Fusosome Compositions for CNS 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 claims priority to U.S. Provisional Application No. 62 / 767,358, filed November 14, 2018, entitled "Fusosome Compositions for CNS Cell Delivery," and U.S. Patent Application Publication No. 62 / 900,064, filed September 13, 2019, entitled "Fusosome Compositions for CNS Cell Delivery," the contents of which are incorporated by reference in their entirety for all purposes.

[0002] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application has been submitted with an electronic Sequence Listing, which is provided as a file entitled 186152003340SeqList.TXT, created on November 14, 2019, and having a size of 819 kilobytes. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. [Background technology]

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

[0004] The present disclosure provides, at least in part, fusosome methods and compositions for in vivo 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.

[0005] Enumeration of Embodiments 1. The following: a) a lipid bilayer containing a fusogen, and b) Below: (i) a payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent of Table 5 or Table 6, wherein optionally, the exogenous agent is set forth in any one of SEQ ID NOs: 134-154, or a functional fragment or functional variant thereof comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 134-154; (ii) a nucleic acid comprising 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 expression of the payload gene in a target cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element, and the target cell is a CNS cell.

[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 reduces expression of the payload gene in a non-target cell relative to an otherwise similar fusosome lacking the NTCSRE; optionally, the target cell is a first type of CNS cell and the non-target cell is a second, different type of CNS cell or a non-CNS cell; and optionally, the target cell is a neuron and the non-target cell is a glial cell (e.g., an oligodendrocyte, an astrocyte, or a microglial cell); or 2. The fusosome of embodiment 1, wherein the target cell is a glial cell (e.g., an oligodendrocyte, an astrocyte, or a microglial cell) and the non-target cell is a neuron.

[0007] 3. The following: a) a lipid bilayer containing a fusogen; and b) Below: (i) a payload gene encoding an exogenous agent, for example, an exogenous agent of Table 5 or Table 6, optionally wherein the exogenous agent is set forth in any one of SEQ ID NOs: 134-154, or a functional fragment or functional variant thereof comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 134-154; (ii) a nucleic acid comprising a promoter operably linked to a payload gene, wherein the promoter is selected from a SYN, NSE, CaMKII, α-tubulin, PDGF, fSST, fNPY, GAD67, DLX5 / 6, VGLUT1, Dock10, ChAT, VAChT, Drd1a, TPH-2, GFAP, EAAT1, GS, CX3CR1, TMEM119, MBP, CNP, or CRFR2β promoter, e.g., by a promoter sequence in Table 3, or by a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0008] 4. The following: a) a lipid bilayer containing a fusogen; and b) Below: (i) a payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent of Table 5 or Table 6, wherein optionally, the exogenous agent is set forth in any one of SEQ ID NOs: 134-154, or a functional fragment or functional variant thereof comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 134-154; (ii) A fusosome comprising a nucleic acid comprising a non-target cell-specific regulatory element (NTCSRE) (e.g., a non-target cell-specific miRNA recognition sequence) operably linked to a payload gene, wherein the NTCSRE reduces expression of the payload gene in a non-target cell or tissue compared to an otherwise similar fusosome lacking the NTCSRE.

[0009] 5. The following: a) a lipid bilayer containing a fusogen; and b) Below: (i) a payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent of Table 5 or Table 6, wherein optionally, the exogenous agent is set forth in any one of SEQ ID NOs: 134-154, or a functional fragment or functional variant thereof comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 134-154; (ii) A fusosome comprising a nucleic acid comprising a negative target cell-specific regulatory element (negative TCSSE) (e.g., a tissue-specific miRNA recognition sequence) operably linked to a payload gene, wherein the negative TCSSE reduces expression of an exogenous agent in a non-target cell or tissue compared to an otherwise similar nucleic acid lacking the negative TCSSE.

[0010] 6. The nucleic acid further comprises a positive target cell-specific regulatory element (e.g., a target cell-specific promoter) operably linked to a payload gene, wherein the positive target cell-specific regulatory element increases expression of the payload gene in a target cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element, wherein the target cell is a first type of CNS cell, and optionally, the non-target cell is a second, different type of CNS cell or a non-CNS cell, and optionally, the target cell is a neuron and the non-target cell is a glial cell (e.g., an oligodendrocyte, an astrocyte, or a microglial cell); or 6. The fusosome of embodiment 4 or 5, wherein the target cell is a glial cell (e.g., an oligodendrocyte, an astrocyte, or a microglial cell) and the non-target cell is a neuron.

[0011] 7. The following: a) a lipid bilayer containing a fusogen; b) a nucleic acid comprising a payload gene encoding an exogenous agent, for example, a payload gene encoding an exogenous agent of Table 5 or Table 6, wherein optionally the exogenous agent is set forth in any one of SEQ ID NOs: 134-154, or a functional fragment or functional variant thereof comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 134-154; c) One or both of the following: (i) a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer; or (ii) a first immunostimulatory protein that is absent or present at reduced levels (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to fusosomes produced from an otherwise similar, unmodified source cell.

[0012] 8. The fusosome of any of the preceding embodiments, which is one or more of the following: i) fusosomes fuse with target cells at a higher rate, 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, than with non-target cells; ii) fusosomes fuse with target cells at a higher rate, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, than with other fusosomes; iii) the fusosomes fuse with the target cells at a rate such that the agent within the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target cells after 24, 48, or 72 hours; iv) the fusosomes deliver nucleic acid, e.g., retroviral nucleic acid, to target cells at a higher rate, 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, than to non-target cells; v) the fusosome delivers a nucleic acid, e.g., a retroviral nucleic acid, to a target cell at a higher rate, e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, than another fusosome; or vi) The fusosomes deliver nucleic acid, e.g., retroviral nucleic acid, to the target cells at a rate such that the agent in the fusosomes is delivered to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the target cells after 24, 48, or 72 hours.

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

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

[0015] 11. The fusosome of any of the preceding embodiments, comprising one or more (e.g., all) of a polymerase (e.g., a reverse transcriptase, e.g., pol or a portion thereof), an integrase (e.g., pol or a portion thereof, e.g., a functional or non-functional variant), a matrix protein (e.g., gag or a portion thereof), a capsid protein (e.g., gag or a portion thereof), a nucleocapsid protein (e.g., gag or a portion thereof), and a protease (e.g., pro).

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

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

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

[0019] 15. The fusosome of any of embodiments 7-14, wherein the nucleic acid, e.g., retroviral vector, 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 increases expression of the payload gene in a target cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element, and wherein the target cell is a CNS cell.

[0020] 16. The nucleic acid, e.g., retroviral 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 reduces expression of the payload gene in a non-target cell or tissue compared to an otherwise similar fusosome lacking the NTCSRE; optionally, the target cell is a first type of CNS cell and the non-target cell is a second, different type of CNS cell or a non-CNS cell; and optionally, the target cell is a neuron and the non-target cell is a glial cell (e.g., an oligodendrocyte, an astrocyte, or a microglial cell); or 16. The fusosome of any one of embodiments 7 to 15, wherein the target cell is a glial cell (eg, an oligodendrocyte, an astrocyte, or a microglial cell) and the non-target cell is a neuron.

[0021] 17. The fusosome of any of embodiments 7-15, wherein the nucleic acid, e.g., retroviral nucleic acid, comprises a negative target cell-specific regulatory element (negative TCSSE) (e.g., a tissue-specific miRNA recognition sequence) operably linked to the payload gene, and the negative TCSSE reduces expression of the exogenous agent in non-target cells or tissues compared to an otherwise similar nucleic acid, e.g., retroviral nucleic acid, lacking the negative TCSSE.

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

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

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

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

[0026] 22. The fusosome of any of the preceding embodiments, wherein the exogenous agent is selected from SYNE1, SETX, FMR1, SLC6A8, UBE3A, SOD1, TDP43, C9orf72, FXN, MECP2, ASPA, or ALDH7A1, or wherein the exogenous agent is selected from TPP1, FUCA1, GALC, HEXA, HEXB, MANBA, ARSA, GNPTAB, or MCOLN1.

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

[0028] 24. The fusosome of any of embodiments 1, 2, 6, 15, 22, or 23, wherein the positive target cell-specific regulatory element comprises a CNS cell-specific promoter, a CNS cell-specific enhancer, a CNS cell-specific splice site, a CNS cell-specific site that extends the half-life of RNA or protein, a CNS cell-specific mRNA export-promoting site, a CNS cell-specific translation-enhancing site, or a CNS cell-specific post-translational modification site.

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

[0030] 26. The fusosome of embodiment 25, wherein the CNS cell-specific promoter comprises a motif from Table 3.

[0031] 27. The fusosome of embodiment 25 or 26, wherein the positive CNS cell-specific regulatory element comprises a promoter selected from SYN, NSE, CaMKII, α-tubulin, PDGF, fSST, fNPY, GAD67, DLX5 / 6, VGLUT1, Dock10, ChAT, VAChT, Drd1a, TPH-2, GFAP, EAAT1, GS, CX3CR1, TMEM119, MBP, CNP, or CRFR2β promoter.

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

[0033] 29. The fusosome of any of embodiments 4 to 6, or 16 to 21, or 28, wherein the negative TCSRE or NTCSRE comprises a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site.

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

[0035] 31. The fusosome of any of embodiments 4 to 6, 16 to 21, or 28 to 30, wherein the negative TCSRE or NTCSRE comprises a non-target cell-specific miRNA recognition sequence bound by a miRNA of Table 4, such as by one or more (e.g., two or more) miR-338-3p, miR-9, miR-125b-5p, miR-342-3p, or miR-124, and optionally the miRNA is or comprises a sequence set forth in any one of SEQ ID NOs: 156 to 162.

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

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

[0038] 34. The fusosome of embodiment 33, wherein the nucleic acid, e.g., a 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., the first 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 tumor formation in target cells.

[0040] 36. The fusosome of any of the preceding embodiments, wherein the nucleic acid, e.g., retroviral nucleic acid, is capable of being integrated into the genome of the target cell.

[0041] 37. The fusosome of embodiment 36, wherein the nucleic acid, such as a retroviral nucleic acid, is an integration-competent lentivirus or an integration-incompetent lentivirus.

[0042] 38. The fusosome of any of the preceding embodiments, wherein the target cell is selected from a CNS cell, a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, a serotonergic neuron, an astrocyte, a microglia, an oligodendrocyte, or a choroid plexus cell.

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

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

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

[0046] 42. A fusosome according to any of embodiments 40 or 41, wherein the positive TCSSE comprises a target cell-specific promoter that is at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, 1000% or more active in target cells than in non-target cells.

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

[0048] 44. The fusosome of any of the preceding embodiments, which does not deliver nucleic acid, e.g., retroviral nucleic acid, to a non-target cell, e.g., a neuron, a glial cell, an antigen-presenting cell, an MHC class II+ cell, a professional antigen-presenting cell, an atypical antigen-presenting cell, a macrophage, a dendritic cell, a myeloid dendritic cell, a plasmacytoid dendritic cell, a CD11c+ cell, a CD11b+ cell, a splenocyte, a B cell, a hepatocyte, an endothelial cell, or a non-cancerous cell.

[0049] 45. The fusosomes of any of the preceding embodiments, wherein, e.g., using quantitative PCR, e.g., using the assay of Example 1, less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, or 0.000001% of the non-target cell type (e.g., one or more neurons, glial cells, antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) comprise nucleic acid, e.g., retroviral nucleic acid.

[0050] 46. ​​The fusosome of any of the preceding embodiments, wherein the target cell comprises 0.00001-10, .0001-10, .001-10, .01-10, .1-10, .5-5, 1-4, 1-3, or 1-2 copies of a nucleic acid, e.g., a retroviral nucleic acid, or portion thereof, per host genome, e.g., wherein the copy number of the nucleic acid, e.g., the retroviral nucleic acid, is assessed following administration in vivo.

[0051] 47. The fusosome of any of the preceding embodiments: less than 10%, 5%, 2.5%, 1%, 0.5%, 0.1%, 0.01% of the non-target cells (e.g., neurons, glial cells, antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells) contain the exogenous agent; or The fusosome of any of the preceding embodiments, wherein the exogenous agent (e.g., protein) is not detectably present in non-target cells, such as neurons, glial cells, antigen-presenting cells, MHC class II+ cells, professional antigen-presenting cells, atypical antigen-presenting cells, macrophages, dendritic cells, myeloid dendritic cells, plasmacytoid dendritic cells, CD11c+ cells, CD11b+ cells, splenocytes, B cells, hepatocytes, endothelial cells, or non-cancerous cells.

[0052] 48. The fusosome of any of the preceding embodiments, wherein the fusosome delivers a nucleic acid, e.g., a retroviral nucleic acid, to a target cell, e.g., a CNS cell, a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, a serotonergic neuron, a glial cell, an astrocyte, a microglial cell, an oligodendrocyte, or a choroid plexus cell.

[0053] 49. The fusosome of any of the preceding embodiments, wherein 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 the target cells (e.g., one or more CNS cells, pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, glial cells, astrocytes, microglial cells, oligodendrocytes, or choroid plexus cells) comprise nucleic acid, e.g., retroviral nucleic acid, e.g., when using quantitative PCR, e.g., when using the assay of Example 3.

[0054] 50. The fusosome of any of the preceding embodiments, wherein 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 the target cells (e.g., CNS cells, pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, glial cells, astrocytes, microglial cells, oligodendrocytes, or choroid plexus cells) comprise the exogenous agent.

[0055] 51. The fusosome of any of the preceding embodiments, wherein upon administration, the ratio of target cells comprising nucleic acid, e.g., retroviral nucleic acid, to non-target cells comprising nucleic acid, e.g., retroviral nucleic acid, is at least 1.5, 2, 3, 4, 5, 10, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by quantitative PCR assay, e.g., using the assays of Examples 1 and 3.

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

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

[0058] 54. The fusosome of any of the preceding embodiments, wherein 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, 25, 50, 100, 500, 1000, 5000, 10,000, e.g., by reverse transcription quantitative PCR assay.

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

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

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

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

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

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

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

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

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

[0068] 64. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation one hour after administration.

[0069] 65. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 2 hours after administration.

[0070] 66. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 4 hours after administration.

[0071] 67. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 8 hours after administration.

[0072] 68. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 12 hours after administration.

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

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

[0075] 71. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 36 hours after administration.

[0076] 72. The fusosomes of any of the preceding embodiments, wherein at least 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the fusosomes are in circulation 48 hours after administration.

[0077] 73. The fusosome of any of the preceding embodiments, which has reduced immunogenicity as measured by a reduced humoral response following one or more administrations of the fusosome to a suitable animal model, e.g., an animal model described herein, compared to a reference fusosome, e.g., an unmodified fusosome that is otherwise similar to the fusosome.

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

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

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

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

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

[0083] 79. The fusosomes of any of the preceding embodiments, wherein fusosomes generated from NMC-HLA-G cells exhibit a reduced rate of lysis, e.g., PBMC-mediated lysis, NK cell-mediated lysis, and / or CD8+ T cell-mediated lysis, at a particular time point compared to fusosomes generated from NMC or NMC-empty vector.

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

[0085] 81. The fusosomes of any of the preceding embodiments, wherein the fusosomes are produced by the method of Example 8, e.g., 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 a retroviral vector derived from NMC-CD47 compared to a vector derived from NMC or an NMC empty vector.

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

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

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

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

[0091] The fusosomes of any of the preceding embodiments, wherein the dose of fusosomes at which 87,200 pg / ml of C3a is present is greater for modified fusosomes (e.g., HEK293-DAF) incubated with the corresponding mouse serum (e.g., HEK-293 DAF mouse serum) than for reference fusosomes (e.g., HEK293 retroviral vector) incubated with the corresponding mouse serum (e.g., HEK293 mouse serum).

[0092] The fusosomes of any of the preceding embodiments, wherein the dose of fusosomes at which 88,200 pg / ml of C3a is present is greater in modified fusosomes (e.g., HEK293-DAF) incubated with naive mouse serum than in reference fusosomes (e.g., HEK293 retroviral vector) incubated with naive mouse serum.

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

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

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

[0096] 92. The fusosome of any of the preceding embodiments, e.g., produced by the method of Example 10 from cells transfected with DNA encoding shRNA targeting MHC class I, and wherein the retroviral vector derived from, e.g., NMC-shMHC class I, results in reduced expression of MHC class I compared to NMC and NMC vector controls.

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

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

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

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

[0101] 97. The fusosomes of any of the preceding embodiments, wherein the modified fusosomes, e.g., those modified by the methods described herein, have reduced serum inactivation (e.g., reduced compared to administration of unmodified fusosomes) following multiple (e.g., more than one, e.g., two or more) administrations of the modified fusosomes.

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

[0103] 99. The fusosomes of any of the preceding embodiments, wherein the measure of the immunogenicity of the fusosomes is, e.g., serum inactivation after multiple doses, e.g., serum inactivation after multiple doses measured as described herein, e.g., as described in Example 12.

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

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

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

[0107] 103. The fusosomes of any of the preceding embodiments, wherein the percentage of cells receiving the exogenous agent is less in fusosomes derived from reference cells (e.g., HEK293) than in modified (e.g., HEK293-HLA-G) fusosomes.

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

[0109] 105. The fusosomes of any of the preceding embodiments, wherein the subject receiving the fusosomes described herein has pre-existing antibodies that bind to and recognize the fusosomes, measured as described herein, e.g., as described in Example 13.

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

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

[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, and wherein the humoral response after multiple (e.g., more than one, e.g., two or more) administrations of the modified fusosome is reduced (e.g., reduced compared to administration of unmodified fusosomes), e.g., as described in Example 14, e.g., as measured as described herein.

[0113] 109. The fusosomes of any of the previous embodiments, wherein the fusosomes are produced by the method of Example 5, 6, 7, or 14, for example, 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 value of the level of anti-fusosome antibodies (e.g., IgM, IgG1, and / or IgG2 antibodies).

[0115] 111. The fusosomes of any of the preceding embodiments, wherein the modified (e.g., NMC-HLA-G) fusosomes have reduced anti-viral IgM or IgG1 / 2 antibody titers (e.g., as measured by fluorescence intensity by FACS) following injection compared to a control, e.g., NMC fusosomes or NMC-empty fusosomes.

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

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

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

[0119] 115. The fusosomes of any of the preceding embodiments, wherein the recipient cells are not targeted by macrophages, or are targeted at below reference levels.

[0120] 116. The fusosomes of any of the preceding embodiments, wherein the phagocytic index, measured as described herein, e.g., as described in Example 16, is similar in recipient cells from fusosome-treated mice and PBS-treated mice.

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

[0122] 118. The fusosome of any of the preceding embodiments, wherein the recipient cells do not induce a PBMC response.

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

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

[0125] 121. The fusosome of any of the preceding embodiments, wherein the recipient cells do not induce a natural killer cell response, or e.g., induce a natural killer cell response that is lower than a reference value.

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

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

[0128] 124. The fusosome of any of the preceding embodiments, wherein the recipient cells do not induce a CD8+ T cell response, or e.g., induce a CD8+ T cell response that is lower than a reference value.

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

[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, comprising a nucleic acid, e.g., a retroviral nucleic acid, encoding one or both of (i) a positive target cell-specific regulatory element operably linked to a nucleic acid encoding an exogenous agent, or (ii) a non-control cell-specific regulatory element or a negative TCSSE operably linked to a nucleic acid encoding an exogenous agent.

[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 for delivering an exogenous agent to a subject (e.g., a human subject), comprising administering to the subject a fusosome of any of embodiments 1 to 127 or a pharmaceutical composition of embodiment 128, thereby delivering the exogenous agent to the subject.

[0134] 130. A method for modulating a function in a subject (e.g., a human subject), a target tissue, or a target cell (e.g., a CNS cell, such as a neuron or glial cell), comprising contacting, e.g., administering to, the subject, the target tissue, or the target cell a fusosome of any of embodiments 1 to 127, or a pharmaceutical composition of embodiment 128.

[0135] 131. The method of embodiment 130, wherein the target tissue or target cell is present in a subject.

[0136] 132. A method of treating a genetic defect in a subject (e.g., a human subject), comprising administering to the subject a fusosome of any of embodiments 1-127, or a pharmaceutical composition of claim 128.

[0137] 133. The method of embodiment 132, wherein the gene defect is a gene defect in Table 5 or Table 6.

[0138] 134. The method of embodiment 132 or 133, wherein the genetic defect is a genetic defect that can be treated by a payload gene encoding an exogenous agent.

[0139] 135. The fusosome of any of embodiments 132-134, wherein the genetic defect is associated with a CNS disease or disorder or a lysosomal disease or disorder, and the method treats the CNS disease or disorder or a lysosomal disease or disorder.

[0140] 136. The method of embodiment 135, wherein the CNS disease or disorder or lysosomal disease or disorder is spinocerebellar ataxia; autosomal recessive, type 1; ataxia with oculomotor apraxia, type 2; fragile X syndrome; cerebral creatine deficiency syndrome 1; Angelman syndrome; amyotrophic lateral sclerosis; Friedreich's ataxia; Rett syndrome; Canavan disease; pyridoxine-dependent epilepsy; Batten disease, fucosidosis; Krabbe disease; Tay-Sachs disease; Sandhoff disease; beta-mannosidosis; metachromatic leukodystrophy; mucolipidosis type IIIa; mucolipidosis type IIIb; or mucolipidosis type IV.

[0141] 137. The fusosome of any of embodiments 1 to 127 or the pharmaceutical composition of embodiment 128 for use in treating a subject (e.g., a human subject) having a genetic defect.

[0142] 138. Use of fusosomes of any of embodiments 1 to 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) having a genetic defect.

[0143] 139. 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 genetic defect.

[0144] 140. A fusosome or pharmaceutical composition for use according to embodiment 137 or 139, or a use according to embodiment 138 or 139, wherein the genetic defect is associated with a CNS disease or disorder or a lysosomal disease or disorder, and the method treats a CNS disease or disorder or a lysosomal disease or disorder.

[0145] 141. Fusosomes or pharmaceutical compositions for use according to embodiment 137, 139 or 140, or the use according to embodiment 138, 139 or 140, wherein the CNS disease or disorder or lysosomal disease or disorder is spinocerebellar ataxia; autosomal recessive, type 1; ataxia with oculomotor apraxia, type 2; fragile X syndrome; cerebral creatine deficiency syndrome 1; Angelman syndrome; amyotrophic lateral sclerosis; Friedreich's ataxia; Rett syndrome; Canavan disease; pyridoxine-dependent epilepsy; Batten disease, fucosidosis; Krabbe disease; Tay-Sachs disease; Sandhoff disease; beta-mannosidosis; metachromatic leukodystrophy; mucolipidosis type IIIa; mucolipidosis type IIIb; or mucolipidosis type IV.

[0146] 142. The following: a) providing a cell containing nucleic acid, e.g., retroviral nucleic acid, and a fusogen; b) culturing the cells under conditions that allow the production of fusosomes; and c) isolating, enriching, or purifying fusosomes from the cells, thereby producing fusosomes.

[0147] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0148] 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 mentioned herein, for example, in any table, are incorporated by reference. Unless otherwise specified, sequence accession numbers specified herein, including in the tables herein, refer to database entries current as of May 15, 2018. When a gene or protein references multiple sequence accession numbers, all sequence variants are included. Additionally, the materials, methods, and examples are merely illustrative and not intended to be limiting.

[0149] 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, there are described herein specific embodiments that are illustrated in the specification, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0150] [Figure 1] Figure 1 quantifies the staining of fusosomes with F-actin dyes. [Figure 2] FIG. 2 is a graph showing the ability of fusosomes and parental cells to polymerize actin over a period of 3, 5, and 24 hours. [Figure 3] FIG. 3 is a table showing size distribution statistics of fusosomes and parent cells as determined by NTA and microscopy. [Figure 4] FIG. 4 is a table showing the average size and volume of fusosomes and parent cells. [Figure 5] FIG. 5 is a series of graphs showing the soluble:insoluble ratios observed for fusosome or cell preparations. [Figure 6]FIG. 6 is a series of graphs showing MvH(CD8)+F fusosome fusion to target or non-target cells, and the absolute amount of target fusion. [Figure 7] FIG. 7 shows the mean fluorescence intensity of 2-NBDG in VSV-G fusosomes. [Figure 8] FIG. 8 shows esterase activity in the cytosol of VSV-G fusosomes. [Figure 9-1] Figures 9A-9B are a series of images showing Cre recombinase delivery by fusosomes, as detected by bioluminescence imaging in mice. (A) Ventral images and luminescence signal overlays of exposed livers and spleens from IV fusosome-treated mice (1x and 3x concentrations). The bottom shows luminescence signal only. (B) Total flux signal of fusosome-targeted spleens and livers; γ-scale is a log10 scale. Mice treated with 3x the fusosome treatment concentration showed significantly greater signal in the spleen than background 72 hours after treatment (p=0.0004). [Figure 9-2] Figures 9A-9B are a series of images showing Cre recombinase delivery by fusosomes, as detected by bioluminescence imaging in mice. (A) Ventral images and luminescence signal overlays of exposed livers and spleens from IV fusosome-treated mice (1x and 3x concentrations). The bottom shows luminescence signal only. (B) Total flux signal of fusosome-targeted spleens and livers; γ-scale is a log10 scale. Mice treated with 3x the fusosome treatment concentration showed significantly greater signal in the spleen than background 72 hours after treatment (p=0.0004). [Figure 10-1]Figures 10A-10B are a series of images showing Cre recombinase delivery to mouse liver and spleen by fusosomes, as detected by bioluminescence imaging. (A) From left to right: Dorsal images and luminescence signal overlays of excised liver, heart, lung, kidney, small intestine, pancreas, and spleen, collected and imaged within 5 minutes of euthanasia. The bottom shows luminescence signal only. (B) Total flux signal of fusosome-targeted spleen and liver, as well as other tissues; γ-scale is a log10 scale. Mice treated with a 3x higher concentration of fusosomes showed significantly greater signal in the spleen compared to the tissue with the lowest signal (heart) (p<0.0001). [Figure 10-2] Figures 10A-10B are a series of images showing Cre recombinase delivery to mouse liver and spleen by fusosomes, as detected by bioluminescence imaging. (A) From left to right: Dorsal images and luminescence signal overlays of excised liver, heart, lung, kidney, small intestine, pancreas, and spleen, collected and imaged within 5 minutes of euthanasia. The bottom shows luminescence signal only. (B) Total flux signal of fusosome-targeted spleen and liver, as well as other tissues; γ-scale is a log10 scale. Mice treated with a 3x higher concentration of fusosomes showed significantly greater signal in the spleen compared to the tissue with the lowest signal (heart) (p<0.0001). [Figure 11] FIG. 11 is a table showing delivery of Cre cargo by NivG+F fusosomes via a non-endocytic pathway. [Figure 12] FIG. 12 is a graph showing the GAPDH:total protein ratio measured by bicinchoninic acid assay in fusosomes and parental cells. [Figure 13] FIG. 13 is a graph showing the lipid:protein ratio measured by the bicinchoninic acid assay in fusosomes and parental cells. [Figure 14] FIG. 14 is a graph showing the protein:DNA ratio measured by the bicinchoninic acid assay in fusosomes and parental cells. [Figure 15]FIG. 15 is a graph showing the lipid:DNA ratio measured by the bicinchoninic acid assay in fusosomes and parental cells. [Figure 16] FIG. 16 is a graph showing the protein levels of the exosomal marker CD63 in exosomes and fusosomes. [Figure 17] FIG. 17 is a graph showing the intensity of the calnexin signal detected in fusosomes and parental cells. [Figure 18] FIG. 18 is a graph showing the lipid:DNA ratios determined for fusosomes and parental cells. [Figure 19-1] 19A-19B are a series of graphs showing the proportion of lipid species as a percentage of total lipid in parental cells, exosomes, and fusosomes. [Figure 19-2] 19A-19B are a series of graphs showing the proportion of lipid species as a percentage of total lipid in parental cells, exosomes, and fusosomes. [Figure 20] FIG. 20 is a series of graphs showing the protein content of parental cells, exosomes, and fusosomes with respect to proteins associated with specific compartments, as indicated. [Figure 21] FIG. 21 is a series of graphs showing the levels of ARRDC1 (left panel) or TSG101 (right panel) as a percentage of the total protein content in parental cells, exosomes, and fusosomes. DETAILED DESCRIPTION OF THE INVENTION

[0151] The present disclosure provides, at least in part, fusosome methods and compositions for in vivo delivery. In some embodiments, the fusosomes contain a combination of elements that promote specificity for target cells, such as one or more retargeted fusogens, a positive target cell-specific regulatory element, and a non-target cell-specific regulatory element. In some embodiments, the fusosome compositions contain one or more modifications that reduce an immune response to the fusosomes.

[0152] I. Definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0153] 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. 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.

[0154] 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, fusosomes contain nucleic acid. In some embodiments, fusosomes are membrane-enclosed preparations. In some embodiments, fusosomes are derived from a source cell.

[0155] As used herein, a "fusosome composition" refers to a composition comprising one or more fusosomes.

[0156] As used herein, "fusogen" refers to an agent or molecule that creates an interaction between two membrane-bound lumens. In embodiments, a fusogen promotes membrane fusion. In other embodiments, a fusogen creates a connection, e.g., a pore, between two lumens (e.g., the lumen of a retroviral vector and the cytoplasm of a target cell). In some embodiments, a fusogen comprises a complex of two or more proteins, e.g., neither protein has solely fusogenic activity. In some embodiments, a fusogen comprises a targeting domain.

[0157] As used herein, "insulator sequence" refers to a nucleotide sequence that blocks an enhancer or prevents heterochromatin spreading. Insulator sequences can be wild-type or mutant.

[0158] As used herein, the term "effective amount" refers to that amount of a pharmaceutical composition sufficient to significantly and positively modify the symptoms and / or condition being treated (e.g., provide a positive clinical response). The effective amount of active ingredient for use in a pharmaceutical composition will vary depending on the particular condition being treated, the severity of the condition, the duration of treatment, the nature of any concurrent therapy, the particular active ingredient(s) used, the particular pharmaceutically acceptable excipients and / or carriers employed, and similar factors according to the knowledge and expertise of the attending physician.

[0159] As used herein, an "exogenous agent" with respect to a virus, VLP, or fusosome refers to an agent that is not contained in or encoded by the corresponding wild-type virus or fusogen produced from a corresponding wild-type source cell. In some embodiments, the exogenous agent is not naturally occurring, such as a protein or nucleic acid having a sequence that is altered (e.g., by insertion, deletion, or substitution) compared to a naturally occurring protein. In some embodiments, the exogenous agent is not naturally occurring in the source cell. In some embodiments, the exogenous agent is naturally present in the source cell but is exogenous to the virus. In some embodiments, the exogenous agent is not naturally occurring in the recipient cell. In some embodiments, the exogenous agent is naturally present in the recipient cell, but not at a desired level or for a desired time. In some embodiments, the exogenous agent comprises RNA or a protein.

[0160] As used herein, the term "pharmaceutically acceptable" refers to excipients, compositions and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0161] As used herein, "promoter" refers to a cis-regulatory DNA sequence that drives transcription of a gene when operably linked to the gene coding sequence. A promoter may contain transcription factor binding sites. In some embodiments, a promoter functions in cooperation with one or more enhancers distal to the gene.

[0162] As used herein, a "positive target cell-specific regulatory element" (or positive TCSRE) refers to a nucleic acid sequence that increases the level of an exogenous agent in a target cell compared to a non-target cell, wherein a nucleic acid encoding the exogenous agent is operably linked to the positive TCSRE. In some embodiments, the positive TCSRE is a functional nucleic acid sequence, e.g., the positive TCSRE can comprise a promoter or enhancer. In some embodiments, the positive TCSRE encodes a functional RNA sequence, e.g., the positive TCSRE can encode a splice site that promotes correct splicing of RNA in a target cell. In some embodiments, the positive TCSRE encodes a functional protein sequence, or the positive TCSRE can encode a protein sequence that promotes correct post-translational modification of a protein. In some embodiments, the positive TCSRE reduces the level or activity of a down-regulator or inhibitor of an exogenous agent.

[0163] As used herein, a "negative target cell-specific regulatory element" (or negative TCSSE) refers to a nucleic acid sequence that reduces the level of an exogenous agent in non-target cells compared to target cells, wherein the nucleic acid encoding the exogenous agent is operably linked to the negative TCSSE. In some embodiments, the negative TCSSE is a functional nucleic acid sequence, e.g., an miRNA recognition site, that causes degradation or inhibition of a retroviral nucleic acid in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, e.g., the nucleic acid encodes an miRNA sequence present in an mRNA encoding the exogenous protein agent, such that the mRNA is degraded or inhibited in the non-target cells. In some embodiments, the negative TCSSE increases the level or activity of a down-regulator or inhibitor of the exogenous agent.

[0164] As used herein, a "non-target cell-specific regulatory element" (or NTCSRE) refers to a nucleic acid sequence that reduces the level of an exogenous agent in non-target cells compared to target cells, wherein the nucleic acid encoding the exogenous agent is operably linked to the NTCSRE. In some embodiments, the NTCSRE is a functional nucleic acid sequence, e.g., an miRNA recognition site, that causes degradation or inhibition of a retroviral nucleic acid in non-target cells. In some embodiments, the nucleic acid sequence encodes a functional RNA sequence, e.g., the nucleic acid encodes an miRNA sequence present in an mRNA encoding an exogenous protein agent, such that the mRNA is degraded or inhibited in the non-target cells. In some embodiments, the NTCSRE increases the level or activity of a down-regulator or inhibitor of an exogenous agent. The terms "negative TCSRE" and "NTCSRE" are used interchangeably herein.

[0165] As used herein, "non-CNS cell-specific regulatory element" refers to a non-target cell-specific regulatory element (NTCSRE), and the target cell is a CNS cell. Thus, the non-CNS cell-specific regulatory element refers to a nucleic acid sequence that reduces the level of an exogenous agent in a non-CNS cell compared to a CNS cell, and the nucleic acid encoding the exogenous agent is operably linked to the non-CNS cell-specific regulatory element.

[0166] As used herein, a "retargeted fusogen" refers to a fusogen that includes a targeting moiety with a sequence that is not part of the naturally occurring form of the fusogen. In embodiments, the fusogen includes a different targeting moiety compared to the targeting moiety of the naturally occurring form of the fusogen. In embodiments, the naturally occurring form of the fusogen lacks a targeting domain, and the retargeted fusogen includes a targeting moiety that is not present in the naturally occurring form of the fusogen. In embodiments, the fusogen is modified to include a targeting moiety. In embodiments, the fusogen includes one or more sequence changes outside of the targeting moiety, e.g., in the transmembrane domain, the fusogenically active domain, or the cytoplasmic domain, compared to the naturally occurring form of the fusogen.

[0167] As used herein, "retroviral nucleic acid" refers to a nucleic acid that comprises at least the minimum sequence requirements for packaging into a retrovirus or retroviral vector, alone or in combination with a helper cell, helper virus, or helper plasmid. 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 TCSSE. In some embodiments, the retroviral nucleic acid comprises one or more of a 5' LTR (e.g., to facilitate integration), a U3 (e.g., to activate viral genome RNA transcription), an R (e.g., a Tat binding region), a U5, a 3' LTR (e.g., to facilitate integration), a packaging site (e.g., psi (Ψ)), or an RRE (e.g., to bind Rev and promote nuclear export). Retroviral nucleic acid can comprise RNA (e.g., when part of a virion) or DNA (e.g., 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, a helper virus, or a helper plasmid containing one or more (eg, all) of gag, pol, and env.

[0168] As used herein, "target cell" refers to a cell type to which it is desired that a fusosome (e.g., a lentiviral vector) deliver an exogenous agent. In embodiments, the target cell is a cell of a particular tissue type or class, e.g., a CNS cell, such as a neuron or glial cell. In some embodiments, the target cell is a diseased cell, e.g., a cancer cell. 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), directs preferential delivery of the exogenous agent to the target cell relative to non-target cells.

[0169] As used herein, "non-target cells" refers to cell types to which it is not desirable for a lentiviral vector to deliver an exogenous agent. In some embodiments, non-target cells are cells of a particular tissue type or class. In some embodiments, non-target cells are non-disease cells, e.g., non-cancerous cells. In some embodiments, a fusogen, e.g., a retargeted fusogen (alone or in combination with a positive TCSRE, NTCSRE, negative TCSRE, or any combination thereof), leads to lower delivery of an exogenous agent to non-target cells compared to target cells.

[0170] As used herein, the terms "treat", "treating", or "treatment" refer to ameliorating a disease or disorder, e.g., the underlying cause of the disorder or at least one of its clinical symptoms, e.g., delaying, preventing, or reducing the onset of the disease or disorder.

[0171] As used herein, "cell biological material" refers to a portion of a cell, including the lumen and cell membrane, or a cell with partial or complete nuclear inactivation. In some embodiments, the cell biological material includes one or more of cytoskeletal components, organelles, and ribosomes. In embodiments, the cell biological material is an enucleated cell, a microvesicle, or a cell ghost.

[0172] II. Fusosomes, e.g., cell-derived fusosomes Fusosomes can take various forms. For example, in some embodiments, the fusosomes described herein are derived from source cells. Fusosomes can be or include, for example, extracellular vesicles, microvesicles, nanovesicles, exosomes, apoptotic bodies (derived from apoptotic cells), microparticles (e.g., derived from platelets), ectosomes (e.g., derived from neutrophils and monocytes in serum), prostatosomes (obtained from prostate cancer cells), cardiosomes (obtained from cardiac cells), or any combination thereof. In some embodiments, fusosomes are naturally released from source cells, and in some embodiments, the source cells are treated to enhance fusosome formation. In some embodiments, fusosomes are about 10-10,000 nm in diameter, e.g., about 30-100 nm in diameter. In some embodiments, fusosomes comprise one or more synthetic lipids.

[0173] In some embodiments, the fusosome is or comprises a virus, e.g., a retrovirus, e.g., a lentivirus. For example, in some embodiments, the amphiphilic lipid bilayer of the fusosome is or comprises a viral envelope. The viral envelope can comprise a fusogen, e.g., a fusogen endogenous to the virus or a pseudotyped fusogen. In some embodiments, the lumen or cavity of the fusosome comprises viral nucleic acid, e.g., a retroviral nucleic acid, e.g., a lentiviral nucleic acid. The viral nucleic acid can be a viral genome. In some embodiments, the fusosome further comprises, e.g., in its cavity or lumen, one or more viral nonstructural proteins.

[0174] Fusosomes can have a variety of properties that facilitate delivery of a payload to a target cell. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid(s) to create a particle capable of fusing with a target cell. In embodiments, these nucleic acid(s) encode a protein having one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0175] Fusosomes can also contain various structures that facilitate delivery of a payload to a target cell. For example, in some embodiments, the fusosome and the source cell together contain sufficient nucleic acid(s) to create a particle capable of fusing with a target cell. In embodiments, these nucleic acid(s) encode proteins with one or more (e.g., all) of the following activities: gag polyprotein activity, polymerase activity, integrase activity, protease activity, and fusogenic activity.

[0176] Fusosomes can also comprise various structures that facilitate delivery of a payload to a target cell. For example, in some embodiments, fusosomes (e.g., viruses, e.g., retroviruses, e.g., lentiviruses) comprise one or more (e.g., all) of the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or non-functional variants), protease, and fusogen. In some embodiments, fusosomes further comprise rev. In some embodiments, one or more of the aforementioned proteins are encoded by the retroviral genome, and in some embodiments, one or more of the aforementioned proteins are provided in trans, e.g., by a helper cell, helper virus, or helper plasmid. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) comprises one or more (e.g., all) of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., lacking a U5 and a functional U3). In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more insulator sequences. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more miRNA recognition sites. In some embodiments, the one or more miRNA recognition sites are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and the WPRE.

[0177] In some embodiments, the fusosomes provided herein are administered to a subject, e.g., a mammal, e.g., a human. In such embodiments, the subject is at risk for, has symptoms of, can be diagnosed with, or can be identified as having a particular disease or condition (e.g., a disease or condition described herein). In one embodiment, the subject has a genetic defect, such as those listed in Table 5 or Table 6. In some embodiments, the fusosomes comprise a nucleic acid sequence encoding an exogenous agent for treating a disease or condition, such as for treating a genetic defect.

[0178] A. Fusosomes produced by the virus. For example, in some embodiments, a fusosome (e.g., a virus, e.g., a retrovirus, e.g., a lentivirus) comprises one or more (e.g., all) of the following proteins: gag polyprotein, polymerase (e.g., pol), integrase (e.g., functional or non-functional variants), protease, and fusogen. In some embodiments, the fusosome further comprises rev. In some embodiments, one or more of the foregoing proteins are encoded by the retroviral genome, and in some embodiments, one or more of the foregoing proteins are provided in trans, for example, by a helper cell, helper virus, or helper plasmid. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) comprises one or more (e.g., all) of the following nucleic acid sequences: a 5' LTR (e.g., comprising a U5 and lacking a functional U3 domain), a Psi packaging element (Psi), a central polypurine tract (cPPT) promoter operably linked to a payload gene, a payload gene (optionally comprising an intron before the open reading frame), a polyA tail sequence, a WPRE, and a 3' LTR (e.g., lacking a U5 and a functional U3). In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more insulator sequences. In some embodiments, the fusosomal nucleic acid (e.g., retroviral nucleic acid) further comprises one or more miRNA recognition sites. In some embodiments, the one or more miRNA recognition sites are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and the WPRE.

[0179] i) Lentiviral components and helper cells In some embodiments, the retroviral nucleic acid comprises one or more (e.g., all) of the following: a 5' promoter (e.g., to control expression of the entire packaged RNA), a 5' LTR (e.g., a U5 containing an R (including a polyadenylation tail signal) and / or a primer activation signal), a primer binding site, a psi packaging signal, an RRE element for nuclear export, a promoter immediately upstream of the transgene to control expression of the transgene, the transgene (or other exogenous agent element), a polypurine tract, and a 3' LTR (e.g., containing a mutated U3, R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator sequence.

[0180] Retroviruses typically replicate by converting their genomic RNA into a linear, double-stranded DNA copy by reverse transcription, followed by covalent integration of the 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 ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0181] In some embodiments, the retrovirus is a gamma retrovirus. In some embodiments, the retrovirus is an epsilon retrovirus. In some embodiments, the retrovirus is an alpha retrovirus. In some embodiments, the retrovirus is a beta retrovirus. In some embodiments, the retrovirus is a delta retrovirus. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the retrovirus is a spumaretrovirus. In some embodiments, the retrovirus is an endogenous retrovirus.

[0182] Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and 2); visna-maedi virus (VMV); caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is used.

[0183] In some embodiments, a vector herein is a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked, e.g., inserted, into the vector nucleic acid molecule. The vector may contain a sequence that directs autonomous replication within a cell, or may contain a sequence sufficient to allow it to integrate into host cell DNA. Useful vectors include plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include replication-defective retroviruses and lentiviruses.

[0184] Viral vectors, for example, can include nucleic acid molecules (e.g., transfer plasmids) that typically contain virally derived nucleic acid elements that facilitate the transfer of the nucleic acid molecule or its integration into a cell's genome or a viral particle that mediates nucleic acid transfer. Viral particles typically contain various viral components, and sometimes host cell components, in addition to the nucleic acid(s). Viral vectors can include, for example, viruses or viral particles that can transfer nucleic acids into cells or transfer the transferred nucleic acid (e.g., as naked DNA). Viral vectors and transfer plasmids can include structural and / or functional genetic elements that are primarily derived from viruses. Retroviral vectors can include viral vectors or plasmids that contain structural and functional genetic elements, or portions thereof, that are primarily derived from retroviruses. Lentiviral vectors can include viral vectors or plasmids that contain structural and functional genetic elements, or portions thereof, that are primarily derived from lentiviruses, including LTRs.

[0185] In embodiments, a lentiviral vector (e.g., a lentiviral expression vector) may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of these elements can be present in the form of RNA in a lentiviral particle or in the form of DNA in a DNA plasmid.

[0186] 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, resulting in a viral vector that is replication-defective. In some embodiments, the vector is capable of transducing target non-dividing host cells and / or integrating its genome into the host genome.

[0187] The structure of wild-type retroviral genomes often includes a 5' long terminal repeat (LTR) and a 3' long terminal repeat (LTR), between which or within which are located a packaging signal that enables genome packaging, a primer binding site, an integration site that allows integration into the host cell genome, and the gag, pol, and env genes that encode packaging components that promote viral particle assembly. More complex retroviruses contain additional features, such as the HIV rev and RRE sequences, which enable efficient export of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells. In proviruses, viral genes are flanked on both ends by regions called long terminal repeats (LTRs). LTRs are involved in proviral integration and transcription. LTRs also function as enhancer-promoter sequences, allowing them to control viral gene expression. Retroviral RNA encapsidation is mediated by the psi sequence located at the 5' end of the viral genome.

[0188] The LTRs themselves are usually similar (e.g., identical) sequences that can be divided into three elements called U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements vary greatly among retroviruses.

[0189] In viral genomes, the transcription initiation site is usually at the boundary between U3 and R of one LTR, and the poly(a) addition (termination) site is at the boundary between R and U5 of the other LTR. U3 contains most of the transcriptional control elements of the provirus, including a promoter and multiple enhancer sequences that respond to cellular and, in some cases, viral transcriptional activator proteins. Some retroviruses contain one or more of the following genes that encode proteins involved in regulating gene expression: tot, rev, tax, and rex.

[0190] Regarding 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 the DNA polymerase, associated RNase H, and reverse transcriptase (RT) including integrase (IN), which mediate genome replication. The env gene encodes the surface (SU) glycoprotein and transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction facilitates infection, for example, by fusion of the viral and cellular membranes.

[0191] In replication-defective retroviral vector genomes, the pol and env regions may be absent or non-functional. The R regions at both ends of the RNA are usually repetitive sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome, respectively.

[0192] Retroviruses may also contain additional genes encoding proteins other than gag, pol, and env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev, and nef (in HIV). EIAV contains an additional gene, S2 (among others). Proteins encoded by additional genes perform various functions, some of which may overlap with functions provided by cellular proteins. For example, in EIAV, tat functions as a transcriptional activator of the viral long terminal repeat (LTR) (Derse and Newbold 1993 Virology 194:530-6; Maury et al. 1994 Virology 200:632-42). tat binds to a stable stem-loop RNA secondary structure called TAR. Rev regulates and coordinates viral gene expression via the Rev response element (RRE) (Martarano et al. 1994 J. Virol. 68:3102-11). The mechanisms of action of these two proteins are thought to be largely similar to those of similar primate viruses. In addition, an EIAV protein, Ttm, has been identified, encoded by the first exon of tat spliced ​​into the env coding sequence at the start of the transmembrane protein.

[0193] In addition to protease, reverse transcriptase, and integrase, non-primate lentiviruses contain a fourth pol gene product that encodes a dUTPase, which may play a role in the ability of these lentiviruses to infect certain non-dividing or slowly dividing cell types.

[0194] In embodiments, a recombinant lentiviral vector (RLV) is a vector that carries sufficient retroviral genetic information to allow packaging of an RNA genome into viral particles capable of infecting target cells in the presence of packaging components. Infection of a target cell can involve reverse transcription and integration into the target cell genome. RLVs typically carry non-viral coding sequences delivered to the target cell by the vector. In embodiments, RLVs are incapable of independent replication to produce infectious retroviral particles within the target cell. RLVs typically lack functional gag-pol and / or env genes and / or other genes involved in replication. Vectors can be configured as split-intron vectors, for example, as described in PCT Patent Application WO 99 / 15683, which is incorporated herein by reference in its entirety.

[0195] In some embodiments, lentiviral vectors comprise a minimal viral genome, e.g., viral vectors that have been engineered to remove non-essential elements and retain essential elements to provide the functions necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98 / 17815, which is incorporated herein by reference in its entirety.

[0196] A minimal lentiviral genome may comprise, for example, (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to produce the lentiviral genome in the source cell may also comprise transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the source cell. These regulatory sequences may include native sequences associated with the transcribed retroviral sequence, e.g., the 5'U3 region, or may include another viral promoter, e.g., a heterologous promoter such as the CMV promoter. Some lentiviral genomes contain additional sequences to promote efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included. Alternatively or in combination, codon optimization can be used; for example, the gene encoding the exogenous agent may be codon-optimized, as described, for example, in WO 01 / 79518, which is incorporated herein by reference in its entirety. Alternative sequences that perform similar or identical functions to the rev / RRE system may also be used. For example, a functional analog of the rev / RRE system is found in the Mason-Pfizer monkey virus. This is known as a CTE, which contains an RRE-type sequence in its genome that is thought to interact with factors in infected cells. The cellular factors can be thought of as rev analogs. Therefore, the CTE can be used as a substitute for the rev / RRE system. In addition, the Rex protein of HTLV-I can functionally replace the Rev protein of HIV-I. Rev and Rex have effects similar to those of IRE-BP.

[0197] In some embodiments, the retroviral nucleic acid (e.g., lentiviral nucleic acid, e.g., primate or non-primate lentiviral nucleic acid) (1) comprises a deleted gag gene, where the deletion in gag removes one or more nucleotides downstream of about 350 or 354 nucleotides of the gag coding sequence; (2) has one or more accessory genes not present in the retroviral nucleic acid; (3) lacks the tat gene but includes a leader sequence between the end of the 5' LTR and the ATG of gag; or (4) is a combination of (1), (2), and (3). In one embodiment, the lentiviral vector comprises all of features (1), (2), and (3). This strategy is described in further detail in International Publication WO 99 / 32646, which is incorporated herein by reference in its entirety.

[0198] In some embodiments, the primate lentiviral minimal system does not require any of the HIV / SIV additional genes vif, vpr, vpx, vpu, tat, rev, and nef for either vector production or transduction of dividing and non-dividing cells, hi some embodiments, the EIAV minimal vector system does not require S2 for either vector production or transduction of dividing and non-dividing cells.

[0199] Deletion of additional genes may allow vectors to be created without genes associated with lentiviral (e.g., HIV) infection disease. Tat, in particular, has been associated with disease. Second, deletion of additional genes allows the vector to package more heterologous DNA. Third, genes with unknown functions, such as S2, can be omitted to reduce the risk of undesired effects. Examples of minimal lentiviral vectors are disclosed in WO 99 / 32646 and WO 98 / 17815.

[0200] In some embodiments, the retroviral nucleic acid lacks at least tat and S2 (if 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.

[0201] In some embodiments, the retroviral nucleic acid comprises vpx. The Vpx polypeptide binds to and induces the degradation of the SAMHD1 restriction factor, degrading free dNTPs in the cytoplasm. Thus, when Vpx degrades SAMHD1 and reverse transcription activity increases, the concentration of free dNTPs in the cytoplasm increases, promoting reverse transcription of the retroviral genome and integration into the target cell genome.

[0202] Different cells use specific codons differently. This codon bias corresponds to the bias in the relative abundance of specific tRNAs in cell types. By changing the codons in the sequence so that they are adjusted to match the relative abundance of the corresponding tRNAs, it is possible to increase expression. Similarly, by intentionally selecting codons whose corresponding tRNAs are known to be rare in a particular cell type, it is possible to decrease expression. Thus, additional translational control is available. Further description of codon optimization can be found, for example, in International Publication No. 99 / 41397, the entire contents of which are incorporated herein by reference.

[0203] Many viruses, including HIV and other lentiviruses, use a number of rare codons, and by changing these to correspond to commonly used mammalian codons, expression of the packaging components in mammalian producer cells can be increased.

[0204] Codon optimization has many other advantages. Due to these sequence changes, nucleotide sequences encoding packaging components may have reduced or eliminated RNA instability sequences (INS). At the same time, the amino acid sequences coding for the packaging components are kept similar enough that the viral components encoded by the sequences remain the same, or at least similar enough so that the functionality of the packaging components is not impaired. In some embodiments, codon optimization also overcomes the Rev / RRE requirement for export, making the optimized sequences Rev-independent. In some embodiments, codon optimization also reduces homologous recombination between different constructs within a vector system (e.g., between overlapping regions in the gag-pol and env open reading frames). In some embodiments, codon optimization results in increased viral titer and / or improved safety.

[0205] In some embodiments, only the codons associated with the INS are codon optimized, while in other embodiments, the entire sequence is codon optimized except for the sequence containing the gag-pol frameshift site.

[0206] The gag-pol gene contains two overlapping reading frames encoding the gag-pol proteins. Expression of both proteins depends on a frameshift during translation. This frameshift occurs as a result of ribosome "slippage" during translation. This slippage is thought to be caused, at least in part, by secondary structures in the RNA that stall the ribosome. Such secondary structures are present downstream of the frameshift site in the gag-pol gene. In HIV, the overlapping region extends from nucleotide 1222 downstream of the start of gag (nucleotide 1 is the A of the gag ATG) to the end of gag (nt 1503). As a result, the 281-bp fragment spanning the frameshift site and the overlapping region of the two reading frames is preferably not codon-optimized. In some embodiments, retaining this fragment may allow for more efficient expression of the gag-pol proteins. In EIAV, the start of the overlap is at nt 1262 (nucleotide 1 is the A of the gag ATG). The end of the overlap is at nt 1461. To ensure that the frameshift site and gag-pol overlap are maintained, the wild-type sequence can be retained from nt 1156 to 1465.

[0207] For example, derivations from optimal codon usage can be made to accommodate convenient restriction sites and conservative amino acid changes can be introduced into the gag-pol protein.

[0208] 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.

[0209] It will be appreciated that the degraded nature of the genetic code allows one of skill in the art to achieve numerous gag-pol sequences. Additionally, many retroviral variants have been described that can be used as starting points for generating codon-optimized gag-pol sequences. Lentiviral genomes can vary considerably. For example, HIV-I has many still-functioning quasi-species. This is also true for EIAV. These variants can be used to enhance specific parts of the transduction process. Examples of HIV-I variants can be found in the HIV database maintained by Los Alamos National Laboratory. Details of EIAV clones can be found in the NCBI database maintained by the National Institutes of Health.

[0210] The strategy of codon-optimized gag-pol sequences can be used in connection with retroviruses such as EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-I, and HIV-2. Additionally, this method can be used to increase expression of genes from HTLV-I, HTLV-2, HFV, HSRV, and human endogenous retroviruses (HERVs), MLV, and other retroviruses.

[0211] As described above, the packaging components of a retroviral vector can include the expression products of the gag, pol, and env genes. Furthermore, packaging can utilize a short sequence of four stem-loops followed by partial sequences from gag and env as a packaging signal. Thus, a deleted gag sequence can be included in the retroviral vector genome (in addition to the complete gag sequence in the packaging construct). In embodiments, a retroviral vector contains a packaging signal that includes 255-360 nucleotides of gag in a vector that still retains the env sequence, or approximately 40 nucleotides of gag in a specific combination of gag and env deletions that are splice donor mutations. In some embodiments, a retroviral vector contains a gag sequence that contains one or more deletions, for example, a gag sequence that includes approximately 360 nucleotides from the N-terminus.

[0212] The retroviral vector, helper cell, helper virus, or helper plasmid can include retroviral structural and accessory proteins, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef proteins, or other retroviral proteins. In some embodiments, the retroviral proteins are derived from the same retrovirus. In some embodiments, the retroviral proteins are derived from more than one retrovirus, e.g., two, three, four, or more retroviruses.

[0213] The gag and pol coding sequences are generally organized as the Gag-Pol precursor of native lentiviruses. The gag sequence encodes the 55 kD Gag precursor protein, also called p55. p55 is cleaved by virally encoded protease 4 (the product of the pol gene) during maturation into four smaller proteins called MA (matrix [p17]), CA (capsid [p24]), NC (nucleocapsid [p9]), and p6. The pol precursor protein is cleaved from Gag by the virally encoded protease and further digested to separate the protease (p10), RT (p50), RNase H (p15), and integrase (p31) activities.

[0214] Native Gag-Pol sequences can be utilized in helper vectors (e.g., helper plasmids or helper viruses) or can be modified. These modifications include chimeric Gag-Pols, in which the Gag and Pol sequences are derived from different viruses (e.g., different species, subspecies, strains, clades, etc.) and / or the sequences have been modified to improve transcription and / or translation and / or reduce recombination.

[0215] In various examples, the retroviral nucleic acid comprises a polynucleotide encoding a 150-250 (e.g., 168) nucleotide portion of the gag protein that (i) contains a mutated INS1 inhibitory sequence that reduces restriction of RNA nuclear export compared to wild-type INS1, (ii) contains a two nucleotide insertion that results in a frameshift and premature termination, and / or (iii) does not contain the INS2, INS3, and INS4 inhibitory sequences of gag.

[0216] In some embodiments, the vectors described herein are hybrid vectors that contain both retroviral (e.g., lentiviral) and non-lentiviral sequences. In some embodiments, the hybrid vectors contain retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging.

[0217] According to certain embodiments, most or all of the viral vector backbone sequences are derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences can be used, and that combined and numerous substitutions and changes in a particular lentiviral sequence can be accommodated without impairing the transfer vector's ability to perform the functions described herein. Various lentiviral vectors are described in Naldini et al. (1996a, 1996b, and 1998), Zufferey et al. (1997), Dull et al., 1998, U.S. Patent No. 6,013,516, and U.S. Patent No. 5,994,136, many of which can be adapted to produce retroviral nucleic acid.

[0218] Long terminal repeats (LTRs) are typically found at both ends of a provirus. LTRs typically contain domains located at the ends of retroviral nucleic acids that, in the context of the natural sequence, are direct repeats and include the U3, R, and U5 regions. LTRs generally promote retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs can contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences for viral genome replication and integration. Viral LTRs are typically divided into three regions, designated U3, R, and U5. The U3 region typically contains enhancer and promoter elements. The U5 region is typically located between the primer binding site and the R region and can contain a polyadenylation sequence. R (repeat) regions can flank the U3 and U5 regions. LTRs are typically composed of U3, R, and U5 regions and can appear at both the 5' and 3' ends of the viral genome. In some embodiments, adjacent to the 5'LTR are sequences for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site).

[0219] The packaging signal 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; pp. 2101-2109. Some retroviral vectors use a minimal packaging signal (psi [Ψ] sequence) for encapsidation of the viral genome.

[0220] In various embodiments, the retroviral nucleic acid comprises a modified 5'LTR and / or 3'LTR. Either or both of the LTRs may contain one or more modifications, including, but not limited to, one or more deletions, insertions, or substitutions. Modifications of the 3'LTR are often made to improve the safety of lentiviral or retroviral systems by rendering the virus replication-defective (e.g., a virus that is unable to fully and efficiently replicate so that infectious virions are not produced (e.g., progeny of a replication-defective lentivirus)).

[0221] In some embodiments, the vector is a self-inactivating (SIN) vector, e.g., a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., 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 the absence of the U3 enhancer-promoter inhibits viral replication. In embodiments, the 3' LTR is modified such that the U5 region has been removed, altered, or replaced, e.g., with an exogenous poly(A) sequence. The 3' LTR, the 5' LTR, or both the 3' and 5' LTRs can be modified LTRs.

[0222] In some embodiments, the U3 region of the 5'LTR is replaced with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include, for example, the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. In some embodiments, the promoter can drive high levels of transcription in a Tat-independent manner. In certain embodiments, heterologous promoters have the additional advantage of controlling how the viral genome is transcribed. For example, heterologous promoters can be inducible so that transcription of all or part of the viral genome occurs only in the presence of an inducer. Inducers include, but are not limited to, one or more compounds under which the host cells are cultured, or physiological conditions such as temperature or pH.

[0223] In some embodiments, the viral vector contains a TAR (transactivation response) element, located, for example, in the R region of the lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required, for example, in embodiments in which the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0224] The R region, e.g., the region within a retroviral LTR that begins at the start of the capping group (i.e., 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.

[0225] Retroviral nucleic acids can also contain FLAP elements, e.g., nucleic acids whose sequences include the central polypurine takeaway and central termination sequence (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173, which are incorporated herein by reference in their entireties. During HIV-1 reverse transcription, central initiation of plus-strand DNA at the central polypurine takeaway (cPPT) and central termination at the central termination sequence (CTS) can lead to the formation of a triple-stranded DNA structure, i.e., a central DNA flap. In some embodiments, the retroviral or lentiviral vector backbone contains one or more FLAP elements upstream or downstream of a gene encoding an exogenous agent. For example, in some embodiments, a transfer plasmid contains a FLAP element, e.g., a FLAP element derived from or isolated from HIV-1.

[0226] In embodiments, the retroviral or lentiviral nucleic acid comprises one or more export elements, such as cis-acting post-transcriptional regulatory elements that regulate the transport of RNA transcripts from the nucleus to the cytoplasm of the cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58:423) and the hepatitis B virus post-transcriptional regulatory element (HPRE), which are incorporated herein by reference in their entireties. Generally, RNA export elements are located within the 3'UTR of a gene and can be inserted in one or more copies.

[0227] In some embodiments, expression of heterologous sequences in viral vectors is increased by incorporating one or more of, for example, a posttranscriptional regulatory element, a polyadenylation site, and a transcription termination signal into the vector. Various posttranscriptional regulatory elements can increase expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864), and the like (Liu et al., 1995, Genes Dev., 9:1766), each of which is incorporated herein by reference in its entirety. In some embodiments, the retroviral nucleic acids described herein include a posttranscriptional regulatory element such as a WPRE or HPRE.

[0228] In some embodiments, the retroviral nucleic acids described herein lack or do not include post-transcriptional regulatory elements such as a WPRE or HPRE.

[0229] Elements directing the 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 contains a polyadenylation sequence 3' to the polynucleotide encoding the exogenous agent. A polyA site can contain a DNA sequence that directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. The polyadenylation sequence promotes mRNA stability and contributes to improved translation efficiency by adding a polyA tail to the 3' end of the coding sequence. Examples of polyA signals that can be used in retroviral nucleic acids include AATAAA, ATTAAA, AGTAAA, bovine growth hormone polyA sequence (BGHpA), rabbit β-globin polyA sequence (rβgpA), or another suitable heterologous or endogenous polyA sequence.

[0230] In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator sequences, eg, an insulator sequence described herein.

[0231] In various embodiments, the vector comprises a promoter operably linked to a polynucleotide encoding an exogenous agent. The vector may have one or more LTRs, where any LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector may also comprise one or more accessory elements to enhance transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., Psi (Ψ) packaging signal, RRE), and / or other elements to increase exogenous gene expression (e.g., poly(A) sequence), and may optionally include a WPRE or HPRE.

[0232] In some embodiments, the lentiviral nucleic acid comprises one or more of the following, all of which are present, e.g., 5' to 3': a promoter (e.g., CMV), an R sequence (e.g., including TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter driving expression of an exogenous agent, a gene encoding the 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), and a U5 signal (e.g., for integration).

[0233] ii) Vectors engineered to remove splice sites Some lentiviral vectors have strong splicing and polyadenylation signals that can be integrated within active genes, leading to the formation of aberrant and sometimes truncated transcripts.

[0234] The mechanism of proto-oncogene activation may involve the generation of chimeric transcripts resulting from the interaction of promoter elements or splice sites contained in the genome of the insertional mutagen with the cellular transcription unit targeted for integration (Gabriel et al. 2009. Nat Med 15:1431-1436; Bokhoven, et al. J Virol 83:283-29). Chimeric fusion transcripts containing vector sequences and cellular mRNAs can be generated by read-through transcription that initiates from the vector sequences and proceeds to adjacent cellular genes, or vice versa.

[0235] In some embodiments, the lentiviral nucleic acids described herein comprise a lentiviral backbone in which at least two of the splice sites have been removed, e.g., to improve the safety profile of the lentiviral vector. The types and methods for identifying such splice sites are described in WO2012156839A2, which is incorporated by reference in its entirety.

[0236] iii) Methods for producing retroviruses Large-scale production of viral particles is often useful to achieve a desired viral titer. Viral particles can be produced by transfecting transfer vectors into packaging cell lines 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.

[0237] In embodiments, the packaging vector is an expression vector or viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Typically, packaging vectors are contained in packaging cells and introduced into cells via transfection, transduction, or infection. Retroviral, e.g., lentiviral, transfer vectors can be introduced into packaging cell lines via transfection, transduction, or infection to generate source cells or cell lines. 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 primary selectable marker, such as neomycin, hygromycin, puromycin, blastocidin, zeocin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug to isolate clones. The selectable marker gene can be physically linked to the encoding gene by the packaging vector, for example, by an IRES or a self-cleaving viral peptide.

[0238] Packaging cell lines include cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replicase (e.g., gag, pol, and env) that are capable of packaging viral particles. Any suitable cell line, e.g., mammalian cells, e.g., human cells, can be used. Suitable cell lines that can be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells.

[0239] Source cell lines include packaging cell lines and cell lines capable of producing recombinant retroviral particles containing transfer vector constructs containing packaging signals. Methods for preparing virus stock solutions are described, for example, by Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and NRL Landau et al. (1992) J. Virol. 66:5110-5113, which are incorporated herein by reference. Infectious virus particles can be collected from packaging cells, for example, by cell lysis or collection of cell culture supernatant. If necessary, the collected virus particles can be concentrated or purified.

[0240] 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 (e.g., gag, pol, and env) capable of packaging viral particles. In some embodiments, the sequences encoding at least two of the gag, pol, and env precursors are on the same plasmid. In some embodiments, the sequences encoding the gag, pol, and env precursors are on different plasmids. In some embodiments, the sequences encoding the gag, pol, and env precursors have the same expression signal, e.g., promoter. In some embodiments, the sequences encoding the gag, pol, and env precursors have different expression signals, e.g., different promoters. In some embodiments, expression of the gag, pol, and env precursors is inducible. In some embodiments, the plasmids encoding the viral structural proteins and replication enzymes are transfected simultaneously or at different times. In some embodiments, the plasmids encoding the viral structural proteins and replication enzymes are transfected simultaneously or at different times from the packaging vector.

[0241] In some embodiments, the source cell line comprises one or more stably integrated viral structural genes. In some embodiments, expression of the stably integrated viral structural genes is inducible.

[0242] In some embodiments, expression of viral structural genes is regulated at the transcriptional level. In some embodiments, expression of viral structural genes is regulated at the translational level. In some embodiments, expression of viral structural genes is regulated at the post-translational level.

[0243] In some embodiments, expression of viral structural genes is regulated by a tetracycline (Tet)-dependent system, in which the Tet-regulated transcriptional repressor (Tet-R) binds to DNA sequences contained in the promoter and represses transcription through steric hindrance (Yao et al., 1998; Jones et al., 2005). Addition of doxycycline (dox) releases Tet-R, allowing transcription. Several other suitable transcriptionally regulated promoters, transcription factors, and small molecule inducers are suitable for regulating transcription of viral structural genes.

[0244] In some embodiments, the third generation lentiviral components, human immunodeficiency virus type 1 (HIV) Rev, Gag / Pol, and envelope under the control of a Tet-regulated promoter and linked to an antibiotic resistance cassette, are separately integrated into the source cell genome. In some embodiments, the source cell has only one copy each of Rev, Gag / Pol, and envelope proteins integrated into its genome.

[0245] In some embodiments, a nucleic acid encoding an exogenous agent (e.g., 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, the nucleic acid encoding the exogenous agent is transfected into a source cell that has Rev, Gag / Pol, and envelope proteins stably integrated into the genome. See, e.g., Milani et al. EMBO Molecular Medicine, 2017, incorporated herein by reference in its entirety.

[0246] In some embodiments, the retroviral nucleic acid described herein is incapable of undergoing reverse transcription. Such nucleic acids, in embodiments, are capable of transiently expressing an exogenous agent. The retrovirus or VLP may comprise a disabled reverse transcriptase protein or may not comprise a reverse transcriptase protein. In embodiments, the retroviral nucleic acid comprises a disabled primer binding site (PBS) and / or att site. In embodiments, one or more viral accessory genes, including rev, tat, vif, nef, vpr, vpu, vpx, and S2, or functional equivalents thereof, are 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.

[0247] v) Strategies for packaging retroviral nucleic acids Typically, modern retroviral vector systems consist of (1) a viral genome with cis-acting vector sequences for transcription, reverse transcription, integration, translation, and packaging of viral RNA into viral particles, and (2) a producer cell line expressing the trans-acting retroviral gene sequences (e.g., gag, pol, and env) necessary for the production of viral particles. Complete separation of the cis- and trans-acting vector sequences ensures that the virus cannot sustain replication for more than one cycle of infection. The generation of live virus can be avoided by several strategies, for example, by minimizing overlap between the cis- and trans-acting sequences to avoid recombination.

[0248] Viral vector particles lacking or containing sequences lacking viral RNA may be the result of removing or excluding viral RNA from the sequence. In one embodiment, this can be achieved by using the endogenous packaging signal binding site on gag. Alternatively, the endogenous packaging signal binding site is on pol. In this embodiment, the delivered RNA 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 can be used to ensure packaging of the delivered RNA. The heterologous sequence may be non-viral or viral, in which case it 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 pol is typically included.

[0249] In one embodiment, gag-pol is modified, and the packaging signal is replaced with the corresponding packaging signal.In this embodiment, the particle can package RNA with the new packaging signal.The advantage of this approach is that it can package RNA sequences that lack viral sequences, such as RNAi.

[0250] Another approach is to rely on the overexpression of the RNA to be packaged. In one embodiment, the RNA to be packaged is overexpressed in the absence of RNA containing a packaging signal. This can result in significant levels of therapeutic RNA being packaged, which is sufficient to transduce cells and produce biological effects.

[0251] 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 comprises a heterologous RNA-binding domain that can recognize a corresponding sequence in an RNA sequence and facilitate packaging of the RNA sequence into a viral vector particle.

[0252] In some embodiments, the heterologous RNA binding domain comprises an RNA binding domain derived from a bacteriophage coat protein, a Rev protein, a protein of the U1 small nuclear ribonucleoprotein particle, a Nova protein, a TFl 11A protein, a TISl protein, a trp RNA-binding decay protein (TRAP), or a pseudouridine synthase.

[0253] In some embodiments, the methods herein include detecting or confirming the absence of a replication-competent retrovirus. The methods may include assessing the RNA level of one or more target genes, such as viral genes, e.g., structural genes or packaging genes, whose gene products are expressed in certain cells infected with a replication-competent retrovirus, such as a gammaretrovirus or lentivirus, but which are not present in the viral vector used to transduce the cells with the heterologous nucleic acid and which are not present and / or expressed, or would not be expected to be present and / or expressed, in cells that do not contain a replication-competent retrovirus. A replication-competent retrovirus may be determined to be present when the RNA level of one or more target genes is higher than a reference value, which can be measured directly or indirectly, for example, from a positive control sample containing the target genes. For a detailed disclosure, see WO2018023094A1.

[0254] vi) Repression of genes encoding exogenous agents in source cells Proteins (over)expressed in source cells may have an indirect or direct effect on the assembly and / or infectivity of vector virions. Incorporation of exogenous agents into vector virions may also affect downstream processing of the vector particle.

[0255] In some embodiments, a tissue-specific promoter is used to restrict expression of the exogenous agent in the source cells. In some embodiments, a heterologous translational control system is used in eukaryotic cell culture to repress translation of the exogenous agent in the source cells. More specifically, the retroviral nucleic acid can contain a binding site operably linked to a gene encoding the exogenous agent, where the binding site can interact with an RNA-binding protein such that translation of the exogenous agent is repressed or prevented in the source cells.

[0256] In some embodiments, the RNA-binding protein is a tryptophan RNA-binding attenuation protein (TRAP), e.g., a bacterial tryptophan RNA-binding attenuation protein. The use of an RNA-binding protein (e.g., a bacterial trp operon regulator protein, tryptophan RNA-binding attenuation protein, TRAP) and the RNA target to which it binds represses or prevents translation of a transgene in a source cell. This system is referred to as the Transgene Repression In vector Production cell system, or TRIP system.

[0257] In embodiments, the placement of a binding site for an RNA-binding protein (e.g., a TRAP binding sequence, tbs) upstream of the NOI translation start codon allows for specific repression of translation of mRNA derived from an internal expression cassette without adversely affecting vector RNA production or stability. The number of nucleotides between the tbs and the translation start codon of the gene encoding the exogenous agent can vary from 0 to 12 nucleotides. The tbs can be placed downstream of an internal ribosome entry site (IRES) to repress translation of the gene encoding the exogenous agent in a multicistronic mRNA.

[0258] vii) Kill switch systems and amplification In some embodiments, polynucleotides or cells harboring genes encoding exogenous agents utilize suicide genes, e.g., inducible suicide genes, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain aspects, the suicide genes are not immunogenic to host cells harboring the exogenous agent. Examples of suicide genes include caspase-9, caspase-8, or cytosine deaminase. Caspase-9 can be activated using specific chemical inducers of dimerization (CIDs).

[0259] In certain embodiments, the vector comprises a gene segment that renders target cells, e.g., immune effector cells, e.g., T cells, susceptible to negative selection in vivo. For example, transduced cells may be eliminated as a result of changes in an individual's in vivo conditions. A negatively selectable phenotype may result from the insertion of a gene that confers sensitivity to an administered agent, e.g., a compound. Negatively selectable genes are known in the art and include, among others: the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 11:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA 89:33 (1992)).

[0260] 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 for the selection of cells with a negatively selectable phenotype in vitro. A positive selection marker can be a gene that, when introduced into a target cell, expresses a dominant phenotype that allows for the positive selection of cells carrying the gene. Genes of this type include, among others, the hygromycin-B phosphotransferase gene (hph), which confers resistance to hygromycin B, the Tn5-derived aminoglycoside phosphotransferase gene (neo or aph), which encodes resistance to the antibiotic G418, dihydrofolate reductase (DHFR), the adenosine deaminase gene (ADA), and multidrug resistance (MDR) genes.

[0261] In some embodiments, the positive selectable marker and negative selectable element are linked such that loss of the negative selectable element necessarily results in loss of the positive selectable marker. For example, positive and negative selectable markers can be fused so that loss of one results in loss of the other. An example of a fusion polynucleotide whose expression product is a polypeptide that confers both the desired positive and negative selection functions is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene produces a polypeptide that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See Lupton SD, et al., Mol. and Cell. Biology 1 1:3374-3378, 1991. Furthermore, in embodiments, the polynucleotide encoding the chimeric receptor is present in a retroviral vector, e.g., the HyTK retroviral vector described in Lupton SD, et al., (1991), supra, that contains a fusion gene, particularly a fusion gene that confers hygromycin B resistance for in vitro positive selection and ganciclovir sensitivity for in vivo negative selection. See also publications PCT U591 / 08442 and PCT / U594 / 05601, which describe the use of bifunctional selectable fusion genes derived from the fusion of a dominant positive selectable marker and a negative selectable marker.

[0262] Suitable positive selectable markers are derived from genes selected from the group consisting of hph, nco, and gpt, and suitable negative selectable markers may be derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. Other suitable markers are bifunctional selectable fusion genes in which the positive selectable marker is derived from hph or neo and the negative selectable marker is derived from a cytosine deaminase or TK gene, or selectable marker.

[0263] viii) Strategies for modulating lentiviral integration Retroviral and lentiviral nucleic acids have been disclosed in which key proteins / sequences are missing or disabled to prevent the retroviral or lentiviral genome from integrating 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) allows the production of integration-deficient retroviral nucleic acids.

[0264] For example, in some embodiments, a retroviral nucleic acid herein comprises a lentiviral integrase containing a mutation that renders the integrase unable to catalyze the integration of the viral genome into the cellular genome. In some embodiments, the mutation is a Type I mutation that directly affects integration, or a Type II mutation that induces a pleiotropic defect that affects virion morphogenesis and / or reverse transcription. An illustrative, non-limiting example of a Type I mutation is a mutation that affects any of three residues involved in the catalytic core domain of integrase: DX 39-58 DX 35 E (residues D64, D116, and E152 of HIV-1 integrase). In certain embodiments, the mutation that renders the integrase unable to catalyze the integration of the viral genome into the cellular genome is a substitution of one or more amino acid residues in the DDE motif of the catalytic core domain of integrase, preferably a substitution of the first aspartic acid residue of the DEE motif with an asparagine residue. In some embodiments, the retroviral vector does not comprise an integrase protein.

[0265] In some embodiments, the retrovirus integrates into an active transcription unit. In some embodiments, the retrovirus does not integrate near the transcription start site, the 5' end of a gene, or a DNAse 1 cleavage site. In some embodiments, retroviral integration does not activate proto-oncogenes or inactivate tumor suppressor genes. In some embodiments, the retrovirus is not genotoxic. In some embodiments, the lentivirus integrates into an intron.

[0266] In some embodiments, the retroviral nucleic acid integrates into the genome of the target cell at a specific copy number. The average copy number can be determined from a single cell, a population of cells, or an individual cell colony. Exemplary methods for determining copy number include polymerase chain reaction (PCR) and flow cytometry.

[0267] In some embodiments, the DNA encoding the exogenous agent is genomically integrated. In some embodiments, the DNA encoding the exogenous agent is episomally maintained. In some embodiments, the ratio of exogenous agent integrated into episomal DNA encoding the exogenous agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, 100.

[0268] In some embodiments, the DNA encoding the exogenous agent is linear. In some embodiments, the DNA encoding the exogenous agent is circular. In some embodiments, the ratio of linear copies to circular copies of the DNA encoding the exogenous agent is at least 0.01, 0.1, 0.5, 1.0, 2, 5, 10, or 100.

[0269] In embodiments, the DNA encoding the exogenous agent is circular and comprises one LTR. In some embodiments, the DNA encoding the exogenous agent is circular and comprises two LTRs. In some embodiments, the ratio of circular, single-LTR DNA encoding the exogenous agent to circular, two-LTR DNA encoding the exogenous agent is at least 0.1, 0.5, 1.0, 2, 5, 10, 20, 50, or 100.

[0270] ix) Episomal virus maintenance In poorly integrated retroviruses, circular cDNA by-products of reverse transcription (e.g., 1-LTR and 2-LTR) can accumulate in the cell nucleus without integrating into the host genome (Yanez-Munoz RJ et al., Nat. Med. 2006, 12:348-353). These intermediates are then ligated at the same frequency (e.g., 10 ) as other exogenous DNA. 3 ~10 5 / cell) and can integrate into cellular DNA.

[0271] 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 scaffold / matrix attachment region (S / MAR) for association with the nuclear matrix.

[0272] 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 origin of replication of the β-globin gene described by Aladjem et al. (Science, 1995, 270:815-819), the consensus sequence derived from an autonomously replicating sequence associated with an alpha-satellite sequence previously isolated from monkey CV-1 cells and human skin fibroblasts as described by Price et al. Journal of Biological Chemistry, 2003, 278(22):19649-59, and the origin of replication of the human c-myc promoter region described by McWinney and Leffak (McWinney C. and Leffak M., Nucleic Acid Research 1990, 18(5):1233-42). In embodiments, the variants substantially retain the ability to initiate replication in eukaryotes. The ability of a particular sequence to initiate replication can be determined by any suitable method, for example, by autonomous replication assays based on bromodeoxyuridine incorporation and density shifts (Araujo, F.D. et al., supra; Frappier, L. et al.).

[0273] In some embodiments, the retroviral nucleic acid comprises a scaffold / matrix attachment region (S / MAR) or a variant thereof, e.g., a non-consensus-like AT-rich DNA element several hundred base pairs in length, which organizes the nuclear DNA of eukaryotic genomes into chromatin domains by periodic attachment to the protein scaffold or matrix of the cell nucleus. They are typically found in non-coding regions such as flanking regions, chromatin boundary regions, and introns. An example of an S / MAR region is the human IFN-γ gene (hIFN-γ ) described by Bode et al. (Bode J. et al., Science, 1992, 255:195-7). large), the 1.8 kbp S / MAR of the human IFN-γ gene, the 0.7 Kbp minimal S / MAR region of the human IFN-γ gene described by Ramezani (Ramezani A. et al., Blood 2003, 101:4717-24). short), Mesner LD et al., Proc Natl Acad Sci USA, 2003, 100:3281-86). In embodiments, functionally equivalent variants of S / MARs are sequences selected based on a set of six rules that have been suggested, together or individually, to contribute to S / MAR function (Kramer et al. (1996) Genomics 33, 305; Singh et al. (1997) Nucl. These rules were merged into the MAR-Wiz computer program, available for free at genomecluster.secs.oakland.edu / MAR-Wiz. In embodiments, variants substantially maintain the same function of the S / MAR from which they are derived, in particular the ability to specifically bind to the nuclear matrix. Those skilled in the art will be able to identify functionally equivalent variants of S / MARs by using, for example, in vitro or in vitro methods described in Mesner et al. (Mesner LD et al., supra). In vivo MAR assays can determine whether a particular variant can specifically bind to the nuclear matrix. In some embodiments, a particular sequence is an S / MAR variant if it exhibits a tendency for DNA strand separation. This property can be determined using specific programs based on equilibrium statistical mechanics methods. The stress-induced duplex destabilization (SIDD) analysis method, as defined by Bode et al. (2005) J. Mol. Biol. 358, 597, "calculates the extent to which an imposed level of superhelical stress reduces the free energy required to open the duplex at each position along the DNA sequence. The results are displayed as a SIDD profile, where sites of strong destabilization appear as deep minima." The SIDD algorithm and mathematical foundations (Bi and Benham (2004) Bioinformatics 20, 1477), as well as analysis of SIDD profiles, can be performed using the freely available internet resource WebSIDD (www.genomecenter.ucdavis.edu / benham).Thus, in some embodiments, a polynucleotide is considered a variant of an S / MAR sequence if it exhibits a similar SIDD profile as the S / MAR.

[0274] B. Cell-derived fusosomes The fusosome composition can be produced from cells in culture, e.g., cultured mammalian cells, e.g., cultured human cells. The cells can be progenitor cells or non-progenitor (e.g., differentiated) cells. The cells can be primary cells or cell lines (e.g., mammalian, e.g., human, cell lines described herein). In embodiments, the cultured cells are progenitor cells, e.g., bone marrow stromal cells, bone marrow-derived adult progenitor cells (MAPCs), endothelial progenitor cells (EPCs), blast cells, intermediate progenitor cells formed in the subventricular zone, neural stem cells, muscle stem cells, satellite cells, liver stem cells, hematopoietic stem cells, bone marrow stromal cells, epidermal stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, progenitor cells, muscle progenitor cells, myoblasts, cardiac myoblasts, neural progenitor cells, glial progenitor cells, neural progenitor cells, or hepatoblasts.

[0275] 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, adolescent skin, blood, bone marrow, adipose tissue, erythropoietic tissue, hematopoietic tissue), myoblasts, parenchymal cells (e.g., hepatocytes), alveolar cells, neurons (e.g., retinal neurons), or the like. The cell may be a retinal progenitor cell, a myeloblast, a myeloid progenitor cell, a thymocyte, a meiocyte, a megakaryoblast, a promegakaryoblast, a melanocyte, a lymphoblast, a myeloid progenitor cell, a normoblast, or a hemangioblast, a progenitor cell (e.g., a cardiac progenitor cell, a satellite cell, a radial glial cell, a bone marrow stromal cell, a pancreatic progenitor cell, an endothelial progenitor cell, a blast cell), or an immortalized cell (e.g., a HeLa, HEK293, HFF-1, MRC-5, WI-38, IMR90, IMR91, PER.C6, HT-1080, or BJ cell).

[0276] The cultured cells may be cells derived from epithelial, connective, muscle, or nervous tissue or cells, as well as combinations thereof. Fusosomes may be produced from cultured cells derived from any eukaryotic (e.g., mammalian) organ system, such as the cardiovascular system (heart, vasculature); digestive system (esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, and anus); endocrine system (hypothalamus, pituitary gland, pineal gland or glands, thyroid gland, parathyroid glands, adrenal glands); 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 (bone, cartilage), and combinations thereof. In embodiments, the cells are derived from highly mitotic tissue (e.g., highly mitotic healthy tissue such as epithelium, embryonic tissue, bone marrow, intestinal crypts, etc.) In embodiments, the tissue sample is a highly metabolic tissue (e.g., skeletal tissue, neural tissue, cardiomyocytes).

[0277] In some embodiments, the cells are derived from young donors, for example, donors under the age of 25, 20, 18, 16, 12, 10, 8, 5, 1. In some embodiments, the cells are derived from fetal tissue.

[0278] In some embodiments, the cells are derived from a subject and are administered to the same subject or a subject with similar genetic characteristics (eg, MHC match).

[0279] In certain embodiments, the cells have average sized telomeres greater than 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length (e.g., 4,000-10,000 nucleotides in length, 6,000-10,000 nucleotides in length).

[0280] In some embodiments, fusosomes are generated from cell clones that have been identified, selected, or chosen based on a desirable phenotype or genotype for use as a source of the fusosome compositions described herein, e.g., cell clones are identified, selected, or chosen based on low mitochondrial mutation burden, long telomere length, differentiation state, or particular genetic characteristics (e.g., genetic characteristics that match the recipient).

[0281] The fusosome compositions described herein can be composed of fusosomes from a single cell or tissue source or from a combination of sources. For example, fusosome compositions can include fusosomes from xenogeneic sources (e.g., animal, tissue culture of cells from the aforementioned species), allogeneic, autologous, specific tissues (e.g., liver, skeletal, nervous, adipose, etc.) that result in different protein concentrations and distributions, or cells in different metabolic states (e.g., glycolytic, respiratory). The compositions can also include fusosomes in different metabolic states, e.g., bound or unbound, as described elsewhere herein.

[0282] In some embodiments, fusosomes are generated from source cells that express a fusogen, e.g., a fusogen described herein. In some embodiments, the fusogen is located in a membrane of the source cell, e.g., a lipid bilayer membrane, e.g., a cell surface membrane, or an intracellular membrane (e.g., a lysosomal membrane). In some embodiments, fusosomes are generated from source cells that have the fusogen located in the cell surface membrane.

[0283] In some embodiments, fusosomes are generated by inducing budding of exosomes, microvesicles, membrane vesicles, extracellular membrane vesicles, plasma membrane vesicles, giant plasma membrane vesicles, apoptotic bodies, mitoparticles, pyrenocytes, lysosomes, or other membrane-enclosed vesicles.

[0284] In some embodiments, fusosomes are generated by inducing cell enucleation. Enucleation can be achieved using assays such as genetic, chemical (e.g., using actinomycin D, see Bayona-Bafaluy et al., "A chemical enucleation method for the transfer of mitochondrial DNA to ρ cells," Nucleic Acids Res. 2003 Aug 15;31(16):e98), or mechanical (e.g., 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 a combination thereof. Enucleation can refer not only to the complete removal of the nucleus, but also to the displacement of the nucleus from its typical location so that the cell contains a nucleus but is non-functional.

[0285] In embodiments, generating fusosomes comprises producing cell ghosts, giant plasma membrane vesicles, or apoptotic bodies. In embodiments, the fusosome composition comprises one or more of cell ghosts, giant plasma membrane vesicles, and apoptotic bodies.

[0286] In some embodiments, fusosomes are generated by inducing cell fragmentation, which can be achieved using the following methods, including but not limited to chemical methods, mechanical methods (e.g., centrifugation (e.g., ultracentrifugation, or density centrifugation), freeze-thawing, or sonication), or combinations thereof.

[0287] In some embodiments, fusosomes can be generated from source cells expressing a fusogen by any one, all, or a combination of the following methods, for example, as described herein. i) induce the budding of mitoses, exosomes, or other membrane-enclosed vesicles; ii) Inducing nuclear inactivation, e.g., enucleation, by any one of the following methods or a combination thereof: a) Genetic methods; b) Chemical methods, for example using actinomycin D; or c) mechanical methods, such as squeezing or suction; or iii) Inducing cell fragmentation, for example, by any one of the following methods or a combination thereof: a) chemical methods; b) Mechanical methods, such as centrifugation (e.g., ultracentrifugation or density centrifugation); freeze-thaw; or sonication.

[0288] i) Modification of cells before fusosome formation In some aspects, modifications are made to cells, such as modifications of a subject, tissue, or cell, prior to fusosome production. Such modifications may be effective, for example, to improve fusion, fusogen expression or activity, cargo structure or function, or target cell structure or function.

[0289] a) Physical modification In some embodiments, cells are physically modified prior to producing fusosomes, e.g., fusogens can be linked to the surface of the cells, as described elsewhere herein.

[0290] In some embodiments, cells are treated with a chemical agent prior to generating fusosomes. For example, cells can be treated with a chemical or lipid fusogen such that the chemical or lipid fusogen interacts non-covalently or covalently with or is embedded within the surface of the cells. In some embodiments, cells are treated with an agent to enhance the fusogenic properties of lipids in the cell membrane.

[0291] In some embodiments, cells are physically modified prior to generating fusosomes to have one or more covalent or non-covalent attachment sites on the cell surface for synthetic or endogenous small molecules or lipids that enhance targeting of fusosomes to organs, tissues, or cell types.

[0292] In embodiments, fusosomes contain increased or decreased levels of endogenous molecules. For example, fusosomes may contain endogenous molecules that are naturally present in naturally occurring source cells, but at higher or lower levels than in fusosomes. In some embodiments, the polypeptide is expressed from an exogenous nucleic acid in the source cells or fusosomes. In some embodiments, the polypeptide is isolated from a source and loaded or bound to the source cells or fusosomes.

[0293] In some embodiments, cells are treated with a chemical agent, e.g., a small molecule, prior to generating fusosomes to increase the expression or activity of an endogenous fusogen in the cells (e.g., in some embodiments, endogenous to the source cell, and in some embodiments, endogenous to the target cell). In some embodiments, the small molecule may increase the expression or activity of a transcriptional activator of the endogenous fusogen. In some embodiments, the small molecule may decrease the expression or activity of a transcriptional repressor of the endogenous fusogen. In some embodiments, the small molecule is an epigenetic modifier that increases the expression of the endogenous fusogen.

[0294] In some embodiments, fusosomes are generated from cells treated with a fusion-stopping compound, e.g., lysophosphatidylcholine. In some embodiments, fusosomes are generated from cells treated with a dissociation reagent that does not cleave fusogens, e.g., Accutase.

[0295] In some embodiments, the source cells are physically modified, for example with a CRISPR activator, before generating fusosomes to add or increase the concentration of fusogens.

[0296] In some embodiments, cells are physically modified to increase or decrease the amount or enhance the structure or function of organelles, such as mitochondria, the Golgi apparatus, the endoplasmic reticulum, intracellular vesicles (lysosomes, autophagosomes, etc.).

[0297] b) Genetic modification In some embodiments, cells are genetically modified prior to producing fusosomes to increase expression of an endogenous fusogen in the cells (e.g., in some embodiments, endogenous to the source cell and, in some embodiments, endogenous to the target cell). In some embodiments, the genetic modification may increase expression or activity of a transcriptional activator of the endogenous fusogen. In some embodiments, the genetic modification may decrease expression or activity of a transcriptional repressor of the endogenous fusogen. In some aspects, the activator or repressor is a nuclease-inactive Cas9 (dCas9) linked to a transcriptional activator or repressor that targets the endogenous fusogen by a guide RNA. In some embodiments, the genetic modification epigenetically modifies the endogenous fusogen gene to increase its expression. In some aspects, the epigenetic activator is a nuclease-inactive Cas9 (dCas9) linked to an epigenetic modifier that targets the endogenous fusogen by a guide RNA.

[0298] In some embodiments, cells are genetically modified before producing fusosomes to increase expression of exogenous fusogens within the cells, e.g., to deliver a transgene. In some aspects, nucleic acids, e.g., DNA, mRNA, or siRNA, are introduced into cells before producing fusosomes to increase or decrease expression of cell surface molecules (proteins, glycans, lipids, or low molecular weight molecules), e.g., for use in organ, tissue, or cell targeting. In some embodiments, the nucleic acid targets a repressor of a fusogen, e.g., an shRNA or siRNA construct. In some embodiments, the nucleic acid encodes an inhibitor of a fusogen repressor.

[0299] In some embodiments, the method includes introducing into the source cell a nucleic acid exogenous to the source cell that encodes a fusogen. The exogenous nucleic acid can be, for example, DNA or RNA. In some embodiments, the exogenous nucleic acid can be, for example, DNA, gDNA, cDNA, RNA, pre-mRNA, mRNA, miRNA, siRNA, etc. In some embodiments, the exogenous DNA can be linear DNA, circular DNA, or an artificial chromosome. In some embodiments, the DNA is maintained episomally. In some embodiments, the DNA is integrated into the genome. The exogenous RNA can be chemically modified RNA, for example, and can include one or more backbone modifications, sugar modifications, non-canonical bases, or caps. Backbone modifications include, for example, phosphorothioates, N3' phosphoramidites, boranophosphates, phosphonoacetates, thio-PACE, morpholinophosphoramidites, or PNAs. Sugar modifications include, for example, 2'-O-Me, 2'F, 2'F-ANA, LNA, UNA, and 2'-O-MOE. Non-canonical bases include, for example, 5-bromo-U and 5-iodo-U, 2,6-diaminopurine, C-5 propynylpyrimidine, difluorotoluene, difluorobenzene, dichlorobenzene, 2-thiouridine, pseudouridine, and dihydrouridine. Caps include, for example, ARCA. Additional modifications are discussed, for example, in Deleavy et al., "Designing Chemically Modified Oligonucleotides for Targeted Gene Silencing," Chemistry & Biology, Volume 19, Issue 8, 24 August 2012, Pages 937-954, which is incorporated herein by reference in its entirety.

[0300] In some aspects, cells are treated with a chemical agent, e.g., a small molecule, prior to generating fusosomes to increase the expression or activity of exogenous fusogens relative to the source cells. In some embodiments, the small molecule may increase the expression or activity of a transcriptional activator of the exogenous fusogen. In some embodiments, the small molecule may decrease the expression or activity of a transcriptional repressor of the exogenous fusogen. In some embodiments, the small molecule is an epigenetic modifier that increases the expression of the exogenous fusogen.

[0301] In some embodiments, the nucleic acid encodes an altered fusogen. For example, a fusogen with tunable fusogenic activity, e.g., activity in a particular cell type, tissue type, or local microenvironment. Such tunable fusogenic activity can include activation and / or initiation of fusogenic activity by low pH, high pH, ​​heat, infrared light, extracellular enzyme activity (eukaryotic or prokaryotic), or exposure to small molecules, proteins, or lipids. In some embodiments, the small molecules, proteins, or lipids are displayed on target cells.

[0302] In some aspects, cells are genetically modified prior to producing fusosomes to alter (i.e., upregulate or downregulate) expression of a signaling pathway (e.g., the Wnt / beta-catenin pathway). In some aspects, cells are genetically modified prior to producing fusosomes to alter (e.g., upregulate or downregulate) expression of one or more genes of interest. In some embodiments, cells are 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. In some embodiments, a nucleic acid, e.g., DNA, mRNA, or siRNA, is introduced into cells prior to producing fusosomes, e.g., 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 represses a signaling pathway, gene, or nucleic acid. In some embodiments, the nucleic acid encodes a transcription factor that upregulates or downregulates a signaling pathway, gene, or nucleic acid. In some embodiments, the activator or repressor is a nuclease-inactive cas9 (dCas9) linked to a transcriptional activator or repressor that targets a signaling pathway, gene, or nucleic acid via a guide RNA. In some embodiments, the genetic modification epigenetically modifies the expression of an endogenous signaling pathway, gene, or nucleic acid. In some embodiments, the epigenetic activator is a nuclease-inactive cas9 (dCas9) linked to an epigenetic modifier that targets a signaling pathway, gene, or nucleic acid via a guide RNA. In some aspects, the cell's DNA is edited prior to generating fusosomes to alter (e.g., upregulate or downregulate) the expression of a signaling pathway (e.g., the Wnt / beta-catenin pathway), gene, or nucleic acid. In some embodiments, the DNA is edited using guide RNA and CRISPR-Cas9 / Cpf1 or other gene editing techniques.

[0303] Cells can be genetically modified using recombinant methods. The nucleic acid sequence encoding the desired gene can be obtained using recombinant methods, for example, by screening a library from cells that express the gene, by introducing the gene from a vector known to contain it, or by directly isolating it from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0304] Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding a gene of interest to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and a promoter useful for expressing the desired nucleic acid sequence.

[0305] In some aspects, cells can be genetically modified with one or more expression regions, e.g., genes. In some embodiments, cells can be genetically modified with exogenous genes (e.g., capable of expressing an exogenous gene product, such as an RNA or polypeptide product) and / or exogenous regulatory nucleic acids. In some embodiments, cells can be genetically modified with exogenous sequences encoding gene products endogenous to the target cell and / or exogenous regulatory nucleic acids capable of regulating expression of endogenous genes. In some embodiments, cells can be genetically modified with exogenous genes and / or regulatory nucleic acids that regulate expression of exogenous genes. In some embodiments, cells can be genetically modified with exogenous genes and / or regulatory nucleic acids that regulate expression of endogenous genes. It will be understood by those skilled in the art that the cells described herein can be genetically modified to express a variety of exogenous genes, encoding proteins or regulatory factors that can act, for example, on gene products of the endogenous or exogenous genome of the target cell. In some embodiments, such genes confer characteristics to fusosomes, e.g., regulating fusion with the target cell. In some embodiments, cells can be genetically modified to express endogenous genes and / or regulatory nucleic acids. In some embodiments, the endogenous genes or regulatory nucleic acids regulate the expression of other endogenous genes. In some embodiments, cells can be genetically modified to express endogenous genes and / or regulatory nucleic acids that are expressed differently (e.g., inducibly, tissue-specifically, constitutively, or at higher or lower levels) than versions of the endogenous genes and / or regulatory nucleic acids on other chromosomes.

[0306] Promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in the region 30–110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0307] 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-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate-early promoter, and Ruth sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0308] Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired or turn off expression when expression is not desired. 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, fusogen expression is upregulated before fusosome formation, for example, 3, 6, 9, 12, 24, 26, 48, 60, or 72 hours before fusosome formation.

[0309] The expression vector introduced into the source may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells desired to be transfected or infected via the viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0310] Reporter genes can be used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes encoding polypeptides that are not present in or expressed by the recipient source and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with the minimal 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0311] In some embodiments, cells can be genetically modified to alter the expression of one or more proteins. The expression of one or more proteins can be altered for a particular time, e.g., the developmental or differentiation state of the source. In some embodiments, fusosomes are produced from a source of cells that have been genetically modified to alter the expression of one or more proteins, e.g., fusogenic or non-fusogenic proteins, that affect fusion activity, structure, or function. The expression of one or more proteins can be restricted to a specific location(s) or can be widespread throughout the source.

[0312] In some embodiments, expression of a fusogenic protein is altered, hi some embodiments, fusosomes are generated from cells that have altered expression of a fusogenic protein, e.g., at least a 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more increase or decrease in fusogenic expression.

[0313] In some embodiments, cells can be engineered to express cytoplasmic enzymes (e.g., proteases, phosphatases, kinases, demethylases, methyltransferases, acetylases) that target fusogenic proteins. In some aspects, the cytoplasmic enzymes affect one or more fusogenic proteins by altering post-translational modifications. Post-translational protein modifications of proteins can affect responsiveness to nutrient availability and redox conditions, as well as protein-protein interactions. In some embodiments, fusosomes contain fusogenic proteins with altered post-translational modifications, e.g., an increase or decrease in post-translational modifications of at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 75%, 80%, 90% or more.

[0314] Methods for introducing modifications into cells include physical, biological, and chemical methods (see, 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. Nature Reviews Genetics. 15:541-555. 2014). Suitable methods for modifying cells for use in generating fusosomes as described herein include, for example, diffusion, osmotic pressure, osmotic pulse, osmotic shock, hypotonic lysis, hypotonic dialysis, ionophoresis, electroporation, sonication, microinjection, calcium precipitation, membrane insertion, lipid-mediated transfection, detergent treatment, viral infection, receptor-mediated endocytosis, use of protein transduction domains, particle firing, membrane fusion, freeze-thaw, mechanical disruption, and filtration.

[0315] Confirmation of the presence of a genetic modification can involve a variety of assays, including molecular biological assays such as Southern and Northern blotting, RT-PCR and PCR, and biochemical assays such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blot) or the assays described herein.

[0316] In some aspects, the present disclosure provides fusosomes comprising (a) a lipid bilayer, (b) a lumen (e.g., comprising the cytosol) surrounded by the lipid bilayer, and (c) an exogenous or overexpressed fusogen (e.g., the fusogen is disposed in the lipid bilayer and the fusosome is derived from a source cell), wherein the fusosome has partial or complete nuclear inactivation (e.g., nucleus removed).

[0317] The present disclosure provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising cytosol, the lumen being surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed within the lipid bilayer; (d) a cargo, e.g., a nucleic acid comprising a payload gene; the fusosomes do not comprise a nucleus; the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; and (i) the plurality of fusosomes is derived from a cell population comprising target cells and non-target cells. or (ii) the plurality of fusosomes fuse at a faster rate with at least 50% of the non-target or reference cells than with the target cells, wherein the target cells are selected from pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, glial cells, astrocytes, microglial cells, oligodendrocytes, or choroid plexus cells.

[0318] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising cytosol, the lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer; and (d) a nucleic acid comprising a payload gene encoding an exogenous agent listed in Table 5 or Table 6, wherein the fusosomes do not comprise a nucleus, and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0319] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising the cytosol, the lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer; and (d) a nucleic acid comprising a payload gene, the nucleic acid comprising an NTCSRE operably linked to the payload gene, the NTCSRE comprising a non-target cell-specific miRNA recognition sequence, e.g., a non-target cell-specific miRNA recognition sequence bound by an miRNA present at higher levels in the non-target cell than in the target cell, e.g., a non-target cell-specific miRNA recognition sequence bound by an miRNA in Table 4; the target cell is a first type of CNS cell; and optionally, the non-target cell is a second, different type of CNS cell or a non-CNS cell; the fusosomes do not comprise a nucleus; and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0320] In some aspects, the miRNA is present in a non-target cell (e.g., a non-target cell described herein) at a level that is at least 10, 100, 1,000, or 10,000 times higher than the level of the miRNA present in the target cell (e.g., a CNS cell). In some embodiments, the miRNA is not detectably present in the target cell (e.g., a CNS cell, e.g., a CNS cell described herein). In some aspects, the miRNA is not present in the target cell (e.g., a CNS cell, e.g., a CNS cell described herein).

[0321] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising cytosol, the lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer; and (d) a nucleic acid comprising a payload gene, the nucleic acid comprising a promoter operably linked to the payload gene, the promoter being a CNS cell-specific promoter, e.g., a promoter specific to a CNS cell, a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, a serotonergic neuron, a glial cell, an astrocyte, a microglial cell, an oligodendrocyte, or a choroid plexus cell; the fusosome composition does not comprise a nucleus; and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0322] In some aspects, the present disclosure provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising the cytosol, the lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer; and (d) a nucleic acid comprising a payload gene, the nucleic acid comprising a promoter having the sequence of a promoter in Table 3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; the fusosomes do not comprise a nucleus; and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0323] The present disclosure, in some aspects, provides a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising cytosol, the lumen surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer; and (d) a nucleic acid comprising: (i) a payload gene; (ii) an NTCSRE operably linked to the payload gene, the NTCSRE comprising, for example, a non-target cell-specific miRNA recognition sequence, e.g., a non-target cell-specific miRNA recognition sequence bound by an miRNA in Table 4; and (iii) optionally, a nucleic acid comprising 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 a payload gene, wherein the positive target cell-specific regulatory element increases expression of the payload gene in a target cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element; the target cell is a first type of CNS cell; optionally, the non-target cell is a second, different type of CNS cell or a non-CNS cell; optionally, the target cell is a neuron and the non-target cell is a glial cell (e.g., an oligodendrocyte, astrocyte, or microglial cell), or the target cell is a glial cell (e.g., an oligodendrocyte, astrocyte, or microglial cell) and the non-target cell is a neuron; the fusosome does not comprise a nucleus; and the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein.

[0324] In some embodiments, one or more of the following are present: i) the fusosomes contain or are contained by the cell biologic; ii) the fusosomes contain enucleated cells; iii) the fusosomes contain inactivated nuclei; iv) the fusosomes are more closely related to target cells than to 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 1-fold, in the assay of Example 42. v) the fusosomes fuse with target cells at a higher rate than other fusosomes, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold, in the assay of Example 42; vi) the agent within the fusosomes fuses with target cells at a higher rate than other fusosomes, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 50-fold, or 100-fold, in the assay of Example 42, at 24, 48, or 72 hours. vii) the fusogen fuses with target cells at a rate such that 70%, 80%, or 90% of the target cells are delivered; and vii) the fusogen delivers at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of the fusogen as measured by the assay of Example 26. viii) the fusosomes contain at least 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies or fewer of the Therapeutic Agent as measured by the assay of Example 88;ix) The ratio of the copy number of the fusogen to the copy number of the therapeutic agent is 1,000,000:1 to 100,000:1, 100,000:1 to 10,000:1, 10,000:1 to 1,000:1, 1,000:1 to 100:1, 100:1 to 50:1, 50:1 to 20:1, 20:1 to 10:1, 10:1 to 5:1, 5:1 to 2:1, 2:1 to 1:1, 1:1 to 1:2, 1:2 to 1:5, 1:5 to 1:10, 1:10 to 1:20, 1:20 to 1:50, 1:50 to 1:100, 1:100 to 1:1,000, 1:1,000 to 1:10,000 x) the fusosomes contain a lipid composition substantially similar to that of the source cells, or one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are present within 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the corresponding lipid levels in the source cells. xi) the fusosomes comprise a proteomic composition similar to that of the source cells, e.g., using the assay of Example 87; xii) the fusosomes comprise a protein to lipid ratio within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, e.g., as measured using the assay of Example 40; xiii) the fusosomes comprise nucleic acid (e.g., phospholipid) content within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, e.g., as measured using the assay of Example 41. xiv) the fusosomes comprise a lipid to nucleic acid (e.g., DNA) ratio that is within 10%, 20%, 30%, 40%, or 50% of the corresponding ratio in the source cells, as measured using the assay of Example 91; xv) the fusosomes have a half-life in a subject, e.g., a mouse, that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of that of a reference cell, e.g., a source cell, as measured using the assay of Example 60;xvi) the fusosomes transport glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (e.g., about 11.6%) more than a negative control, e.g., an otherwise similar fusosome, in the absence of glucose, as measured, e.g., using the assay of Example 50; xvii) the fusosomes transport glucose (e.g., labeled glucose, e.g., 2-NBDG) across the membrane, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% (e.g., about 11.6%) more than a reference cell, e.g., a negative control, e.g., an otherwise similar fusosome, as measured, e.g., using the assay of Example 51; xviii) fusosomes contain an esterase activity in their lumen that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the esterase activity in the source cells or mouse embryonic fibroblasts; xviii) fusosomes have a metabolic activity level that is within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the citrate synthase activity in a reference cell, e.g., a source cell, e.g., as described in Example 53. xix) the fusosomes comprise a respiration level (e.g., oxygen consumption rate) within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the respiration level in a reference cell, e.g., a source cell, e.g., as described in Example 54; xx) the fusosomes comprise a respiration level (e.g., oxygen consumption rate) within 18,000, 17,000, 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, or 10,000, e.g., using the assay of Example 55. or the fusosomes comprise an annexin-V staining level of 0 that is at least 5%, 10%, 20%, 30%, 40%, or 50% less than the annexin-V staining level of otherwise similar fusosomes treated with menadione in the assay of Example 55, or the fusosomes comprise an annexin-V staining level that is at least 5%, 10%, 20%, 30%, 40%, or 50% less than the annexin-V staining level of macrophages treated with menadione in the assay of Example 55;xxi) the fusosomes have miRNA content levels of at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the source cells, e.g., by the assay of Example 33; xxii) the fusosomes have miRNA content levels of within or greater than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the source cells, e.g., 1% to 2%, 2% of the source cells, e.g., by the assay of Example 38. xxiii) the fusosomes have an LPS level of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001%, or lower than the LPS content of the source cells, as measured, for example, by mass spectrometry, e.g., in Example 39; xxiv) the fusosomes have an LPS level of less than 5%, 1%, 0.5%, 0.01%, 0.005%, 0.0001%, 0.00001%, or lower than the LPS content of the source cells, as measured, for example, by mass spectrometry, e.g., in Example 39; xxv) the fusosomes are capable of signaling, e.g., extracellular signaling, e.g., AKT phosphorylation in response to insulin, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG), in response to insulin, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more than a negative control, e.g., otherwise similar fusosomes, in the absence of insulin, e.g., using the assay of Example 49; xxv) the fusosomes are capable of signaling, e.g., extracellular signaling, e.g., AKT phosphorylation in response to insulin, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG) in response to insulin, at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more than an otherwise similar fusosome; When administered to a target tissue, e.g., liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, a subject, e.g., a mouse, at least 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosomes in the population to which the fusosomes were administered are present in the target tissue 24, 48, or 72 hours later, e.g., by the assay of Example 64;xxvi) the fusosomes have a juxtacrine signaling level induced by a reference cell, e.g., a source cell or bone marrow mesenchymal cells (BMSCs), that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher, e.g., by the assay of Example 56; xxvii) the fusosomes have a juxtacrine signaling level induced by a reference cell, e.g., a source cell or bone marrow mesenchymal cells (BMSCs), that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher, e.g., by the assay of Example 57. xxviii) the fusosomes polymerize actin at a level within 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of polymerized actin in a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 58; xxix) the fusosomes polymerize actin at a level within about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the membrane potential of a reference cell, e.g., a source cell or a C2C12 cell, e.g., by the assay of Example 59. 0%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of the membrane potential of the source cells or of cells of the same type as the source cells, e.g., using the assay of Example 44 (e.g., the source cells are neutrophils, lymphocytes, B cells, macrophages, or NK cells). xxxi) the fusosomes are capable of extravasation from blood vessels with an extravasation rate of 0%, or 90%; xxxi) the fusosomes are capable of crossing cell membranes, e.g., endothelial cell membranes or the blood-brain barrier; xxxii) the fusosomes are capable of secreting protein at a rate that is at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than reference cells, e.g., using the assay of Example 48; xxxiii) the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards;xxxiv) the fusosomes have been produced in accordance with Good Manufacturing Practice (GMP); xxxv) the fusosomes have pathogen levels below a predetermined standard, e.g., are substantially free of pathogens; xxxiv) the fusosomes have contaminant levels below a predetermined standard, e.g., are substantially free of contaminants; xxxvii) the fusosomes have low immunogenicity, e.g., as described herein; xxxviii) the source cells are selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells; or xxxix) the source cells are other than 293 cells, HEK cells, human endothelial or epithelial cells, monocytes, macrophages, dendritic cells, or stem cells;

[0325] The present disclosure also provides, in some aspects, fusosomes comprising: a) a lipid bilayer and a lumen that is miscible with an aqueous solution, e.g., water (the fusosome is derived from a source cell); b) an exogenous or overexpressed fusogen disposed in the lipid bilayer; and c) an organelle, e.g., a therapeutically effective number of organelles, disposed in the lumen.

[0326] In some embodiments, one or more of the following are present: i) the source cell is selected from an endothelial cell, a macrophage, a neutrophil, a granulocyte, a leukocyte, a stem cell (e.g., a mesenchymal stem cell, a bone marrow stem cell, an induced pluripotent stem cell, an embryonic stem cell), a myeloblast, a myoblast, a hepatocyte, or a neuron, e.g., a retinal neuron; ii) the organelle is selected from a Golgi apparatus, a lysosome, an endoplasmic reticulum, a mitochondrion, a vacuole, an endosome, an acrosome, an autophagosome, a centriole, a glycosome, a glyoxysodium, a phosphodiesterase ... iii) the fusosome has a size greater than 5 um, 10 um, 20 um, 50 um, or 100 um; iv) the fusosome, or composition or preparation comprising a plurality of fusosomes, has a density other than 1.08 g / ml to 1.12 g / ml, e.g., the fusosome has a density greater ... vii) the source cells are not transformed or immortalized; viii) the source cells are transformed or immortalized using a method other than adenovirus-mediated immortalization, e.g., immortalized by spontaneous mutation or telomerase expression; ix) the fusogen is other than VSVG, a SNARE protein, or a secretory granule protein; x) the fusosomes do not comprise Cre or GFP, e.g., EGFP; xi) the fusosomes further comprise an exogenous protein other than Cre or GFP, e.g., EGFP; xii) the fusosomes further comprise an exogenous nucleic acid (e.g., RNA, e.g., mRNA, miRNA, or siRNA) or an exogenous protein (e.g., an antibody), e.g., in the lumen; or xiii) the fusosomes do not comprise mitochondria.

[0327] The present disclosure also provides, in some aspects, fusosomes comprising (a) a lipid bilayer, (b) a lumen (e.g., comprising the cytosol) surrounded by the lipid bilayer, (c) an exogenous or overexpressed fusogen (e.g., the fusogen is disposed in the lipid bilayer), and (d) a functional nucleus, wherein the fusosome is derived from a source cell.

[0328] In some embodiments, one or more of the following are present: i) the source cell is other than a dendritic cell or a tumor cell, e.g., the source cell is an endothelial cell, a macrophage, a neutrophil, a granulocyte, a leukocyte, a stem cell (e.g., a mesenchymal stem cell, a bone marrow stem cell, an induced pluripotent stem cell, an embryonic stem cell), a myeloblast, a myoblast, a hepatocyte, or a neuron, e.g., a retinal neuron; ii) the fusogen is other than a fusion glycoprotein; iii) the fusogen is a mammalian protein other than fatelin beta; iv) the fusosomes have low immunogenicity, e.g., as described herein; v) the fusosomes meet pharmaceutical or Good Manufacturing Practice (GMP) standards; vi) the fusosomes were manufactured in accordance with Good Manufacturing Practice (GMP); vii) the fusosomes have pathogen levels below a predetermined standard, e.g., are substantially free of pathogens; or viii) the fusosomes have contaminant levels below a predetermined standard, e.g., are substantially free of contaminants.

[0329] The present disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising (a) a lipid bilayer, (b) a lumen comprising the cytosol, the lumen surrounded by the lipid bilayer, (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer, and (d) cargo, wherein the fusosomes do not comprise a nucleus, the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein, and the plurality of fusosomes, when contacted with a target cell population in the presence of an endocytosis inhibitor and when contacted with a reference target cell population not treated with the endocytosis inhibitor, deliver the cargo to at least 30% of the number of cells in the target cell population compared to the reference target cell population.

[0330] The present disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising (a) a lipid bilayer, (b) a lumen comprising a cytosol, the lumen surrounded by the lipid bilayer, (c) an exogenous or overexpressed retargeted fusogen disposed in the lipid bilayer, and (d) cargo, wherein the fusosomes do not comprise a nucleus, the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein, and (i) when the plurality of fusosomes contact a cell population comprising target cells and non-target cells, the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more target cells than non-target cells, or (ii) the plurality of fusosomes fuse with target cells at a rate at least 50% higher than non-target cells.

[0331] The disclosure also provides, in some aspects, a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising (a) a lipid bilayer, (b) an inner lumen surrounded by the lipid bilayer, (c) an exogenous or overexpressed fusogen disposed in the lipid bilayer, and (d) a cargo, wherein the fusosomes do not comprise a nucleus and comprise one or more (e.g., at least 2, 3, 4, or 5) of the following: i) at least 1,000 copies of the fusogen; ii) the fusosomes contain the therapeutic agent in a copy number of at least 1,000 copies; iii) the fusosomes contain lipids, and one or more of CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG are within 75% of the corresponding lipid levels in the source cells; iv) the fusosomes contain a proteomic composition similar to the proteomic composition of the source cells. v) the fusosomes are capable of transducing a signal, e.g., an extracellular signal, e.g., AKT phosphorylation in response to insulin, or uptake of glucose (e.g., labeled glucose, e.g., 2-NBDG) in response to insulin, e.g., in the absence of insulin, that is at least 10% greater than a negative control, e.g., an otherwise similar fusosome; vi) the fusosomes are targeted to a tissue, e.g., the liver, lung, heart, spleen, pancreas, gastrointestinal tract, kidney, testis, ovary, brain, reproductive organs, central nervous system, peripheral nervous system, skeletal muscle, endothelium, inner ear, or eye, and when administered to a subject, e.g., a mouse, at least 0.1%, or 10%, of the fusosomes in the population of administered fusosomes are present in the target tissue 24 hours later; or the source cell is selected from neutrophils, granulocytes, mesenchymal stem cells, bone marrow stem cells, induced pluripotent stem cells, embryonic stem cells, myeloblasts, myoblasts, hepatocytes, or neurons, e.g., retinal neuronal cells.

[0332] In embodiments, one or more of the following are true: i) the source cells are other than 293 cells; ii) the source cells are not transformed or immortalized; iii) the source cells are transformed or immortalized using a method other than adenovirus-mediated immortalization, e.g., immortalization by spontaneous mutation or telomerase expression; iv) the fusogen is other than VSVG, a SNARE protein, or a secretory granule protein; 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); or vii) the fusosomes do not contain mitochondria.

[0333] In embodiments, one or more of the following are true: i) the source cells are other than 293 cells or HEK cells; ii) the source cells are not transformed or immortalized; iii) the source cells are transformed or immortalized using a method other than adenovirus-mediated immortalization, for example, a method that immortalizes by spontaneous mutation or telomerase expression; iv) the fusogen is not a viral fusogen; or v) the fusosomes have a size other than 40 to 150 nm, for example, a size greater than 150 nm, 200 nm, 300 nm, 400 nm, or 500 nm.

[0334] In embodiments, one or more of the following are present: i) the therapeutic agent is a soluble protein expressed by the source cells; ii) the fusogen is other than TAT, TAT-HA2, HA-2, gp41, Alzheimer's beta-amyloid peptide, Sendai virus protein, or amphipathic net-negative peptide (WAE 11); iii) the fusogen is a mammalian fusogen; iv) the fusosomes contain in their lumen a polypeptide selected from an enzyme, an antibody, or an antiviral polypeptide; v) the fusosomes do not contain an exogenous therapeutic transmembrane protein; or vi) the fusosomes do not contain CD63 or GLUT4, or the fusosomes contain 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 10% or less (e.g., about 0.048% or less) of CD63, as determined, for example, according to the method described in Example 89.

[0335] In embodiments, the fusosomes i) do not contain virus, are not infectious, or do not propagate in host cells; ii) are not viral vectors; iii) are not VLPs (virus-like particles); iv) do not contain viral structural proteins, e.g., proteins derived from gag, e.g., viral capsid proteins, e.g., viral capsule proteins, e.g., viral nucleocapsid proteins, or the amount of viral capsid proteins is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, e.g., by mass spectrometry, e.g., using the assay of Example 93; v) do not contain viral matrix proteins; vi) do not contain viral nonstructural proteins; e.g., po l or a fragment or variant thereof, viral reverse transcriptase protein, viral integrase protein, or viral protease protein; vii) do not contain viral nucleic acid; e.g., viral RNA or viral DNA; viii) contain less than 10, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000,000, or 1,000,000,000 copies of viral structural protein per vesicle; or ix) the fusosome is not a virosome.

[0336] In some embodiments, the fusosomes comprise (or are identified as comprising) less than about 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the viral capsid protein (e.g., about 0.05% of the viral capsid protein). In embodiments, the viral capsid protein is a complex of rabbit endogenous lentivirus (RELIK) capsid and cyclophilin A. In embodiments, the viral capsid protein:total protein ratio is (or is identified to be) about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1.

[0337] In some embodiments, the fusosomes do not contain (or are identified as not containing) gag protein, or a fragment or variant thereof, or the amount of gag protein, or a fragment or variant thereof, is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of the total protein, e.g., by the assay of Example 93.

[0338] In embodiments, the ratio of the copy number of the fusogen to the copy number of the viral structural protein on the fusosome is at least 1,000,000:1, 100,000:1, 10,000:1, 1,000:1, 100:1, 50:1, 20:1, 10:1, 5:1, or 1:1, or between 100:1 and 50:1, 50:1 and 20:1, 20:1 and 10:1, 10:1 and 5:1, or 1: 1. In embodiments, the ratio of the copy number of the fusogen to the copy number of the viral matrix protein on the fusosome 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.

[0339] In embodiments, one or more of the following are selected from: i) the fusosome does not comprise a water-immiscible droplet; ii) the fusosome comprises an aqueous lumen and a hydrophilic exterior; iii) the fusogen is a protein fusogen; or iv) the organelle is selected from a mitochondrion, Golgi apparatus, lysosome, endoplasmic reticulum, vacuole, endosome, acrosome, autophagosome, centriole, glycosome, glyoxysome, hydromenosome, melanosome, mitosome, cnidocyst, peroxisome, proteasome, vesicle, and granule.

[0340] In embodiments, one or more of the following: i) the fusogen is a mammalian fusogen or a viral fusogen; ii) the fusosomes are not produced by loading a therapeutic or diagnostic agent into the fusosomes; iii) the source cells are not loaded with a therapeutic or diagnostic agent; iv) the fusosomes do not contain doxorubicin, dexamethasone, cyclodextrin, polyethylene glycol, microRNA, e.g., miR125, VEGF receptor, ICAM-1, E-selectin, iron oxide, fluorescent protein, e.g., GFP or RFP, nanoparticles, or RNase, or do not contain exogenous forms of any of the foregoing; or v) the fusosomes further contain an exogenous therapeutic agent having one or more post-translational modifications, e.g., glycosylation.

[0341] In embodiments, the fusosomes are unilamellar or multilamellar.

[0342] In embodiments, one or more of the following: i) the fusosomes are not exosomes; ii) the fusosomes are microvesicles; iii) the fusosomes contain a non-mammalian fusogen; iv) the fusosomes are engineered to incorporate a fusogen; v) the fusosomes contain an exogenous fusogen; vi) the fusosomes have a size of at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the population of fusosomes has an average size of at least 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm; vi i) fusosomes comprise one or more organelles, e.g., mitochondria, Golgi apparatus, lysosomes, endoplasmic reticulum, vacuoles, endosomes, acrosomes, autophagosomes, centrioles, glycosomes, glyoxysomes, hydrogenosomes, melanosomes, mitosomes, cnidosomes, peroxisomes, proteasomes, vesicles, and stress granules; viii) fusosomes comprise the cytoskeleton or a component thereof, e.g., actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; ix) fusosomes, or compositions or preparations comprising multiple fusosomes, are described, e.g., in Thery In the sucrose gradient centrifugation assay described in [Isolation and characterization of exosomes from cell culture supernatants and biological fluids] Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22, the exosomes do not have a buoyant density of 1.08-1.22 g / ml, or at least 1.18-1.25 g / ml, or 1.05-1.x) the lipid bilayers are enriched for ceramide or sphingomyelin, or a combination thereof, compared to the source cells, or the lipid bilayers are not enriched (e.g., depleted) for glycolipids, free fatty acids, or phosphatidylserine, or a combination thereof, compared to the source cells; xi) the fusosomes contain phosphatidylserine (PS) or CD40 ligand, or both PS and CD40 ligand, as measured, for example, by the assay of Example 92; xii) the fusosomes are enriched for PS compared to the source cells, as measured, for example, by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosome population are positive for PS. xiii) the fusosomes are substantially free of acetylcholinesterase (AChE) or contain less than 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 / μg protein, e.g., by the assay of Example 52; xiv) the fusosomes are free of tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., TSG101, CHMP4A-B, or VPS4B), or ... CD63, CD9, or CD81), or tetraspanin family proteins (e.g., CD63, CD9, or CD81), ESCRT-related proteins (e.g., CD63, CD9, or CD81), or tetraspanin family proteins (e.g., CD63, CD9, or CD8 ), 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, or is substantially free of or contains less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5%, or 10% of any individual exosome marker protein and / or ...x) fusosomes contain 5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% total exosome marker protein of any of the above proteins, or are not enriched in any one or more of these proteins compared to source cells, e.g., by the assay of Example 89; or xv) fusosomes contain 500, 250, 100, 50, 20, 10, 5, or 1 ng of exosome marker protein. xvi) fusosomes enriched in one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasome proteins, or one or more mitochondrial proteins, or any combination thereof, e.g., 500, 250, 100, 50, 20, 10, 5, or 1 ng of calnexin; There is less than calnexin / μg total protein, or the fusosomes contain 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin per total protein on a ng / μg basis compared to the source cells, using, for example, the assay of Examples 37 or 90, or the average calnexin content in fusosomes is about 1×10 -4 , 1.5×10 -4 , 2 × 10 -4 , 2.1×10 -4 , 2.2 × 10 -4 , 2.3 × 10 -4 , 2.4 × 10 -4 , 2.43 × 10 -4 , 2.5×10 -4 , 2.6×10 -4 , 2.7 × 10 -4 , 2.8×10 -4 , 2.9 × 10 -4 , 3×10 -4 , 3.5×10 -4 , or 4 x 10 -4xvii) the fusosomes contain an exogenous agent (e.g., an exogenous protein, mRNA, or siRNA), e.g., as measured using the assay of Example 34; or xviii) the fusosomes can be immobilized on a mica surface for at least 30 minutes by atomic force microscopy, e.g., as measured by the assay of Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442.

[0343] In embodiments, one or more of the following apply: i) the fusosomes are exosomes; ii) the fusosomes are not microvesicles; iii) the size of the fusosomes is less than 80 nm, 100 nm, 200 nm, 500 nm, 1000 nm, 1200 nm, 1400 nm, or 1500 nm, or the average size of a 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; v) the fusosomes do not contain the cytoskeleton or components thereof, e.g., actin, Arp2 / 3, formin, coronin, dystrophin, keratin, myosin, or tubulin; vi) the fusosome, or a composition or preparation comprising a plurality of fusosomes, is described, for example, in Thery et al., "Isolation and characterization of exosomes from cell culture supernatants and biological fluids." Curr. The buoyant density is 1.08 to 1.22 g / ml in a sucrose gradient centrifugation assay as described in Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22; vii) the lipid bilayers are not enriched (e.g., depleted) in ceramide or sphingomyelin, or a combination thereof, compared to the source cells, or the lipid bilayers are enriched in glycolipids, free fatty acids, or phosphatidylserine, or a combination thereof, compared to the source cells; viii) the fusosomes are free of phosphatidylserine (PS) or CD40 ligand, or both PS and CD40 ligand, or are depleted relative to the source cells, as measured, for example, by the assay of Example 92; and ix) the fusosomes are not enriched (e.g., depleted) in PS compared to the source cells, as measured, for example, by Kanada M, et al. (2015) Different fates of biomolecules delivered to target cells via extracellular vesicles.For example, less than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the fusosome population is positive for PS by the assay of Proc Natl Acad Sci USA 112:E1433-E1442. x) the fusosomes substantially comprise acetylcholinesterase (AChE), e.g., comprising at least less than 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 / μg protein, e.g., by the assay of Example 52; xi) the fusosomes are free of any of the following proteins: a tetraspanin family protein (e.g., CD63, CD9, or CD81), an ESCRT-related protein (e.g., TSG101, CHMP4A-B, or VPS4B), Alix, TSG101, MHCI, MHCII, GP96, actinin-4, mitofilin, syntenin-1, TSG101, ADAM10, EHD4, syntenin-1, xii) fusosomes contain TSG101, EHD1, flotillin-1, heat shock 70-kDa proteins (HSC70 / HSP73, HSP70 / HSP72), or any combination thereof, e.g., less than 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 5%, or 10% of an individual exosome marker protein and / or 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% of the total exosome marker protein of any of the above proteins, or are enriched in any one or more of these proteins relative to the source cells, e.g., by the assay of Example 89; xii) fusosomes contain 500, 250, 100, 50, 20, 10, 5, or 1 ng A level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) less than GAPDH / μg total protein, or, for example, a level of GAPDH per total protein in ng / ug using the assay of Example 36, at least 1%, 2.xiii) the fusosomes are not enriched (e.g., depleted) in one or more endoplasmic reticulum proteins (e.g., calnexin), one or more proteasome proteins, or one or more mitochondrial proteins, or any combination thereof, e.g., 500, 250, 100, 50, 20, 10, 5, or 1 ng of calnexin. There is less than calnexin / µg total protein, or the fusosomes contain 1%, 2.5%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less calnexin per total protein on a ng / µg basis compared to the source cells, e.g., using the assay of Example 90, or the average calnexin content in fusosomes is 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 x 10 -4or the fusosomes contain about 70%, 75%, 80%, 85%, 88%, 90%, 95%, 99% or more less calnexin per total protein than the parent cells; or xiv) the fusosomes cannot be immobilized on a mica surface for at least 30 minutes by atomic force microscopy, e.g., as assayed by Kanada M, et al. (2015) Differential fates of biomolecules delivered to target cells via extracellular vesicles. Proc Natl Acad Sci USA 112:E1433-E1442.

[0344] In embodiments, one or more of the following: 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 do not contain viral proteins, such as viral structural proteins, such as capsid proteins or viral matrix proteins; v) the fusosomes do not contain capsid proteins from enveloped viruses; vi) the fusosomes do not contain nucleocapsid proteins; or vii) the fusogen is not a viral fusogen.

[0345] In embodiments, the fusosome comprises the cytosol.

[0346] In embodiments, one or more of the following are true: i) the fusosomes or source cells do not form teratomas when transplanted into a subject, e.g., by the assay of Example 65; ii) the fusosomes are capable of chemotaxis, e.g., 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more, than a reference cell, e.g., a macrophage, e.g., using the assay of Example 45; iii) the fusosomes are capable of homing, e.g., to a site of injury, e.g., using the assay of Example 46, where the fusosomes or cell biological material are from a human cell, e.g., the source cell is a neutrophil; iv) the fusosomes are capable of phagocytosis, e.g., phagocytosis by the fusosomes is detectable within 0.5, 1, 2, 3, 4, 5, or 6 hours using the assay of Example 47, e.g., where the source cell is a macrophage.

[0347] In embodiments, the fusosomes or fusosome compositions retain one, two, three, four, five, six, or more of any of the above characteristics for 5 days or less, e.g., 4 days or less, 3 days or less, 2 days or less, 1 day or less, e.g., for about 12 to 72 hours, after administration to a subject, e.g., a human subject.

[0348] In embodiments, fusosomes have one or more of the following characteristics: a) contain one or more endogenous proteins, e.g., membrane proteins or cytosolic proteins, from the source cell; b) contain at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different proteins; c) contain at least 1, 2, 5, 10, 20, 50, or 100 different glycoproteins; d) at least 10%, 20%, 30%, 40%, or 50% of the proteins in the fusosomes are endogenous. e) comprises at least 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000 different RNAs; or f) comprises at least 2, 3, 4, 5, 10, or 20 different lipids selected from, for example, CL, Cer, DAG, HexCer, LPA, LPC, LPE, LPG, LPI, LPS, PA, PC, PE, PG, PI, PS, CE, SM, and TAG.

[0349] In embodiments, the fusosomes are engineered to have one, two, three, four, five or more of the following properties, or the fusosomes are not naturally occurring cells, or the nuclei do not naturally have one, two, three, four, five or more of the following properties: a) partial nuclear inactivation results in at least a 50%, 60%, 70%, 80%, 90% or more reduction in nuclear function, e.g., a reduction in transcription or DNA replication, or both, as measured by the assays of Example 24 for transcription and Example 25 for DNA replication; b) the fusosomes are unable to transcribe or transcriptional activity is reduced, e.g., as measured by the assays of Example 24 for DNA replication. c) the fusosomes are incapable of nuclear DNA replication or have nuclear DNA replication that is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the reference cells, e.g., source cells, using the assay; c) the fusosomes are incapable of nuclear DNA replication or have nuclear DNA replication that is less than 1%, 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the reference cells, e.g., source cells, using the assay of Example 25; d) the fusosomes lack chromatin or have chromatin content that is less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of that of the reference cells, e.g., source cells, using the assay of Example 32.e) the fusosomes lack a nuclear membrane, e.g., by the assay of Example 31, or have less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the amount of nuclear membrane of a reference cell, e.g., a source cell or a Jurkat cell; f) the fusosomes lack a functional nuclear pore complex, e.g., by the assay of Example 31, or have at least 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% reduced nuclear import or export activity, or lack one or more nuclear pore proteins, e.g., NUP98 or importin 7; g) the fusosomes do not contain histones. or the histone levels are less than 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the histone levels (e.g., H1, H2a, H2b, H3, or H4) of the source cell, e.g., by the assay of Example 32; h) the fusosomes contain less than 20, 10, 5, 4, 3, 2, or 1 chromosome; i) nuclear function has been eliminated; j) the fusosomes are enucleated mammalian cells; k) the nucleus has been removed or inactivated, e.g., by mechanical force extrusion, radiation, or chemical ablation; or l) the fusosomes are derived from mammalian cells whose DNA has been completely or partially removed during interphase or mitosis.

[0350] In embodiments, the fusosomes contain mtDNA or vector DNA. In embodiments, the fusosomes do not contain DNA.

[0351] In embodiments, the source cells are primary cells, immortalized cells, or cell lines (e.g., myeloblast cell lines, e.g., C2C12). In embodiments, the fusosomes are derived from source cells with an altered genome, e.g., reduced 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 treated with anti-inflammatory signals. In embodiments, the source cells are derived from cell cultures treated with immunosuppressants. In embodiments, the source cells are substantially non-immunogenic, e.g., using the assays described herein. In embodiments, the source cells comprise an exogenous agent, e.g., a therapeutic agent. In embodiments, the source cells are recombinant cells.

[0352] In embodiments, the fusosomes further comprise an exogenous agent, e.g., a therapeutic agent, e.g., a protein or nucleic acid (e.g., DNA, a chromosome (e.g., a human artificial chromosome), RNA, e.g., mRNA or miRNA). In embodiments, the exogenous agent is at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000, In embodiments, fusosomes may contain, for example, 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 copies per fusosome. In embodiments, fusosomes have altered, e.g., increased or decreased, levels of one or more endogenous molecules, e.g., proteins or nucleic acids, by, for example, treating mammalian cells with siRNA or gene editing enzymes. In embodiments, the endogenous agent may be present in an amount of, for example, at least 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000, 100,000,000, 500,000, In embodiments, the endogenous molecule (e.g., RNA or protein) is present (e.g., contained by fusosomes) at an average level of 10, 20, 50, 100, 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 500,000, or 1,000,000 copies per fusosome. In embodiments, the endogenous molecule (e.g., RNA or protein) is present at an average level of at least 1, 2, 3, 4, 5, 10, 20, 50, 100, 50 ... 3 , 5.0×10 3 , 10 4, 5.0×10 4 , 10 5 , 5.0×10 5 , 10 6 , 5.0×10 6 , 1.0×10 7 , 5.0×10 7 , or 1.0 × 10 8 It is present in high concentrations.

[0353] In embodiments, the active agent is selected from a protein, a 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), a polypeptide, a nucleic acid (e.g., DNA, a chromosome, or RNA, e.g., mRNA, siRNA, or miRNA), or a small molecule. In embodiments, the exogenous agent comprises a site-specific nuclease, e.g., a Cas9 molecule, a TALEN, or a ZFN.

[0354] In embodiments, the fusogen is a viral fusogen, e.g., HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, e.g., 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, fusosomes fuse with target cells at the surface of the target cells. 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, e.g., oleic acid, glycerol monooleate, glycerides, diacylglycerol, or modified unsaturated fatty acids. In embodiments, the fusogen is a chemical fusogen, e.g., PEG. In embodiments, the fusogen is a small molecule fusogen, e.g., 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 incorporated, e.g., expressed in source cells under conditions that allow the fusogen to bind to the lipid bilayer.

[0355] In embodiments, the fusosomes bind to a target cell. In embodiments, the target cell is other than a HeLa cell, or the target cell is not transformed or immortalized.

[0356] In some embodiments comprising a fusosome composition, the fusosomes are the same. In some embodiments, the fusosomes are different. In some embodiments, the fusosomes are derived from one or more source cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the fusosomes have a diameter within 10%, 20%, 30%, 40%, or 50% of the average diameter of the fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the fusosomes have a volume within 10%, 20%, 30%, 40%, or 50% of the average volume of the fusosomes in the fusosome composition. In some embodiments, the fusosome composition has a variation in size distribution of less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%, within 10%, 50%, or 90% of the source cell population's variation in size distribution, based on, for example, Example 28. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a copy number of fusogen within 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the average fusogen copy number among fusosomes in the fusosome composition. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the plurality of fusosomes have a copy number of a therapeutic agent that is within 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the average copy number of a therapeutic agent in fusosomes in the fusosome composition. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15In some embodiments, the fusosome 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.

[0357] In some embodiments, the fusosome composition delivers cargo to at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cell population of a target cell population compared to a reference target cell population.

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

[0359] In some embodiments, less than 10% of the cargo enters the cell by endocytosis.

[0360] In some embodiments, the inhibitor of endocytosis is a lysosomal acidification inhibitor, e.g., bafilomycin A1. In some embodiments, the inhibitor of endocytosis is a dynamin inhibitor, e.g., dynasoar.

[0361] In some embodiments, the target cell population is at a physiological pH (eg, 7.3-7.5, eg, 7.38-7.42).

[0362] In some embodiments, the cargo delivered is determined using an endocytosis inhibition assay, such as the assay of Example 80.

[0363] In some embodiments, the cargo enters the cell via a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., an inhibitor of endocytosis, e.g., a macropinocytosis inhibitor, e.g., 5-(N-ethyl-N-isopropyl)amiloride (EIPA) at a concentration of, e.g., 25 μM), or an actin-independent pathway (e.g., an inhibitor of endocytosis, e.g., an inhibitor of actin polymerization, e.g., latrunculin B at a concentration of, e.g., 6 μM).

[0364] In some embodiments, the plurality of fusosomes further comprises a targeting moiety, hi embodiments, the targeting moiety is comprised by the fusogen or by a separate molecule.

[0365] In some embodiments, when multiple fusosomes contact a cell population comprising target cells and non-target cells, the cargo is present in at least 10-fold greater abundance in the target cells than in the non-target cells.

[0366] In some embodiments, when a plurality of fusosomes contact a cell population comprising target cells and non-target cells, the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold more abundant in the target cells than in the non-target cells, and / or the cargo is present in at least 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold more abundant in the target cells than in the reference cells.

[0367] In some embodiments, multiple fusosomes fuse with target cells at a rate 50% higher than with non-target cells.

[0368] In some embodiments, upon contact with a target cell population, the fusosomes deliver cargo to a target cell location other than an endosome or lysosome, e.g., the cytosol, hi embodiments, less than 50%, 40%, 30%, 20%, or 10% of the cargo is delivered to endosomes or lysosomes.

[0369] In some embodiments, the plurality of fusosomes comprises exosomes, microvesicles, or a combination thereof.

[0370] 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, while 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.

[0371] In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises a mammalian fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises a viral fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) is a protein fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) is Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, and comprises a sequence selected from Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or derivatives thereof.

[0372] In some embodiments, the fusogen (e.g., a retargeted fusogen) is active at a pH of 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. In some embodiments, the fusogen (e.g., a retargeted fusogen) is not active at a pH of 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0373] In some embodiments, the fusogen is present in a copy number of at least 1, 2, 5, or 10 copies per fusosome.

[0374] In some embodiments, the fusogen (e.g., a retargeted fusogen) is Nipah virus protein G, measles protein H, Tupaia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, including Sendai virus HN protein, rubulavirus HN protein, abulavirus HN protein, or a derivative thereof. In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0375] In some embodiments, the cargo comprises an exogenous protein or exogenous nucleic acid. In some embodiments, the cargo comprises or encodes a cytosolic protein. In some embodiments, the cargo comprises or encodes a membrane protein. In some embodiments, the cargo comprises a therapeutic agent. In some embodiments, the cargo is present at a copy number of at least 1, 2, 5, 10, 20, 50, 100, or 200 copies per fusosome (e.g., up to about 1,000 copies per fusosome). In some embodiments, the ratio of the copy number of the fusogen (e.g., the retargeted fusogen) to the copy number of the cargo is 1000:1 to 1:1, or 500:1 to 1:1, or 250:1 to 1:1, or 150:1 to 1:1, or 100:1 to 1:1, or 75:1 to 1:1, 50:1 to 1:1, 25:1 to 1:1, 20:1 to 1:1, or 15:1 to 1:1, or 10:1 to 1:1, or 5:1 to 1:1, or 2:1 to 1:1, or 1:1 to 1:2.

[0376] In some embodiments, the fusosome composition comprises a viral capsid protein or a DNA-integrating polypeptide. In some embodiments, the cargo comprises a viral genome.

[0377] In some embodiments, fusosomes can deliver nucleic acids to target cells, eg, for gene therapy, eg, to stably modify the genome of the target cell.

[0378] In some embodiments, the fusosome composition does not contain viral nucleocapsid protein, e.g., the amount of viral nucleocapsid protein is less than 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% of total protein by mass spectrometry, e.g., using the assay of Example 93.

[0379] In embodiments, the fusosome composition comprises at least 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In embodiments, the fusosome composition comprises at least 10 ml, 20 ml, 50 ml, 100 ml, 200 ml, 500 ml, 1 L, 2 L, 5 L, 10 L, 20 L, or 50 L.

[0380] In embodiments, the fusosomes are derived from, e.g., mammalian cells having a genome modified to reduce immunogenicity (e.g., to remove MHC proteins or MHC complexes, e.g., by genome editing). In embodiments, the source cells are derived from a cell culture treated with an anti-inflammatory signal. In embodiments, the method further comprises contacting the source cells of step a) with an immunosuppressant or anti-inflammatory signal, e.g., before or after inactivating the nucleus, e.g., enucleating the cells.

[0381] In one aspect, provided herein is a fusosome composition comprising a plurality of fusosomes derived from a source cell, the plurality of fusosomes comprising: (a) a lipid bilayer; (b) a lumen comprising the cytosol, the lumen being surrounded by the lipid bilayer; (c) an exogenous or overexpressed fusogen disposed within the lipid bilayer; and (d) a cargo; the fusosomes do not comprise a nucleus; the amount of viral capsid protein in the fusosome composition is less than 1% of the total protein; and the plurality of fusosomes, when contacted with a target cell population in the presence of an inhibitor of endocytosis and when contacted with a reference target cell population not treated with the inhibitor of endocytosis, deliver the cargo to at least 30% more cells in the target cell population compared to a reference target cell population.

[0382] In embodiments, the fusosome composition delivers cargo to at least 40%, 50%, 60%, 70%, or 80% of the cells in a target cell population compared to a reference target cell population or a non-target cell population, or delivers, for example, at least 40%, 50%, 60%, 70%, or 80% of the cargo to a target cell population compared to a reference target cell population or a non-target cell population. In some embodiments, less than 10% of the cargo enters cells by endocytosis. In some embodiments, the inhibitor of endocytosis is a lysosomal acidification inhibitor, e.g., bafilomycin A1. In embodiments, the delivered cargo is determined using an endocytosis inhibition assay, e.g., the assay of Example 80. In embodiments, the cargo enters the cell via a dynamin-independent pathway or a lysosomal acidification-independent pathway, a macropinocytosis-independent pathway (e.g., an inhibitor of endocytosis, e.g., a macropinocytosis inhibitor, e.g., 5-(N-ethyl-N-isopropyl)amiloride (EIPA) at a concentration of, e.g., 25 μM), or an actin-independent pathway (e.g., an inhibitor of endocytosis, e.g., an inhibitor of actin polymerization, e.g., latrunculin B at a concentration of, e.g., 6 μM).

[0383] C. fusogenes and pseudotyping In some embodiments, the fusosomes described herein (e.g., including vesicles or portions of cells) contain one or more fusogens, e.g., to facilitate fusion of the fusosome with a membrane, e.g., a cell membrane. These compositions may also include surface modifications, either during or after synthesis, to include one or more fusogens. Surface modifications may include membrane modifications, e.g., the insertion of lipids or proteins into the membrane.

[0384] In some embodiments, fusosomes contain one or more fusogens on their outer surface (e.g., incorporated into the cell membrane) to target specific cell or tissue types (e.g., CNS cells). In some aspects, the specific cell types targeted by the one or more fusogens are CNS cells, pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, glial cells, astrocytes, microglial cells, oligodendrocytes, or choroid plexus cells. Fusosomes may contain a targeting domain. Fusogens include, but are not limited to, protein-based, lipid-based, and chemical-based fusogens. Fusogens may bind to a partner, e.g., a feature on the surface of a target cell. In some embodiments, the partner on the surface of a target cell is a target cell moiety. In certain embodiments, the fusogen is a fusogen or retargeting fusogen that binds to target cells among CNS cells, pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, glial cells, astrocytes, microglial cells, oligodendrocytes, or choroid plexus cells. In some embodiments, fusogen-containing fusosomes will integrate their membranes into the lipid bilayer of target cells. In some embodiments, one or more fusogens described herein can be included in fusosomes.

[0385] The fusosomes (eg, retroviral vectors) described herein can comprise a fusogen, eg, an endogenous fusogen or a pseudotyped fusogen.

[0386] i) Protein fusogens In some embodiments, the fusogen comprises a protein (e.g., a glycoprotein), a lipid, or a small molecule. The fusogen can be, for example, a mammalian fusogen or a viral fusogen. In some embodiments, the fusogen comprises a protein fusogen, e.g., a mammalian protein or a homolog of a mammalian protein (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a non-mammalian protein such as a viral protein or a homolog of a viral protein (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity), a naturally occurring protein or a derivative of a naturally occurring protein, a synthetic protein, a fragment thereof, a variant thereof, a protein fusion, 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 protein, or a homolog thereof, a fragment thereof, a variant thereof, or a protein fusion comprising one or more proteins or fragments thereof.

[0387] In embodiments, the fusogen is a viral fusogen, e.g., HA, HIV-1 ENV, HHV-4, gp120, or VSV-G. In embodiments, the fusogen is a mammalian fusogen, e.g., 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, fusosomes fuse with target cells at the surface of the target cells. 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, e.g., oleic acid, glycerol monooleate, glycerides, diacylglycerol, or modified unsaturated fatty acids. In embodiments, the fusogen is a chemical fusogen, e.g., PEG. In embodiments, the fusogen is a small molecule fusogen, e.g., 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 incorporated, e.g., expressed in source cells under conditions that allow the fusogen to bind to the lipid bilayer.

[0388] In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises a mammalian fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) comprises a viral fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) is a protein fusogen. In some embodiments, the fusogen (e.g., the retargeted fusogen) is Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, and comprises a sequence selected from Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein, or derivatives thereof.

[0389] In some embodiments, the fusogen (e.g., a retargeted fusogen) is active at a pH of 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. In some embodiments, the fusogen (e.g., a retargeted fusogen) is not active at a pH of 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0390] In some embodiments, the fusogen is present in a copy number of at least 1, 2, 5, or 10 copies per fusosome.

[0391] In some embodiments, the fusogen (e.g., a retargeted fusogen) is Nipah virus protein G, measles protein H, Tupaia paramyxovirus H protein, paramyxovirus G protein, paramyxovirus H protein, paramyxovirus HN protein, morbillivirus H protein, respirovirus HN protein, including Sendai virus HN protein, rubulavirus HN protein, abulavirus HN protein, or a derivative thereof. In some embodiments, the fusogen (e.g., a retargeted fusogen) comprises a sequence selected from Nipah virus F and G proteins, measles virus F and H proteins, Tupaia paramyxovirus F and H proteins, paramyxovirus F and G proteins or F and H proteins or F and HN proteins, Hendra virus F and G proteins, Henipavirus F and G proteins, Morbillivirus F and H proteins, Respirovirus F and HN proteins, Sendai virus F and HN proteins, Rubulavirus F and HN proteins, or Avulavirus F and HN proteins, or derivatives thereof, or any combination thereof.

[0392] Non-mammalian fusogens include viral fusogens, their homologs, fragments, 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 α-helical hairpins with a central coiled-coil structure. Class I viral fusion proteins include proteins with a central post-fusion six-helix bundle. Class I viral fusion proteins include influenza HA, parainfluenza F, HIV Env, Ebola GP, orthomyxovirus hemagglutinin, paramyxovirus F proteins (e.g., measles (Katoh et al. BMC Biotechnology 2010, 10:37)), retrovirus ENV proteins, and filovirus and coronavirus fusogens. In embodiments, class II viral fusogens, such as dengue E glycoprotein, have the structural characteristics of a beta sheet that refolds to form an elongated ectodomain resulting in a trimer of hairpins. In embodiments, class II viral fusogens lack a central coiled-coil. Class II viral fusogens are found in alphaviruses (e.g., E1 protein) and flaviviruses (e.g., E glycoprotein). Class II viral fusogens include fusogens from Semliki Forest virus, Sinbis virus, rubella virus, and dengue virus. In embodiments, class III viral fusogens, such as vesicular stomatitis virus G glycoprotein, combine structural features found in classes I and II. In embodiments, class III viral fusogens contain an alpha helix (e.g., the protein folds back to form a six-helix bundle, similar to class I viral fusogens) and a beta sheet with an amphipathic fusion peptide at its terminus, reminiscent of class II viral 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 encoded by non-enveloped reovirus (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). In embodiments, the Class IV viral fusogen is small enough that it does not form hairpins (doi:10.1146 / annurev-cellbio-101512-122422, doi:10.1016 / j.devcel.2007.12.008).

[0393] 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, and EIV, native env proteins include gp41 and gp120. In some embodiments, the viral env proteins expressed by the source cells described herein are encoded on a vector separate from the viral gag and pol genes, as previously described.

[0394] 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 virus envelope. Similarly, RNA viruses (e.g., Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, etc.) can be used. In addition to RNA virus families such as Arenaviridae and Reoviridae, genes encoding envelopes derived from DNA viruses (Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) can be used. Representative examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

[0395] In some embodiments, envelope proteins for display on fusosomes include, but are not limited to, any of the following sources: influenza A, such as H1N1, H1N2, H3N2, and H5N1 (avian influenza), influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, lyssaviruses, such as monkeypox, mononegavirus, and rabies virus, Lagos bat virus, Mokola virus, Duvenhage virus, European bat virus 1 and 2, Australian bat virus, ephemerovirus, vesiculovirus, vesicular stomatitis virus (VSV), herpesviruses, such as herpes simplex virus 1 and 2, and varicella zoster. cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, murine gammaherpesvirus, arenaviruses such as Argentine hemorrhagic fever virus, Bunyaviridae such as Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), and Crimean-Congo hemorrhagic fever virus, Filoviridae (filoviruses) including hantaviruses, viruses causing hemorrhagic renal syndrome, Rift Valley fever virus, Ebola hemorrhagic fever, and Marburg hemorrhagic fever, Flaviviridae including Kyasanur Forest disease virus, Paramyxoviridae such as Omsk hemorrhagic fever virus, viruses causing tick-borne encephalitis, Hendra virus, and Nipah virus, Variola Alphaviruses such as variola major and variola minor (smallpox), Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any encephalitis-causing virus.

[0396] In some embodiments, the source cells described herein produce fusosomes pseudotyped with VSV-G glycoprotein, eg, recombinant retroviruses, eg, lentiviruses.

[0397] Fusosomes or pseudotyped viruses generally have one or more modifications in their envelope proteins, for example, the envelope protein is replaced with the envelope protein from another virus.For example, HIV can be pseudotyped with the fusion protein from a rhabdovirus, such as the vesicular stomatitis virus G protein (VSV-G) envelope protein, and HIV envelope protein (encoded by env gene) usually targets the virus to CD4+ presenting cells, allowing HIV to infect a wider range of cells.In some embodiments, lentivirus envelope protein is pseudotyped with VSV-G.In one embodiment, source cells produce recombinant retroviruses, such as lentiviruses, pseudotyped with VSV-G envelope glycoprotein.

[0398] Furthermore, fusogens or viral envelope proteins can be modified or engineered to contain polypeptide sequences that enable the transduction vector to target and infect host cells outside its normal range, or more specifically, restrict transduction to a cell or tissue type. For example, fusogens or envelope proteins can be linked in-frame to targeting sequences such as receptor ligands, antibodies (using recombinant antibody-type molecules such as antigen-binding portions of antibodies or single-chain antibodies), and polypeptide moieties or modifications thereof that, when displayed on the transduction vector coat, facilitate direct delivery of virion particles to the desired target cells (e.g., when glycosylation sites are present in the targeting sequence). Furthermore, envelope proteins can further contain sequences that modulate cellular function. Modulating cellular function with transduction vectors can increase or decrease the transduction efficiency of specific cell types in a mixed cell population. For example, stem cells can be more specifically transduced with envelope sequences containing ligands or binding partners that specifically bind to stem cells but not other cell types found in blood or bone marrow. Non-limiting examples include stem cell factor (SCF) and Flt-3 ligand. Other examples include, for example, antibodies (e.g., cell type-specific single chain antibodies) and essentially any antigen (including receptor) that binds to tissues such as lung, liver, pancreas, heart, endothelial, smooth, breast, prostate, epithelial, vascular cancers, etc.

[0399] Protein fusogens or viral envelope proteins can be retargeted by mutating amino acid residues in the fusogen protein or the targeting protein (e.g., hemagglutinin protein). In some embodiments, the fusogen is randomly mutated. In some embodiments, the fusogen is rationally mutated. In some embodiments, the fusogen is subjected to directed evolution. In some embodiments, the fusogen is truncated and only a subset of the peptides is used in the retroviral vector or VLP. For example, amino acid residues in the measles hemagglutinin protein can be mutated to change the protein's binding properties and redirect fusion (doi:10.1038 / nbt942, Molecular Therapy vol.16 no.8, 1427-1436 Aug.2008, doi:10.1038 / nbt1060, DOI:10.1128 / JVI.76.7.3558-3563.2002, DOI:10.1128 / JVI.75.17.8016-8020.2001, doi:10.1073pnas.0604993103).

[0400] In some embodiments, a protein fusogen or viral envelope protein is retargeted by i) mutating amino acids in the native fusogen protein sequence or viral envelope protein sequence, and / or ii) engineering the fusogen protein or viral envelope protein to include a polypeptide sequence that enables the fusogen or viral envelope protein to target, fuse with, or infect host cells outside its normal range.

[0401] In some embodiments, fusosomes contain one or more fusogens on their outer surface (e.g., 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, fusogen-containing fusosomes will integrate their membranes into the lipid bilayer of target cells.

[0402] In some embodiments, the fusogen is a paramyxovirus fusogen. In some embodiments, the fusogen is Nipah virus protein F, measles virus F protein, Tupaia paramyxovirus F protein, paramyxovirus F protein, Hendra virus F protein, Henipavirus F protein, Morbillivirus F protein, Respirovirus F protein, Sendai virus F protein, Rubulavirus F protein, or Avulavirus F protein.

[0403] In some embodiments, the fusogen is a Poxviridae fusogen.

[0404] Additional exemplary fusogens are disclosed in U.S. Pat. No. 9,695,446, U.S. Pat. App. Pub. No. 2004 / 0028687, U.S. Pat. No. 6,416,997, U.S. Pat. No. 7,329,807, U.S. Pat. App. Pub. No. 2017 / 0112773, U.S. Pat. App. Pub. No. 2009 / 0202622, WO 2006 / 027202, and U.S. Pat. App. Pub. No. 2004 / 0009604, the contents of all of which are incorporated herein by reference.

[0405] In some embodiments, a fusogen described herein comprises an amino acid sequence in Table 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of such a sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a length of 100, 200, 300, 400, 500, or 600 amino acids. 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 encode an amino acid sequence of Table 1, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto over a length of 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids.

[0406] In some embodiments, a fusogen described herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of said sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a length of 100, 200, 300, 400, 500, or 600 amino acids. For example, in some embodiments, a fusogen described herein comprises 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 encode an amino acid sequence set forth in any one of SEQ ID NOs: 1-57, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a length of 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids. [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

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

[0407] In some embodiments, a fusogen described herein comprises an amino acid sequence in Table 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of such a sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a length of 100, 200, 300, 400, 500, or 600 amino acids. 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 encode an amino acid sequence in Table 2, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto over a length of 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids.

[0408] In some embodiments, a fusogen described herein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 58-133, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion of said sequence, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a length of 100, 200, 300, 400, 500, or 600 amino acids. For example, in some embodiments, a fusogen described herein comprises 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 encode an amino acid sequence set forth in any one of SEQ ID NOs: 58-133, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a portion thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity over a length of 40, 50, 60, 80, 100, 200, 300, 400, 500, or 600 amino acids. [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

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

[0409] ii) Lipid fusogens In some embodiments, the fusosomes may be treated with a fusogenic lipid, such as a saturated fatty acid. In some embodiments, the saturated fatty acid has 10-14 carbons. In some embodiments, the saturated fatty acid has a longer chain carboxylic acid. In some embodiments, the saturated fatty acid is a monoester.

[0410] In some embodiments, the fusosomes can be treated with unsaturated fatty acids. In some embodiments, the unsaturated fatty acids include C16-C18 unsaturated fatty acids. In some embodiments, the unsaturated fatty acids include oleic acid, glycerol monooleate, glycerides, diacylglycerols, modified unsaturated fatty acids, and any combination thereof.

[0411] Without wishing to be bound by theory, in some embodiments, negatively curved lipids promote membrane fusion. In some aspects, fusosomes contain one or more negatively curved lipids in their membranes, e.g., negatively curved lipids exogenous to the source cells. In embodiments, negatively curved lipids or their precursors are added to the source cells or to the medium containing the fusosomes. In embodiments, the source cells are engineered to express or overexpress one or more lipid synthesis genes. The negatively curved lipids can be, for example, diacylglycerol (DAG), cholesterol, phosphatidic acid (PA), phosphatidylethanolamine (PE), or fatty acids (FA).

[0412] Without wishing to be bound by theory, in some embodiments, positively curved lipids inhibit membrane fusion. In some embodiments, fusosomes contain reduced levels of one or more positively curved lipids, e.g., exogenous positively curved lipids, in the membrane. In embodiments, the levels are reduced by inhibiting lipid synthesis, e.g., by knocking out or knocking down lipid synthesis genes in the source cells. The positively curved lipid can be, for example, lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), or monoacylglycerol (MAG).

[0413] iii) Chemical Fusogens In some embodiments, the fusosomes may be treated with a fusogenic chemical, hi some embodiments, the fusogenic chemical is polyethylene glycol (PEG) or a derivative thereof.

[0414] In some embodiments, the chemical fusogen induces local dehydration between the two membranes, which leads to unfavorable molecular packing of the bilayer. In some embodiments, the chemical fusogen induces dehydration in the nearby region of the lipid bilayer, causing displacement of aqueous molecules between the membranes and allowing interaction between the two membranes.

[0415] In some embodiments, the chemical fusogen is a cation. Some non-limiting examples of cations include Ca, Mg, Mn, Zn, La, Sr, and H.

[0416] In some embodiments, chemical fusogens bind to target membranes by modifying surface polarity, which alters hydration-dependent intermembrane repulsion.

[0417] In some embodiments, the chemical fusogen is soluble and lipid soluble. Some non-limiting examples include oleoylglycerol, dioleoylglycerol, trioleoylglycerol, and variants and derivatives thereof.

[0418] In some embodiments, the chemical fusogen is a water-soluble chemical. Some non-limiting examples include polyethylene glycol, dimethyl sulfoxide, and variants and derivatives thereof.

[0419] In some embodiments, the chemical fusogen is a small organic molecule. A non-limiting example includes n-hexyl bromide.

[0420] In some embodiments, the chemical fusogen does not alter the composition, cell viability, or ion transport properties of the fusogen or target membrane.

[0421] In some embodiments, the chemical fusogen is a hormone or vitamin. Some non-limiting examples include abscisic acid, retinol (vitamin A1), tocopherol (vitamin E), and variants and derivatives thereof.

[0422] In some embodiments, fusosomes contain an agent that stabilizes actin and polymerized actin. Without 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, neural Wiskott-Aldrich syndrome protein (N-WASP), or formin. See, e.g., Langmuir. 2011 Aug 16;27(16):10061-71 and Wen et al., Nat Commun. 2016 Aug 31;7. In embodiments, fusosomes contain actin that is exogenous or overexpressed relative to the source cell, e.g., wild-type actin or actin containing a mutation that promotes polymerization. In embodiments, fusosomes contain ATP or phosphocreatine, e.g., exogenous ATP or phosphocreatine. iv) Small molecule fusogens

[0423] In some embodiments, fusosomes can be treated with fusogenic small molecules, some non-limiting examples of which include nonsteroidal anti-inflammatory drugs (NSAIDs) such as halothane, meloxicam, piroxicam, tenoxicam, and chlorpromazine.

[0424] In some embodiments, the small molecule fusogen may be present in micelle-like aggregates or may be aggregate-free.

[0425] v) Fusogen modification In some embodiments, the fusogen is linked to a cleavable protein. In some cases, the cleavable protein can be cleaved by exposure to a protease. The engineered fusion protein can bind to any domain of a transmembrane protein. The engineered fusion protein can be linked to a protein domain located within the intermembrane space by a cleavage peptide. The cleavage peptide can be cleaved by one or a combination of intermembrane proteases (e.g., HTRA2 / OMI, which requires a nonpolar aliphatic amino acid at position P1 (preferably valine, isoleucine, or methionine) and a hydrophilic residue at positions P2 and P3 (preferably arginine)).

[0426] In some embodiments, the fusogen is linked to an affinity tag. In some embodiments, the affinity tag aids in the separation and isolation of fusosomes. In some embodiments, the affinity tag is cleavable. In some embodiments, the affinity tag is non-covalently linked to the fusogen. In some embodiments, the affinity tag is present on the fusosome and separate from the fusogen.

[0427] In some embodiments, the fusogen protein is engineered to include a proteolytic sequence, such as a mitochondrial or cytosolic degradation sequence, by any method known in the art or described herein. The fusogen can be engineered to include, but is not limited to, a proteolytic sequence, such as a caspase 2 protein sequence (e.g., Val-Asp-Val-Ala-Asp-|- (SEQ ID NO: 155)) or other proteolytic sequence (see, e.g., Gasteiger et al., *The Proteomics Protocols Handbook*; 2005:571-607), a modified proteolytic sequence having at least 75%, 80%, 85%, 90%, 95% or more identity to a wild-type proteolytic sequence, a cytoplasmic proteolytic sequence (e.g., ubiquitin), or a modified cytoplasmic proteolytic sequence having at least 75%, 80%, 85%, 90%, 95% or more identity to a wild-type proteolytic product. In some embodiments, the composition comprises mitochondria in a source cell or chondrisomes that contain a protein modified with a proteolytic sequence, e.g., at least 75%, 80%, 85%, 90%, 95% or more identity to a wild-type proteolytic sequence, a cytosolic proteolytic sequence (e.g., ubiquitin), or a modified cytosolic proteolytic sequence that has at least 75%, 80%, 85%, 90%, 95% or more identity to the wild-type proteolytic sequence.

[0428] In some embodiments, fusogens can be modified with protease domains that recognize specific proteins, e.g., overexpression of proteases, e.g., engineered fusion proteins with protease activity, e.g., proteases or protease domains derived from proteases, e.g., MMPs, mitochondrial processing peptidases, mitochondrial intermediate peptidases, inner membrane peptidases.

[0429] Alfonzo,JD&Soll,D.Mitochondrial tRNA import-the challenge to understand has just begun.Biological Chemistry 390:717-722.2009;Langer,T.et al.Characterization of Peptides Released from Mitochondria.THE JOURNAL OF BIOLOGICAL CHEMISTRY.Vol.280,No.4.2691-2699,2005;Vliegh,P.et al.Synthetic therapeutic peptides:science and market.Drug Discovery Today.15(1 / 2).2010;Quiros PMm et al.,New roles for mitochondrial proteases in health, aging and disease.Nature Reviews Molecular Cell Biology.V16,2015;Weber-Lotfi,F.et al.DNA import competence and mitochondrial genetics.Biopolymers and See Cell.Vol.30.N 1.71-73,2014.

[0430] III. Positive Target Cell-Specific Regulatory Elements In some embodiments, the fusosomes described herein, e.g., viruses, e.g., retroviruses, comprise a nucleic acid (e.g., a gene encoding an exogenous agent), e.g., a retroviral nucleic acid, that comprises a positive target cell-specific regulatory element, such as a tissue-specific promoter, a tissue-specific enhancer, a tissue-specific splice site, a tissue-specific site that extends the half-life of an RNA or protein, a tissue-specific mRNA export-facilitating site, a tissue-specific translation-enhancing site, or a tissue-specific post-translational modification site.

[0431] In some aspects, the fusosomes described herein, e.g., viruses, e.g., retroviruses, comprise nucleic acid, e.g., retroviral nucleic acid, which may include regions, e.g., origins of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, polyadenylation sequences, and untranslated regions, such as 5' and 3' untranslated regions, that can interact with host cell proteins to effect transcription and translation and direct, increase, regulate, or control the transcription or expression of an operably linked polynucleotide. Such elements may vary 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.

[0432] In certain embodiments, a control element is capable of directing, increasing, regulating, or controlling the transcription or expression of an operably linked polynucleotide in a cell-specific manner. In certain embodiments, a nucleic acid, e.g., a retroviral nucleic acid, comprises one or more expression control sequences that are specific for a particular cell, cell type, or cell lineage, e.g., a target cell, i.e., expression of a polynucleotide operably linked to a particular cell, cell type, or cell lineage-specific expression control sequence is expressed in the target cell but not (or expressed at a lower level) in non-target cells.

[0433] In certain embodiments, a nucleic acid, eg, a retroviral nucleic acid, can include exogenous, endogenous, or heterologous regulatory sequences, such as a promoter and / or an enhancer.

[0434] In embodiments, a promoter contains a recognition site for binding of an RNA polymerase, which initiates and transcribes a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells contain an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins and / or another sequence, a CNCAAT region, found approximately 70-80 bases upstream from the start of transcription, where N can be any nucleotide.

[0435] In embodiments, an enhancer comprises a segment of DNA containing sequences that can provide enhanced transcription and, in some cases, can function independently of orientation relative to another regulatory sequence. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. In some embodiments, a promoter / enhancer segment of DNA comprises sequences that can provide both promoter and enhancer function.

[0436] Exemplary ubiquitous expression control sequences include the cytomegalovirus (CMV) immediate early promoter, the 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, the H5, P7.5, and P11 promoters from vaccinia virus, elongation factor 1-alpha (E1) promoter, and the HIV-1 virus (HIV-2) promoter. F1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 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 These include 26 loci (Orions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative control region deletion, and d1587rev primer binding site substitution (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)).

[0437] In some embodiments, a promoter may be paired with a heterologous gene to confer the regulatory function of that promoter on the heterologous gene. In some embodiments, cis-regulatory elements from a promoter of a first gene may be linked to segments of a promoter of a different gene to create a chimeric promoter with properties of both promoters.

[0438] In some aspects, the promoter is a tissue-specific promoter, e.g., a promoter that drives expression in CNS cells, such as pan-neuronal cells, GABAergic neurons, glutamatergic neurons, cholinergic neurons, dopaminergic neurons, serotonergic neurons, astrocytes, microglia, oligodendrocytes, or choroid plexus cells. Various suitable CNS cell-specific promoters are listed in Table 3 below. In some embodiments, the fusosomes (e.g., viral vectors) described herein comprise in their nucleic acid a promoter having the sequence of a promoter in Table 3, or a transcriptionally active fragment thereof, or a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the fusosomes (e.g., viral vectors) described herein comprise in their nucleic acid a promoter having a transcription factor binding site derived from a region within 3 kb of the transcription start site of a gene listed in Table 3. In some embodiments, the fusosomes (e.g., viral vectors) described herein comprise in their nucleic acid a region within 2.5 kb, 2 kb, 1.5 kb, 1 kb, or 0.5 kb immediately upstream of the transcription start site of a gene listed in Table 3, or a transcriptionally active fragment thereof, or a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. [Table 3]

[0439] In some embodiments, the CNS cell-specific promoter is described in Hioki et al., Ther. 2007 Jun;14(11):872-82 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the SYN, NSE, CaMKII, tubulin, or PDGF promoter. In some aspects, the CNS cell-specific promoter is a promoter described in Nathanson et al., Front. Neural Circuits, 2009, 3:19. doi:10.3389 / neuro.04.019.2009 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the fSST or fNPY promoter. In some aspects, the CNS cell-specific promoter is a promoter described in Delzor et al., Hum Gene Ther Methods. 2012 Aug;23(4):242-54 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the GAD67 or DLX5 / 6 promoter. In some aspects, the CNS cell-specific promoter is a promoter described in Egashira et al., Sci Rep. 2018 Oct 11;8(1):15156 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the VGLUT1 or Dock10 promoter. In some embodiments, the CNS cell-specific promoter is a promoter described in Naciff et al., J. Neurochem., 1999 Jan;72(1):17-28 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the ChAT promoter. In some aspects, the CNS cell-specific promoter is the VAChT promoter. In some embodiments, the CNS cell-specific promoter is a promoter described in Delzor et al., Hum Gene Ther Methods. 2012 Aug;23(4):242-254, which is incorporated by reference herein in its entirety, e.g., the CNS cell-specific promoter is the Drd1a promoter.In some embodiments, the CNS cell-specific promoter is a promoter described in Benzekhroufa et al., Gene Ther. 2009 May;16(5):681-8 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the TPH-2 promoter. In some embodiments, the CNS cell-specific promoter is a promoter described in Merienne et al., Gene Ther. 2015 Oct;22(10):830-9 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the GFAP, EAAT1, or GS promoter. In some aspects, the CNS cell-specific promoter is a promoter described in the Immgen consortium (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the CX3CR1 promoter. In some embodiments, the CNS cell-specific promoter is the TMEM119 promoter. In some aspects, the CNS cell-specific promoter is a promoter described in McIver et al., J Neurosci Res. 2005 Nov 1;82(3):397-403 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the MBP promoter. In some embodiments, the CNS cell-specific promoter is a promoter described in Kagiava et al., J Gene Med. 2014 Nov-Dec;16(11-12):364-73 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the MBP or CNP promoter. In some aspects, the CNS cell-specific promoter is a promoter described in Regev et al., Proc Natl Acad Sci US A. 2010 Mar 2;107(9):4424-9 (incorporated herein by reference in its entirety), e.g., the CNS cell-specific promoter is the CRFR2β promoter. In some embodiments, the CNS cell-specific promoter is a transcriptionally active fragment of any of the foregoing.In some embodiments, the CNS cell-specific promoter is a variant having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the foregoing.

[0440] An internal ribosome entry site (IRES) typically facilitates direct internal ribosome entry to the start codon (e.g., ATG) of a cistron (protein-encoding region), thereby resulting in cap-independent translation of the gene. See, e.g., Jackson et al. (1990) Trends Biochem Sci 15(12):477-83, and Jackson and Kaminski. (1995) RNA 1(10):985-1000. In certain embodiments, a vector contains one or more exogenous genes encoding one or more exogenous agents. In certain embodiments, to achieve efficient translation of each of multiple exogenous protein agents, polynucleotide sequences can be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

[0441] As used herein, nucleic acids, e.g., retroviral nucleic acids, may also contain one or more Kozak sequences, e.g., short nucleotide sequences that facilitate initial binding of mRNA to the small ribosomal subunit and increase translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is a purine (A or G) (Kozak, (1986) Cell. 44(2):283-92, and Kozak, (1987) Nucleic Acids Res. 15(20):8125-48).

[0442] Promoters Responsive to Heterologous Transcription Factors and Inducers In some embodiments, the nucleic acid, retroviral nucleic acid, comprises elements that allow for conditional expression of the exogenous agent, e.g., any type of conditional expression, including, but not limited to, inducible expression, repressible expression, cell type-specific expression, or tissue-specific expression. In some embodiments, to achieve conditional expression of the exogenous agent, expression is controlled by subjecting the cell, tissue, or organism to a treatment or condition that causes expression of the exogenous agent or that increases or decreases expression of the exogenous agent.

[0443] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionine promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0444] Expression of a transgene 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 manner, and in other cases, the inducer molecule activates or represses gene expression in an all-or-nothing manner.

[0445] A commonly used inducible promoter / system is the tetracycline (Tet)-regulated system. The Tet system is based on the coexpression of two elements in each target cell: (i) a tetracycline-responsive element containing repeats of the Tet operator sequence (TetO) fused to a minimal promoter and connected to a gene of interest (e.g., a gene encoding an exogenous agent) and (ii) a fusion protein of a transcriptional transactivator (tTA), a Tet repressor (TetR), and the transactivation domain of the VP16 protein from herpes simplex virus. In the first described version, transgene expression was active in the absence of tetracycline or its potent analog, doxycycline (Dox) (referred to as the Tet-OFF system), but modification of four amino acids within the transactivator protein resulted in a reverse tTA (rtTA) that binds to TetO only in the presence of Dox (the Tet-ON system). In some embodiments, the VP16 domain in the transactivator was replaced with a minimal activation domain, potential splice donor and acceptor sites were removed, and the protein was codon-optimized, resulting in an improved transactivator variant, rtTA2S-M2, that is sensitive to Dox and has reduced baseline activity. Furthermore, various Tet-responsive promoter elements have been generated, including modifying the TetO 36 nucleotide interval from the adjacent operator to enhance regulation. Additional modifications may help further reduce basal activity and increase the dynamic range of expression. For example, the pTet-T11 (abbreviated as T1I) variant exhibits a high dynamic range and low background activity.

[0446] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the nucleic acid, e.g., a retroviral nucleic acid, comprises at least one (typically two) site(s) for recombination mediated by a site-specific recombinase, e.g., one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or excision or integration proteins, enzymes, cofactors, or related proteins involved in the recombination reaction, including mutants, derivatives (e.g., fusion proteins comprising the recombination protein sequence or fragments thereof), fragments, and variants thereof. 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.

[0447] Riboswitches that regulate the expression of exogenous agents Some of the compositions and methods provided herein include one or more riboswitches or polynucleotides containing one or more riboswitches. Riboswitches are a common feature in bacteria that regulate gene expression and provide a means for RNA control of biological functions. Riboswitches can reside in the 5' untranslated region of mRNA and can enable regulatory control of gene expression through the binding of small molecule ligands that induce or inhibit riboswitch activity. In some embodiments, riboswitches control gene products involved in the production of small molecule ligands. Riboswitches typically operate in cis, although riboswitches that operate in trans have been identified. Natural riboswitches are composed of two domains: an aptamer domain that binds to a ligand through a three-dimensional folded RNA structure, and a function switching domain that induces or inhibits riboswitch activity based on the presence or absence of a ligand. Thus, there are two ligand-sensitive conformations that can be achieved by a riboswitch, representing an on state and an off state (Garst et al., 2011). Function switching domains can affect polynucleotide expression by regulating internal ribosome entry sites, pre-mRNA splice donor accessibility in retroviral gene constructs, translation, transcription termination, transcript degradation, miRNA expression, or shRNA expression (Dambach and Winkler 2009). Aptamers and function switching domains can be used as modular components, enabling synthetic RNA devices to control gene expression as native aptamers, mutated / evolved native aptamers, or fully synthetic aptamers identified from screening random RNA libraries (McKeague et al. 2016).

[0448] The purine riboswitch family is one of the largest families, with over 500 sequences identified (Mandal et al., 2003; U.S. Patent Application Publication No. 20080269258; and WO2006055351). Purine riboswitches share a similar architecture consisting of three conserved helical / stem structures (PI, P2, P3) and intervening loop / junction elements (J1-2, L2, J2-3, L3, J3-1). The aptamer domain of the purine family of riboswitches spontaneously changes its affinity / regulation with various purine compounds, including adenine, guanine, adenosine, guanosine, deoxyadenosine, and deoxyguanosine, depending on the sequence (Kim et al., 2007).

[0449] In some embodiments, a nucleic acid, such as a retroviral nucleic acid described herein, comprises a promoter and a polynucleotide encoding an exogenous agent operably linked to a riboswitch. The riboswitch comprises one or more, e.g., all of the following: a.) an aptamer domain, e.g., an aptamer domain capable of binding to a nucleoside analog antiviral drug and exhibiting reduced binding to guanine or 2'-deoxyguanosine compared to the nucleoside analog antiviral drug; b.) a function switching domain, e.g., a function switching domain capable of regulating expression of an exogenous agent, wherein binding of the nucleoside analog by the aptamer domain induces or represses the expression-regulating activity of the function switching domain, thereby regulating expression of the exogenous agent. In some embodiments, the exogenous agent can be a polypeptide, miRNA, or shRNA. For example, in one embodiment, the riboswitch is operably linked to a nucleic acid encoding a chimeric antigen receptor (CAR). In a non-limiting example provided herein, the exogenous gene encodes one or more engineered signaling polypeptides. For example, the target polynucleotide encoding the riboswitch and one or more engineered signaling polynucleotides can be found in the genome of a source cell, a replication-incompetent recombinant retroviral particle, a T cell and / or a NK cell.

[0450] An aptamer domain, for example, can be used as a modular component and combined with any of the function switching domains to affect RNA transcripts. In any of the embodiments disclosed herein, the riboswitch can affect RNA transcripts by regulating 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 function switching domain can control the 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 in its entirety). In any of the embodiments disclosed herein, the presence or absence of a small molecule ligand allows the riboswitch to affect RNA transcripts. In some embodiments, the riboswitch can include a ribozyme. A riboswitch with a ribozyme can inhibit or enhance transcript degradation of a target polynucleotide in the presence of a small molecule ligand. In some embodiments, the ribozyme can be of 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).

[0451] IV. Non-target cell-specific regulatory elements In some embodiments, the non-target cell-specific regulatory element or negative TCSSE comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site.

[0452] In some embodiments, the non-target cells contain endogenous miRNA. In some embodiments, the fusosomes described herein, such as viruses, e.g., retroviruses, contain nucleic acids, e.g., retroviral nucleic acids (e.g., genes encoding exogenous agents), that may contain a recognition sequence for the miRNA. Thus, when the nucleic acid, e.g., retroviral nucleic acid, enters the non-target cells, the miRNA may downregulate the expression of the exogenous agent. This helps to increase the specificity between target cells and non-target cells.

[0453] In some embodiments, miRNAs are small, 20-22 nucleotide non-coding RNAs that are typically excised from approximately 70 nucleotide foldback RNA precursor structures known as pre-miRNAs. Generally, miRNAs negatively regulate targets in one of two ways, depending on the degree of complementarity between the miRNA and the target. First, miRNAs that bind to protein-coding mRNA sequences with perfect or near-perfect complementarity typically induce the RNA-mediated interference (RNAi) pathway. MiRNAs that exert their regulatory effects by binding to imperfectly complementary sites within the 3' untranslated region (UTR) of mRNA targets typically repress target gene expression post-transcriptionally, apparently at the translational level, via a RISC complex similar to, or perhaps the same as, that used in the RNAi pathway. Consistent with translational control, miRNAs using this mechanism reduce the protein levels of target genes, while the mRNA levels of these genes are minimally affected. MiRNAs (e.g., naturally occurring miRNAs or artificially designed miRNAs) can specifically target any mRNA sequence. For example, in one embodiment, one skilled in the art can design a short hairpin RNA construct that is expressed as a human miRNA (e.g., miR-30 or miR-21) primary transcript. This design adds a Drosha processing site to the hairpin structure and has been shown to significantly improve knockdown efficiency (Pusch et al., 2004). The hairpin stem consists of a 22-nt dsRNA (e.g., antisense, perfectly complementary to the target of interest) and a 15-19 nt loop derived from a human miR. Adding the miR loop 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 traditional shRNA designs that do not use microRNAs. Increased Drosha and Dicer processing increases siRNA / miRNA production and enhances the efficacy of the expressed hairpin.

[0454] 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, including developmental timing, cell differentiation, proliferation, apoptosis, carcinogenesis, insulin secretion, and cholesterol biosynthesis. (See 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 analyses have shown that miRNAs have distinct expression profiles in different tissues. Computational methods were used to analyze the expression of approximately 7,000 predicted human miRNA targets. Data suggest that miRNA expression contributes broadly to the tissue specificity of mRNA expression in many human tissues. (See Sood et al. 2006 PNAS USA 103(8):2746-51)

[0455] Thus, miRNA-based approaches can be used to restrict the expression of exogenous agents to target cell populations by using endogenous microRNA species to silence expression of the exogenous agents in non-target cell types. MicroRNAs induce sequence-specific post-transcriptional gene silencing in many organisms by inhibiting messenger RNA (mRNA) translation or causing mRNA degradation. See, e.g., Brown et al. 2006 Nature Med. 12(5):585-91. and WO 2007 / 000668, each of which is incorporated herein by reference in its entirety. In some embodiments, a nucleic acid, e.g., a retroviral nucleic acid, comprises one or more (e.g., multiple) tissue-specific miRNA recognition sequences. In some embodiments, the tissue-specific miRNA recognition sequence is approximately 20-25, 21-24, or 23 nucleotides in length. In embodiments, the tissue-specific miRNA recognition sequence has perfect complementarity to a miRNA present in non-target cells. In some embodiments, the exogenous agent does not include GFP, e.g., does not include a fluorescent protein, e.g., does not include a reporter protein. In some embodiments, the off-target cells are not hematopoietic cells and / or the miRNA is not present in hematopoietic cells.

[0456] In some embodiments, methods described herein include tissue-specific expression of an exogenous agent in a target cell, comprising contacting a plurality of fusosomes, e.g., viral, e.g., retroviral vectors, 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 preferentially, e.g., restricted to, the target cell.

[0457] For example, a nucleic acid, e.g., a retroviral nucleic acid, can contain at least one miRNA recognition sequence operably linked to a nucleotide sequence having a corresponding miRNA in a non-target cell, e.g., a hematopoietic progenitor cell (HSPC), hematopoietic stem cell (HSC), which prevents or reduces expression of the nucleotide sequence in the non-target cell but does not prevent or reduce expression in a target cell, e.g., a differentiated cell. In some embodiments, the nucleic acid, e.g., a retroviral nucleic acid, contains at least one miRNA sequence targeting an miRNA that is present in an effective amount in the non-target cell (e.g., the concentration of the endogenous miRNA is sufficient to reduce or prevent expression of the transgene) and also contains a transgene. In embodiments, the miRNA used in this system is highly expressed in non-target cells, such as HSPCs and HSCs, but not in differentiated progeny, e.g., of the myeloid and lymphoid lineages, preventing or reducing expression of the transgene in a susceptible stem cell population while maintaining expression and therapeutic effect in the target cell.

[0458] In some embodiments, the negative TSCRE or NTSCRE comprises a miRNA recognition site. Exemplary miRNAs are provided in Table 4. In some embodiments, the nucleic acid (e.g., a fusomal or retroviral nucleic acid) comprises a sequence complementary to or having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementarity to a miRNA in Table 4. In some embodiments, the nucleic acid (e.g., a fusomal or retroviral nucleic acid) comprises a sequence that is fully complementary to a seed sequence within an endogenous miRNA, e.g., a miRNA in Table 4. In some embodiments, the miRNA comprises a sequence set forth in any one of SEQ ID NOs: 156-162. In embodiments, the seed sequence is at least 6, 7, 8, 9, or 10 nucleotides in length. [Table 4]

[0459] In some embodiments, the negative TSCRE or NTSCRE comprises an miRNA recognition site for a miRNA described herein. Exemplary miRNAs include those found in Butovsky et al., Nat Neurosci. 2014 Jan;17(1):131-43 (incorporated herein by reference in its entirety), such as miR-338-3p, miR-9, miR-125b-5p, or miR-342-3p. Further exemplary miRNAs are found in Delzor et al., Curr. Drug Targets, 2013 Oct;14(11):1336-46 (incorporated herein by reference in its entirety), such as miR-124.

[0460] 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 neuron, e.g., a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, or a serotonergic neuron. In some aspects, the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE), e.g., the NTSCRE comprises an miRNA recognition site for an miRNA expressed in glial cells (e.g., astrocytes, microglial cells, or oligodendrocytes).

[0461] 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 glial cell, e.g., an astrocyte, a microglia, or an oligodendrocyte. In some aspects, the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE), e.g., the NTSCRE comprises an miRNA recognition site for an miRNA expressed in a neuron, e.g., a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, or a serotonergic neuron.

[0462] In some embodiments, the negative TSCRE or NTSCRE comprises an 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;Chen et al.Science.2004 Jan 2;303(5654):83-6;Barad et al.Genome Res.2004 Dec;14(12):2486-2494;Krichevsky et al.,RNA.2003 Oct;9(10):1274-81;Kasashima et al.Biochem Biophys Res Commun.2004 Sep 17;322(2):403-10;Houbaviy et al.,Dev Cell.2003 Aug;5(2):351-8;Lagos-Quintana et al.,Curr Biol.2002 Apr 30;12(9):735-9; Calin et al., Proc Natl Acad Sci US A. 2004 Mar 2;101(9):2999-3004; Sempere et al. Genome Biol. 2004;5(3):R13; Metzler et al., Genes Chromosomes Cancer. 2004 Feb;39(2):167-9; Calin et al., Proc Natl Acad Sci US A. 2002 Nov 26;99(24):15524-9; Mansfield et al. Nat Genet. 2004 Oct;36(10):1079-83; Michael et al. Mol Cancer Res. 2003 Oct;1(12):882-91; and those found at www.miRNA.org. 2004 Oct;36(10):1079-83; Michael et al. Mol Cancer Res. 2003 Oct;1(12):882-91; and at www.miRNA.org.

[0463] 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, miR-9 9a, miR-183, miR-19b, miR-92, miR-9, miR-130b, miR-21, miR-30b, miR-16, miR-99a, miR-212 , miR-30c, miR-213, miR-20, miR-155, miR-152, miR-139, miR-30b, miR-7, miR-30c, miR-18, mi R-137, miR-219, miR-1d, miR-178, miR-24, miR-122a, miR-215, miR-124a, miR-190, miR-149, miR-193, let-7a, miR-132, miR-27a, miR-9*, miR-200b, miR-266, miR-153, miR-135, miR-206, The miRNA recognition site includes a miRNA recognition site for a miRNA selected from 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.

[0464] In some embodiments, the nucleic acid (e.g., a 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; 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; in some embodiments, the first miRNA recognition site and the second miRNA recognition site 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 an miRNA in Table 4. In some embodiments, one or more miRNA recognition sites on a fusosomal nucleic acid (e.g., a 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 (e.g., a retroviral nucleic acid) are located downstream of the polyA tail sequence, e.g., between the polyA tail sequence and the WPRE. In some embodiments, one or more miRNA recognition sites on a fusosomal nucleic acid (e.g., a retroviral nucleic acid) are located downstream of the WPRE.

[0465] V. Immune regulation In some embodiments, the fusosomes, e.g., retroviral vectors or VLPs described herein comprise elevated CD47. See, e.g., U.S. Patent No. 9,050,269, incorporated herein by reference in its entirety. In some embodiments, the fusosomes, e.g., retroviral vectors or VLPs described herein comprise elevated complement regulatory proteins. See, e.g., Spanish Patent No. 2627445 T3 and U.S. Patent No. 6,790,641, each of which is incorporated herein by reference in its entirety. In some embodiments, the fusosomes, e.g., retroviral vectors or VLPs described herein lack or comprise reduced levels of an MHC protein, e.g., MHC-1 class 1 or class II. See, e.g., U.S. Patent Application Publication No. 20170165348, incorporated herein by reference in its entirety.

[0466] Fusosomes, such as retroviral vectors or VLPs, can be recognized by the subject's immune system. In the case of enveloped viral vector particles (e.g., retroviral vector particles), membrane-associated proteins displayed on the surface of the viral envelope can be recognized, and the viral particles themselves can be neutralized. Furthermore, upon infection of target cells, the viral envelope can be integrated with the cell membrane, resulting in viral envelope proteins being displayed on the surface of the cell or remaining closely associated with the surface of the cell. Therefore, the immune system can also target cells infected with viral vector particles. Both effects can lead to a decrease in the effectiveness of exogenous agent delivery by viral vectors.

[0467] The viral particle envelope is usually derived from the membrane of the source cell, and therefore membrane proteins expressed in the cell membrane from which the viral particle buds can be incorporated into the viral envelope. immunoregulatory protein CD47

[0468] The internalization of extracellular material into cells is generally carried out by a process called endocytosis (Rabinovitch, 1995, Trends Cell Biol. 5(3):85-7; Silverstein, 1995, Trends Cell Biol. 5(3):141-2; Swanson et al., 1995, Trends Cell Biol. 5(3):89-93; Allen et al., 1996, J. Exp. Med. 184(2):627-37). Endocytosis is divided into two general categories: phagocytosis, which involves the uptake of particles, and pinocytosis, which involves the uptake of fluids and solutes.

[0469] Based on studies using knockout mice lacking the membrane receptor CD47, professional phagocytes have been shown to distinguish between self and non-self (Oldenborg et al., 2000, Science 288(5473):2051-4). CD47 is a ubiquitous member of the Ig superfamily that interacts with the immunosuppressive receptor SIRP alpha (signal-regulatory receptor protein alpha) 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 CD47-SIRPα interactions appear to inactivate autologous macrophages in mice, a severe reduction in CD47 (perhaps 90%) is seen on human blood cells derived from several Rh genotypes that show little or no evidence of anemia (Mouro-Chanteloup et al., 2003, Blood 101(1):338-344) and little or no evidence of enhanced cellular interactions with phagocytic monocytes (Arndt et al., 2004, Br. J. Haematol. 125(3):412-4).

[0470] In some embodiments, fusosomes, e.g., retroviral vectors or VLPs (e.g., viral particles having a radius of less than about 1 μm, about 400 nm, or about 150 nm), comprise at least a biologically active portion of CD47, e.g., on an exposed surface of the fusosome, e.g., retroviral vector or VLP. In some aspects, the fusosome, e.g., retroviral vector (e.g., lentivirus) or VLP comprises a lipid coat. In embodiments, the amount of biologically active CD47 in the fusosome, e.g., retroviral vector or VLP, is about 20-250, 20-50, 50-100, 100-150, 150-200, or 200-250 molecules / μm 2 In some embodiments, the CD47 is human CD47.

[0471] The methods described herein can include avoiding phagocytosis of a particle by a phagocyte. The methods can include expressing at least one peptide comprising at least a biologically active portion of CD47 in a fusosome, e.g., a retroviral vector or VLP, such that a CD47-containing fusosome, e.g., a retroviral vector or VLP, is exposed to a phagocyte, and the fusosome, e.g., viral particle, avoids phagocytosis by the phagocyte or exhibits reduced phagocytosis compared to an otherwise similar, unmodified fusosome, e.g., a retroviral vector or VLP. In some embodiments, the half-life of the fusosome, e.g., retroviral vector or VLP, in a subject is extended compared to an otherwise similar, unmodified fusosome, e.g., a retroviral vector or VLP.

[0472] MHC deletion Major histocompatibility complex class I (MHC-I) is a host cell membrane protein that can be incorporated into the viral envelope. Because it is highly polymorphic in nature, it is a major target of the body's immune response (McDevitt HO (2000) Annu. Rev. Immunol. 18:1-17). MHC-I molecules exposed on the plasma membrane of source cells may be incorporated into the viral particle envelope during vector budding. These MHC-I molecules, derived from source cells and incorporated into viral particles, can then be transferred to the plasma membrane of target cells. Alternatively, MHC-I molecules may remain closely associated with the target cell membrane as a result of the tendency of viral particles to absorb and remain bound to the target cell membrane.

[0473] The presence of exogenous MHC-I molecules on or near the plasma membrane of transduced cells can elicit an alloreactive immune response in a subject. This can lead to immune-mediated killing or phagocytosis of the transduced cells, either through ex vivo gene transfer followed by administration of the transduced cells to a subject, or through direct in vivo administration of viral particles. Furthermore, when MHC-I bearing viral particles are administered in vivo into the bloodstream, the viral particles can be neutralized by pre-existing MHC-I-specific antibodies before reaching their target cells.

[0474] Thus, in some embodiments, the source cells are modified (e.g., genetically engineered) to reduce MHC-I expression on the cell surface. In embodiments, the source comprises a genetically engineered disruption of the gene encoding β2-microglobulin (β2M). In embodiments, the source cells comprise a genetically engineered disruption of one or more genes encoding the MHC-I α chain. The cells may comprise a genetically engineered disruption of all copies of the gene encoding β2-microglobulin. The cells may comprise a genetically engineered disruption of all copies of the gene encoding the MHC-I α chain. The cells may comprise both a genetically engineered disruption of the gene encoding β2-microglobulin and a genetically engineered disruption of the gene encoding the 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 so that the immune response to MHC-I is reduced to a therapeutically relevant extent. In some embodiments, the enveloped viral vector particles are substantially devoid of surface-exposed MHC-I molecules.

[0475] HLA-G / E overexpression In some embodiments, the retroviral vector or VLP displays on its envelope a tolerogenic protein, e.g., an ILT-2 or ILT-4 agonist, e.g., HLA-E or HLA-G, or any other ILT-2 or ILT-4 agonist. In some embodiments, the retroviral vector or VLP has increased expression of HLA-E, HLA-G, ILT-2, or ILT-4 compared to a reference retrovirus, e.g., an unmodified retrovirus that is otherwise similar to the retrovirus.

[0476] In some embodiments, the retroviral composition has reduced MHC class I compared to unmodified retrovirus and increased HLA-G compared to unmodified retrovirus.

[0477] In some embodiments, the retroviral vector or VLP results in increased expression of HLA-G or HLA-E, e.g., an increase in HLA-G or HLA-E expression of 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, compared to a reference cellular retrovirus, e.g., an unmodified retrovirus that is otherwise similar to the retrovirus, wherein HLA-G or HLA-E expression is assayed in vitro using flow cytometry, e.g., FACS.

[0478] In some embodiments, retroviruses with increased HLA-G expression exhibit reduced immunogenicity, as measured by reduced immune cell infiltration, for example, in a teratoma formation assay.

[0479] Complement regulatory proteins Complement activity is normally controlled by several complement regulatory proteins (CRPs). These proteins prevent pseudoinflammation and host tissue damage. One group of proteins, including CD55 / decay-accelerating factor (DAF) and CD46 / membrane cofactor protein (MCP), inhibits the C3 / C5 convertases of the classical and alternative pathways. Another set of proteins, including CD59, regulates MAC assembly. CRPs have been used to prevent rejection of xenotransplant tissues and have also been shown to protect viruses and viral vectors from complement inactivation.

[0480] Membrane-located complement regulators include, for example, decay-accelerating factor (DAF) or CD55, factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), complement receptor 1 (CD35), membrane cofactor protein (MCP) or CD46, and CD59 (protectin) (e.g., to prevent formation ...

Claims

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

2. The fusosome of claim 1, wherein the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE) operably linked to the payload gene, the NTCSRE reducing expression of the payload gene in a non-target cell compared to an otherwise similar fusosome lacking the NTCSRE, and the target cell is a first type of CNS cell and the non-target cell is a second, different type of CNS cell or a non-CNS cell.

3. below: a) a lipid bilayer comprising a retargeted paramyxovirus fusogen, said fusogen being a viral envelope protein; and b) Below: (i) a payload gene encoding an exogenous agent; and (ii) a promoter operably linked to the payload gene, wherein the promoter is selected from SYN, NSE, CaMKII, a-tubulin, PDGF, fSST, fNPY, GAD67, DLX5 / 6, VGLUT1, Dock10, ChAT, VAChT, Drd1a, TPH-2, GFAP, EAAT1, GS, CX3CR1, TMEM119, MBP, CNP, or CRFR2β promoter; A fusosome, wherein the retargeted paramyxoviral fusogen is linked to a targeting domain, the targeting domain being an antibody, an antigen-binding portion of an antibody, or a receptor ligand, and the retargeted paramyxoviral fusogen results in preferential delivery of the exogenous agent to CNS cells over non-CNS cells.

4. below: (i) the fusosome further comprises a first exogenous or overexpressed immunosuppressive protein on the lipid bilayer; and / or (ii) a first immunostimulatory protein is absent from the fusosome or the fusosome further comprises a first immunostimulatory protein 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.

5. The fusosome of any one of claims 1 to 4, wherein the payload gene is a gene that treats a lysosomal storage disease or disorder, or a disease or disorder of the CNS, and the disease or disorder is a genetic defect.

6. the fusosome comprises (i) and (ii); the fusosome comprises (i) and further comprises a second exogenous or overexpressed immunosuppressive protein on the lipid bilayer; or the fusosomes comprise (ii) and further comprise a second immunostimulatory protein that is absent or present at reduced levels, the reduced levels being at least 10% reduced compared to fusosomes produced from an otherwise similar unmodified source cell; The fusosome according to claim 4.

7. the nucleic acid further comprises a positive target cell-specific regulatory element operably linked to the payload gene, the positive target cell-specific regulatory element increasing expression of the payload gene in a target cell relative to an otherwise similar fusosome lacking the positive target cell-specific regulatory element, the target cell being a CNS cell; and / or the nucleic acid further comprises a non-target cell-specific regulatory element (NTCSRE) operably linked to the payload gene, the NTCSRE decreasing expression of the payload gene in a non-target cell or tissue relative to an otherwise similar fusosome lacking the NTCSRE, the target cell being a first type of CNS cell and the non-target cell being a second, different type of CNS cell or a non-CNS cell. The fusosome of claim 3.

8. the target cells are neurons and the non-target cells are glial cells, the glial cells being oligodendrocytes, astrocytes or microglial cells; or The fusosome of claim 6 or 7, wherein the target cell is a glial cell, the glial cell being an oligodendrocyte, an astrocyte or a microglial cell, and the non-target cell is a neuron.

9. the immunosuppressive protein is a complement regulatory protein or CD47; and / or The immunostimulatory protein is an MHC I or MHC II protein; A fusosome according to any one of claims 4 and 6 to 8.

10. The fusosome of any one of claims 1 to 9, wherein the payload gene is selected from SYNE1, SETX, FMR1, SLC6A8, UBE3A, SOD1, TDP43, C9orf72, FXN, MECP2, ASPA, ALDH7A1, TPP1, FUCA1, GALC, HEXA, HEXB, MANBA, ARSA, GNPTAB, or MCOLN1.

11. The fusogen of any one of claims 1 to 10, wherein the CNS target cell is a neuron or a glial cell, and the CNS target cell is a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, a serotonergic neuron, a glial cell, an astrocyte, a microglial cell, an oligodendrocyte, or a choroid plexus cell.

12. The fusosome of any one of claims 1 to 11, wherein the paramyxovirus fusogen comprises a sequence selected from paramyxovirus F and G proteins, or F and H proteins, or F and HN proteins.

13. 13. The fusosome of any of claims 1 to 12, wherein the paramyxovirus 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.

14. The fusosome of any one of claims 1, 2, or 7, wherein the positive target cell-specific regulatory element comprises a CNS cell-specific promoter, a CNS cell-specific enhancer, a CNS cell-specific splice site, a CNS cell-specific site that extends the half-life of an RNA or protein, a CNS cell-specific mRNA export promoting site, a CNS cell-specific translation enhancing site, or a CNS cell-specific post-translational modification site.

15. The fusosome of any one of claims 1, 2, 4, 5, or 7 to 14, wherein the positive target cell-specific regulatory element comprises a CNS cell-specific promoter.

16. 16. The fusosome of claim 15, wherein the positive CNS cell-specific regulatory element comprises a promoter selected from SYN, NSE, CaMKII, a-tubulin, PDGF, fSST, fNPY, GAD67, DLX5 / 6, VGLUT1, Dock10, ChAT, VAChT, Drd1a, TPH-2, GFAP, EAAT1, GS, CX3CR1, TMEM119, MBP, CNP, or CRFR2β promoter.

17. the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, a non-target cell-specific protease recognition site, a non-target cell-specific ubiquitin ligase site, a non-target cell-specific transcriptional repression site, or a non-target cell-specific epigenetic repression site; and / or The NTCSRE comprises a tissue-specific miRNA recognition sequence, a tissue-specific protease recognition site, a tissue-specific ubiquitin ligase site, a tissue-specific transcriptional repression site, or a tissue-specific epigenetic repression site; A fusosome according to claim 2, 4 or 7.

18. 18. The fusosome of claim 2, 4, 7, or 17, wherein the NTCSRE comprises a non-target cell-specific miRNA recognition sequence, the miRNA recognition sequence being bound by one or more of miR-338-3p, miR-9, miR-125b-5p, miR-342-3p, or miR-124, and the miRNA is or comprises a sequence set forth in any one of SEQ ID NOs: 156-162.

19. The fusosome of claim 17 or claim 18, wherein the NTCSRE is located or encoded within a transcribed region encoding the exogenous agent, and the RNA produced by the transcribed region comprises a miRNA recognition sequence within a UTR or coding region.

20. The fusosome of any one of claims 1 to 19, wherein the nucleic acid comprises one or more insulator sequences.

21. The fusosome of claim 20, 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.

22. The fusosome according to any one of claims 1 to 21, wherein the fusosome is a retroviral vector particle, and the retroviral vector particle is a lentiviral vector particle.

23. A pharmaceutical composition comprising the fusosome of any one of claims 1 to 22 and a pharma- ceutically acceptable carrier, diluent, or excipient.

24. A fusosome composition comprising a plurality of fusosomes according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23 for use in a method of delivering an exogenous agent to a subject.

25. 25. The fusosome composition or pharmaceutical composition of claim 24, wherein the exogenous agent is delivered to a CNS cell of the subject.

26. A fusosome composition comprising a plurality of fusosomes according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23 for use in a method of modulating function in a subject or in CNS tissue or cells of a subject.

27. the CNS cell is a neuron or a glial cell, the CNS cell being a pan-neuronal cell, a GABAergic neuron, a glutamatergic neuron, a cholinergic neuron, a dopaminergic neuron, a serotonergic neuron, a glial cell, an astrocyte, a microglial cell, an oligodendrocyte, or a choroid plexus cell; and / or the CNS tissue or the CNS cell is present in the subject; 27. The fusosome composition or pharmaceutical composition of claim 26.

28. A composition comprising a fusosome according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23 for use in the treatment of a subject having a CNS disease or disorder or a lysosomal disease or disorder.

29. 24. Use of a fusosome according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23 for the manufacture of a medicament for use in treating a subject having a CNS disease or disorder or a lysosomal disease or disorder.

30. 29. The composition or pharmaceutical composition for use according to claim 28, wherein the CNS disease or disorder or lysosomal disease or disorder is caused by a genetic defect.

31. 31. The composition or pharmaceutical composition for use according to claim 28 or claim 30, wherein the disease or disorder is selected from spinocerebellar ataxia; autosomal recessive, type 1; ataxia with oculomotor apraxia, type 2; fragile X syndrome; cerebral creatine deficiency syndrome 1; Angelman syndrome; amyotrophic lateral sclerosis; Friedreich's ataxia; Rett syndrome; Canavan disease; pyridoxine-dependent epilepsy; Batten disease, fucosidosis; Krabbe disease; Tay-Sachs disease; Sandhoff disease; beta-mannosidosis; metachromatic leukodystrophy; mucolipidosis type IIIa; mucolipidosis type IIIb; or mucolipidosis type IV.

32. A composition comprising a fusosome according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 23 for use in treating a subject having a genetic defect.

33. Use of a fusosome according to any of claims 1 to 22 or a pharmaceutical composition according to claim 23 for the manufacture of a medicament for use in treating a subject having a genetic defect.

34. The fusosome composition or pharmaceutical composition of any one of claims 24 to 27, or the composition or pharmaceutical composition of any one of claims 28 and 30 to 32, wherein the subject is a human subject.

35. 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 for producing fusosomes according to any one of claims 1 to 22, comprising: c) isolating, enriching or purifying fusosomes from said cells, thereby producing fusosomes.

36. 30. The use according to claim 29, wherein the CNS disease or disorder or lysosomal disease or disorder is caused by a genetic defect.

37. 37. The use of claim 29 or 36, wherein the disease or disorder is selected from spinocerebellar ataxia; autosomal recessive, type 1; ataxia with oculomotor apraxia, type 2; fragile X syndrome; cerebral creatine deficiency syndrome 1; Angelman syndrome; amyotrophic lateral sclerosis; Friedreich's ataxia; Rett syndrome; Canavan disease; pyridoxine-dependent epilepsy; Batten disease, fucosidosis; Krabbe disease; Tay-Sachs disease; Sandhoff disease; beta-mannosidosis; metachromatic leukodystrophy; mucolipidosis type IIIa; mucolipidosis type IIIb; or mucolipidosis type IV.

38. 34. The use of claim 29 or 33, wherein the subject is a human subject.

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