from an otherwise similar unmodified source cell.

3. the immunosuppressive protein is a complement regulatory protein or CD47; and / or The immunostimulatory protein is an MHC I or MHC II protein; The fusosome of claim 2.

4. The fusosome of any one of claims 1 to 3, wherein the exogenous agent is a chimeric antigen receptor (CAR) or a chimeric autoantibody receptor (CAAR).

5. The fusosome of any one of claims 1 to 4, wherein the exogenous agent is a CAR, and the CAR targets an antigen that is a B cell antigen, an autoantigen, an antigen characteristic of a neoplastic cell, or an antigen characteristic of an infectious disease.

6. The fusosome according to any one of claims 1 to 5, wherein the T cell is a CD4+ T cell, a CD8+ T cell, an alpha beta T cell, a gamma delta T cell, a naive T cell, an effector T cell, a cytotoxic T cell, a CD8+ cytotoxic T cell, a regulatory T cell, a thymus-derived regulatory T cell, a peripherally-derived regulatory T cell, a CD4+ Foxp3+ regulatory T cell, or a CD4+ FoxP3-1 type regulatory T (Tr1) cell, a helper T cell, a CD4+ helper T cell, a Th1 cell, a Th2 cell, a Th3 cell, a Th9 cell, a Th17 cell, a Th22 cell, or a T follicular helper (Tfh) cell, a memory T cell, a stem cell memory T cell, a central memory T cell, or an effector memory T cell, a NKT cell, or a mucosa-associated invariant T (MAIT) cell.

7. The fusosome according to any one of claims 1 to 6, wherein the T cell is a Treg.

8. The fusosome according to any one of claims 1 to 7, wherein the fusogen is a viral envelope protein.

9. The fusosome of any one of claims 1 to 8, wherein the fusogen is a paramyxovirus fusogen and comprises a sequence selected from paramyxovirus fusogen F and G proteins, or F and H proteins, or F and HN proteins.

10. 10. The fusogen of claim 9, wherein the fusogen comprises sequences selected from Nipah virus F and G proteins, Measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Abulavirus F and HN proteins, or any combination thereof.

11. The fusosome of claim 10, wherein the fusogen comprises sequences of Nipah virus F and G proteins.

12. The fusosome according to any one of claims 1 to 11, wherein the positive target cell-specific regulatory element comprises a T cell-specific promoter, a T cell-specific enhancer, a T cell-specific splice site, a T cell-specific site that extends the half-life of an 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.

13. The fusosome of any one of claims 1 to 12, wherein the positive target cell-specific regulatory element comprises a T cell-specific promoter.

14. The T cell specific promoter CD3δ promoter, CD2 promoter, tyrosine-protein kinase Lck promoter, ICOS promoter, CD28 promoter, TCF7 promoter, or TCR Vβ8.1 promoter, or the CD8 promoter or the GZMB promoter, or CD4 promoter The fusosome of claim 13 .

15. The fusosome of claim 13, wherein the T cell specific promoter is an IL2RA promoter, a LRRC32 promoter, a FOXP3 promoter, or an IKZF2 promoter.

16. The fusosome of any one of claims 1 to 15, wherein the nucleic acid comprises one or more insulator sequences.

17. The fusosome of claim 16, wherein the nucleic acid comprises two insulator sequences, the two insulator sequences comprising a first insulator sequence upstream of the payload gene and a second insulator sequence downstream of the payload gene, the first insulator sequence and the second insulator sequence comprising the same or different sequences.

18. The fusosome according to any one of claims 1 to 17, wherein the fusosome is a retroviral vector particle.

19. The fusosome according to any one of claims 1 to 18, wherein the fusosome is a lentiviral vector particle.

20. A fusosome according to any one of claims 1 to 19, wherein the nucleic acid comprises a non-target cell-specific regulatory element (NTCSSE), a non-T cell-specific regulatory element operably linked to the payload gene, the NTCSSE reducing expression of the payload gene in the non-T cell or tissue compared to an otherwise similar fusosome lacking the NTCSSE.

21. A pharmaceutical composition comprising the fusosomes of any one of claims 1 to 20 and a pharma- ceutically acceptable carrier, diluent, or excipient.

22. A fusosome composition comprising a plurality of fusosomes according to any one of claims 1 to 20 or the pharmaceutical composition of claim 21 for use in a method of delivering an exogenous agent to a subject.

23. The fusosome composition or pharmaceutical composition for use according to claim 22, wherein the exogenous agent is delivered to T cells of the subject.

24. A fusosome composition comprising a plurality of fusosomes according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 for use in a method of modulating function in a subject or in T cells of a subject.

25. The fusosome composition or pharmaceutical composition for use according to claim 24, wherein 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, CD8+ cytotoxic T cells, regulatory T cells, thymus-derived regulatory T cells, peripherally-derived regulatory T cells, CD4+ Foxp3+ regulatory T cells, or CD4+ FoxP3-1 type regulatory T (Tr1) cells, helper T cells, 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, stem cell memory T cells, central memory T cells, or effector memory T cells, NKT cells, or mucosa-associated invariant T (MAIT) cells.

26. The fusosome composition or pharmaceutical composition for use according to claim 24 or claim 25, wherein the T cells are regulatory T cells (Treg).

27. The fusosome composition or pharmaceutical composition for use according to any one of claims 24 to 26, wherein the T cells are present in a subject.

28. A composition comprising a fusosome according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 for use in treating a disease or disorder in a subject.

29. Use of a fusosome according to any one of claims 1 to 20 or a pharmaceutical composition according to claim 21 for the manufacture of a medicament for use in treating a disease or disorder in a subject.

30. 29. The composition or pharmaceutical composition for use according to claim 28, wherein the disease or disorder is cancer, an autoimmune disease, an infectious disease, a metabolic disease, a neurodegenerative disease, or a genetic disease.

31. 31. The composition or pharmaceutical composition for use according to claim 28 or claim 30, wherein the disease or disorder is 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, pemphigoid, pemphigus, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, type 1 diabetes, or ulcerative colitis.

32. The fusosome composition or pharmaceutical composition of any one of claims 22 to 27, or the composition or pharmaceutical composition of any one of claims 28, 30 and 31, wherein the subject is a human subject.

33. below: a) providing a cell comprising said nucleic acid and said fusogen; b) culturing said cells under conditions allowing the production of fusosomes; and A method of producing fusosomes according to any one of claims 1 to 20, comprising c) isolating, enriching or purifying fusosomes from said cells, thereby producing fusosomes.

34. 30. The use according to claim 29, wherein the disease or disorder is cancer, an autoimmune disease, an infectious disease, a metabolic disease, a neurodegenerative disease, or a genetic disease.

35. 35. The use of claim 29 or claim 34, wherein the disease or disorder is 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, pemphigoid, pemphigus, rheumatoid arthritis, organ transplantation, systemic lupus erythematosus, type 1 diabetes, or ulcerative colitis.

36. 36. The use according to any one of claims 29, 34 and 35, wherein the subject is a human subject.

Images